Abstract:
Therapeutic methods for inhibiting the growth of preneoplastic/neoplastic vertebrate cells that abnormally express MN protein are disclosed. Screening assays are provided for identifying compounds, preferably membrane-impermeant compounds, which inhibit the enzymatic activity of MN protein/polypeptides and that are useful for treating patients with preneoplastic/neoplastic disease. Further methods are disclosed for the preparation of positively-charged, membrane-impermeant heterocyclic sulfonamide CA inhibitors with high affinity for the membrane-bound carbonic anhydrase CA IX. Preferred CA IX-specific inhibitors are aromatic and heterocylic sulfonamides, preferably that are membrane-impermeant. Particularly preferred CA IX-specific inhibitors are pyridinium derivatives of such aromatic and heterocyclic sulfonamides. The CA IX-specific inhibitors of the invention can also be used diagnostically/prognostically for preneoplastic/neoplastic disease, and for imaging use, for example, to detect precancerous cells, tumors and/or metastases. The CA IX-specific inhibitors can be labelled or conjugated to radioisotopes for radiotherapy. The CA IX-specific inhibitors may be combined with conventional therapeutic anti-cancer drugs, with other different inhibitors of cancer-related pathways, with bioreductive drugs, or with radiotherapy to enhance the efficiency of each treatment. The CA IX-specific inhibitors may also be combined with CA IX-specific antibodies, preferably monoclonal antibodies or biologically active antibody fragments, more preferably humanized or fully human CA IX monoclonal antibodies or biologically active fragments or such monoclonal antibodies. Still further, the CA IX-specific inhibitors can be used for gene therapy coupled to vectors for targeted delivery to preneoplastic/neoplastic cells expressing CA IX on their surfaces.

Description:
[0001]    This application claims priority from U.S. Provisional Application Nos. 60/429,089 (filed on Nov. 26, 2002), 60/489,473 (filed on Jul. 22, 2003) and 60/515,140 (filed on Oct. 28, 2003). 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention is in the general area of medical genetics and in the fields of chemistry, biochemical engineering, and oncology. More specifically, it relates to the use of organic and inorganic compounds, preferably aromatic and heterocyclic sulfonamides, to treat preneoplastic and/or neoplastic diseases by specifically inhibiting the carbonic anhydrase activity of the oncoprotein now known alternatively as the MN protein, the MN/CA IX isoenzyme, the MN/G250 protein or simply MN/CA IX or CA IX or MN. The present invention also relates to methods of treating preneoplastic and/or neoplastic diseases characterized by MN/CA IX overexpression by administering cell membrane-impermeant, inhibitors of MN/CA IX, preferably pyridinium derivatives of aromatic and heterocyclic sulfonamides. The invention further concerns diagnostic/prognostic methods including imaging methods, for preneoplastic/neoplastic diseases, using the disclosed potent CA IX-specific inhibitors, and gene therapy with vectors conjugated to said inhibitors.  
         BACKGROUND OF THE INVENTION  
         [0003]    The instant inventors, Dr. Silvia Pastorekova and Dr. Jaromir Pastorek, with Dr. Jan Zavada [“Zavada et al.”], discovered MN/CA IX, a cancer related cell surface protein originally named MN. [73, 123; Zavada et al., U.S. Pat. No. 5,387,676 (Feb. 7, 1995).] Zavada et al., WO 93/18152 (published Sep. 16, 1993) and Zavada et al., WO 95/34650 (published Dec. 21, 1995) disclosed the discovery of the MN gene and protein and the strong association of MN gene expression and tumorigenicity led to the creation of methods that are both diagnostic/prognostic and therapeutic for cancer and precancerous conditions. Zavada et al. disclosed further aspects of the MN/CA IX protein and the MN/CA9 gene in Zavada et al., WO 00/24913 (published May 4, 2000).  
           [0004]    Zavada et al. cloned and sequenced the MN cDNA and gene, and revealed that MN belongs to a carbonic anhydrase family of enzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and proton [66, 72]. MN protein (renamed to carbonic anhydrase IX, CA IX) is composed of an extracellular part containing a N-terminal proteoglycan-like region and a catalytically active carbonic anhydrase domain. It is anchored in the plasma membrane by a single transmembrane region and a short intracytoplasmic tail.  
           [0005]    Expression of CA IX is restricted to only few normal tissues [74], but is tightly associated with tumors [123]. It is also regulated by cell density in vitro [52] and is strongly induced by tumor hypoxia both in vitro and in vivo [121]. Numerous clinical papers describe the value of CA IX as an indicator of poor prognosis. All CA IX-related studies performed so far support the assumption made in the original Zavada et al., U.S. Pat. No. 5,387,676 that CA IX is useful as a diagnostic and/or prognostic tumor marker and as a therapeutic target.  
           [0006]    MN/CA IX consists of an N-terminal proteoglycan-like domain that is unique among the CAs, a highly active CA catalytic domain, a single transmembrane region and a short intracytoplasmic tail [66, 72, 74, 116]. CA IX is particularly interesting for its ectopic expression in a multitude of carcinomas derived from cervix uteri, ovarian, kidney, lung, esophagus, breast, colon, endometrial, bladder, colorectal, prostate, among many other human carcinomas, contrasting with its restricted expression in normal tissues, namely in the epithelia of the gastrointestinal tract [8, 11, 21, 35, 41, 48, 50, 51, 56, 66, 72, 74, 86, 110, 111, 113, 116, 121, 122].  
           [0007]    Uemura et al. [112] reported in 1997 that the G250 antigen was identical to MN/CA IX, years after MN/CA IX had been discovered and sequenced by Zavada et al. {[73, 123]; see also Pastorek et al. [72] and Opavsky et al. [66]}. Uemura et al. [112] stated: “Sequence analysis and database searching revealed that G250 antigen is identical to MN a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994).” 
         MN/CA9 and MN/CA IX—Sequence Similarities  
         [0008]    FIGS.  1 A-C shows the full-length MN/CA9 cDNA sequence of 1522 base pairs (bps) [SEQ ID NO: 1], and the full-length MN/CA IX amino acid (aa) sequence of 459 aa [SEQ ID NO: 2]. FIGS.  2 A-F provides the 10,898 bp genomic sequence of MN/CA9 [SEQ ID NO: 3].  
           [0009]    Computer analysis of the MN cDNA sequence was carried out using DNASIS and PROSIS (Pharmacia Software packages). GenBank, EMBL, Protein Identification Resource and SWISS-PROT databases were searched for all possible sequence similarities. In addition, a search for proteins sharing sequence similarities with MN was performed in the MIPS databank with the FastA program [75].  
           [0010]    The proteoglycan-like domain [aa 53-111; SEQ ID NO: 4] which is between the signal peptide and the CA domain, shows significant homology (38% identity and 44% positivity) with a keratan sulphate attachment domain of a human large aggregating proteoglycan aggrecan [28].  
           [0011]    The CA domain [aa 135-391; SEQ ID NO: 5] is spread over 265 aa and shows 38.9% amino acid identity with the human CA VI isoenzyme [5]. The homology between MN/CA IX and other isoenzymes is as follows: 35.2% with CA II in a 261 aa overlap [63], 31.8% with CA I in a 261 aa overlap [71, 31.6% with CA IV in a 266 aa overlap [65], and 30.5% with CA III in a 259 aa overlap [55].  
           [0012]    In addition to the CA domain, MN/CA IX has acquired both N-terminal and C-terminal extensions that are unrelated to the other CA isoenzymes. The amino acid sequence of the C-terminal part, consisting of the transmembrane anchor and the intracytoplasmic tail, shows no significant homology to any known protein sequence.  
           [0013]    The MN gene (MN/CA9 or CA9) was clearly found to be a novel sequence derived from the human genome. The overall sequence homology between the cDNA MN/CA9 sequence and cDNA sequences encoding different CA isoenzymes is in a homology range of 48-50% which is considered by ones in the art to be low. Therefore, the MN/CA9 cDNA sequence is not closely related to any CA cDNA sequences.  
           [0014]    Very few normal tissues have been found to express MN protein to any significant degree. Those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, and some other normal tissues of the alimentary tract. Paradoxically, MN gene expression has been found to be lost or reduced in carcinomas and other preneoplastic/neoplastic diseases in some tissues that normally express MN, e.g., gastric mucosa.  
         CA IX and Hypoxia  
         [0015]    Strong association between CA IX expression and intratumoral hypoxia (either measured by microelectrodes, or detected by incorporation of a hypoxic marker pimonidazole, or by evaluation of extent of necrosis) has been demonstrated in the cervical, breast, head and neck, bladder and non-small cell lung carcinomas (NSCLC) [8, 11, 21, 35, 48, 56, 111, 122]. Moreover, in NSCLC and breast carcinomas, correlation between CA IX and a constellation of proteins involved in angiogenesis, apoptosis inhibition and cell-cell adhesion disruption has been observed, possibly contributing to strong relationship of this enzyme to a poor clinical outcome [8]. Hypoxia is linked with acidification of extracellular milieu that facilitates tumor invasion and CA IX is believed to play a role in this process via its catalytic activity [86]. Thus, inhibition of MN/CA IX by specific inhibitors is considered to constitute a novel approach to the treatment of cancers in which CA IX is expressed.  
         CAIs  
         [0016]    Teicher et al. [106] reported that acetazolamide—the prototypical CA inhibitor (CAI)—functions as a modulator in anticancer therapies, in combination with different cytotoxic agents, such as alkylating agents; nucleoside analogs; platinum derivatives, among other such agents, to suppress tumor metastasis and to reduce the invasive capacity of several renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). Such studies demonstrate that CAIs may be used in the management of tumors that overexpress one or more CA isozymes. It was hypothesized that the anticancer effects of acetazolamide (alone or in combination with such drugs) might be due to the acidification of the intratumoral environment ensuing after CA inhibition, although other mechanisms of action of this drug were not excluded [20]. Chegwidden et al. 2001 hypothesized that the in vitro inhibition of growth in cell cultures, of human lymphoma cells with two other potent, clinically used sulfonamide CAIs, methazolamide and ethoxzolamide, is probably due to a reduced provision of bicarbonate for nucleotide synthesis (HCO 3   −  is the substrate of carbamoyl phosphate synthetase II) as a consequence of CA inhibition [20].  
           [0017]    All the six classical CAIs (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and dichlorophenamide) used in clinical medicine or as diagnostic tools, show some tumor growth inhibitory properties [18, 78, 101, 102].  
           [0018]    The inventors, Dr. Claudia Supuran and Dr. Andrea Scozzafava, reported the design and in vitro antitumor activity of several classes of sulfonamide CAIs, shown to act as nanomolar inhibitors against the classical isozymes known to possess critical physiological roles, such as CA I, CA II and CA IV. Those compounds were also shown to exert potent inhibition of cell growth in several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines, with GI 50  values of 10-75 nM in some cases [77, 91, 92, 100].  
           [0019]    Wingo et al. reported that three classic sulfonamide drugs (acetozolamide, ethoxzolamide and methoxzolamide) inhibited CA IX carbonic anhydrase activity with values of K I  in the nanomolar range [116]. However, until the present invention, no systematic structure-activity relationship study of sulfonamide inhibition of CA IX, alone or in comparison to other CA isozymes had been been performed.  
           [0020]    Certain pyridinium derivatives of aromatic/heterocyclic sulfonamides have shown nanomolar affinities both for CA II, as well as CA IV, and more importantly, they were unable to cross the plasma membranes in vivo [17].  
           [0021]    Sterling et al. [85] investigated the functional and physical relationship between the downregulated in adenoma bicarbonate transporter and CA II, by using membrane-impermeant sulfonamide inhibitors (in addition to the classical inhibitors such as acetazolamide), which could clearly discriminate between the contribution of the cytosolic and membrane-associated isozymes in these physiological processes.  
         CAS  
         [0022]    Carbonic anhydrases (CAs) form a large family of genes encoding zinc metalloenzymes of great physiological importance. As catalysts of reversible hydration of carbon dioxide, these enzymes participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, formation of aqueous humor, cerebrospinal fluid, saliva and gastric acid [reviewed in Dodgson et al. (27)]. CAs are widely distributed in different living organisms. In higher vertebrates, including humans, 14 different CA isozymes or CA-related proteins (CARP) have been described, with very different subcellular localization and tissue distribution [40, 93, 95, 94, 102]. Basically, there are several cytosolic forms (CA I-III, CA VII), four membrane-bound isozymes (CA IV, CA IX, CA XII and CA XIV), one mitochondrial form (CA V) as well as a secreted CA isozyme, CA VI [40, 93, 94, 95, 102].  
           [0023]    It has been shown that some tumor cells predominantly express only some membrane-associated CA isozymes, such as CA IX and CA XII [2, 67, 68, 78, 87, 93, 95]. Occasionally, nuclear localization of some isoenzymes has been noted [64, 69, 70]. Not much is presently known about the cellular localization of the other isozymes.  
           [0024]    CAs and CA-related proteins show extensive diversity in their tissue distribution, levels, and putative or established biological functions [105]. Some of the CAs are expressed in almost all tissues (CA II), while the expression of others appears to be more restricted (e.g., CA VI and CA VII in salivary glands [32, 69, 71]. The CAs and CA-related proteins also differ in kinetic properties and susceptibility to inhibitors [82].  
           [0025]    Most of the clinically used sulfonamides mentioned above are systemically acting inhibitors showing several undesired side effects due to inhibition of many of the different CA isozymes present in the target tissue/organ (14 isoforms are presently known in humans) [93, 94, 95, 102]. Therefore, many attempts to design and synthesize new sulfonamides were recently reported, in order to avoid such side effects [13, 17, 42, 62, 80, 99, 100]. At least four CA isozymes (CA IV, CA IX, CA XII and CA XIV) are associated to cell membranes, with the enzyme active site generally oriented extracellularly [93, 94, 95, 102]. Some of these isozymes were shown to play pivotal physiological roles (such as for example CA IV and XII in the eye, lungs and kidneys, CA IX in the gastric mucosa and many tumor cells) [3, 18, 22, 29, 49, 67, 68, 83, 93, 94, 95, 102], whereas the function of other such isozymes (CA XIV) is for the moment less well understood [93, 95]. Due to the extracellular location of these isozymes, if membrane-impermeant CA inhibitors (CAIs) could be designed, only membrane-associated CAs would be affected.  
           [0026]    The first approach towards introducing the membrane-impermeability to CAIs from the historical point of view was that of attaching aromatic/heterocyclic sulfonamides to polymers, such as polyethyleneglycol, aminoethyldextran, or dextran [39, 60, 107]. Such compounds, possessing molecular weights in the range of 3.5-99 kDa, prepared in that way, showed indeed membrane-impermeability due to their high molecular weights, and selectively inhibited in vivo only CA IV and not the cytosolic isozymes (primarily CA II), being used in several renal and pulmonary physiological studies [39, 60, 107]. Due to their macromolecular nature, such inhibitors could not be developed as drugs/diagnostic tools, since in vivo they induced potent allergic reactions [39, 60, 93, 95, 107]. A second approach for achieving membrane-impermeability is that of using highly polar, salt-like compounds. Only one such sulfonamide has until recently been used in physiological studies, QAS (quaternary ammonium sulphanilamide), which has been reported to inhibit only extracellular CAs in a variety of arthropods (such as the crab  Callinectes sapidus ) and fish [57]. The main draw-back of QAS is its high toxicity in higher vertebrates [57].  
           [0027]    Enzyme activity of carbonic anhydrases (including that of CA IX) can be efficiently blocked by sulfonamide inhibitors. That fact has been therapeutically exploited in diseases caused by excessive activities of certain CA isoforms (e.g. CA II in glaucoma). There is also an experimental evidence that sulfonamides may block tumor cell proliferation and invasion in vitro and tumor growth in vivo, but the targets of those sulfonamides have not been identified yet. However, the sulfonamides available so far indiscriminately inhibit various CA isoenzymes (14 are presently known in humans) that are localized in different subcellular compartments and play diverse biological roles. This lack of selectivity compromises the clinical utilization of these compounds (due to undesired side effects caused by concurrent inhibition of many CA isoforms) and represents a main drawback also for the sulfonamide application against CA IX in anticancer therapy.  
           [0028]    Thus, there is a need in the art for membrane-impermeant, potent CA IX inhibitors, which would become doubly selective inhibitors for CA IX. The inventors have previously made and described some of the membrane-impermeant molecules described here; however, they were characterized only for their ability to inhibit CA I, CA II and CA IV. While others have studied effects of selective inhibition of extracellular CA by membrane impermeant agents in retinal prigmented epithelia or muscle [34, 120], these agents have not been characterized for their ability to inhibit CA IX. Since CA IX is one of the few extracellular carbonic anhydrases, a membrane-impermeant selective inhibitor of CA IX would be doubly selective for this enzyme and thereby avoid side effects associated with nonspecific CA inhibition.  
         SUMMARY OF THE INVENTION  
         [0029]    The inventors approached the problem of lack of selectivity of CAIs by taking advantage of features that distinguish CA IX from the other CA isoforms. First of all, CA IX is an integral plasma membrane protein with an active site exposed on the extracellular side. In this respect, it is similar to some CAs (CA IV, CA XII and CA XIV) but differs from all other isoforms. Among these membrane-bound isoenzymes, CA IX shows some differences in the amino acid sequence of the catalytic domain that may influence the topology of the active site cavity and hence the interaction with sulfonamides. In addition, unlike the other CA isoforms, CA IX is expressed preferentially in hypoxic areas of tumors with poor prognosis.  
           [0030]    The inventors evaluated inhibition profiles of CA IX with a series of aromatic and heterocyclic compounds and found that some of them inhibit CA IX more efficiently than the other widely distributed isoforms CA I, II and IV. Several nanomolar CA IX inhibitors have been detected both among the aromatic and the heterocyclic compounds. This finding is very promising for the design of CA IX-specific inhibitors by modification of their physico-chemical properties such as charge, size and bioreductivity to conform the characteristic properties of CA IX.  
           [0031]    The inventors found that some of the more bulky compounds that strongly inhibited CA IX were very weak inhibitors of CA I, II and IV, possibly due to the fact that the CA IX active site cavity is larger than that of the other investigated isoenzymes. The compounds of such type, identified by screening as disclosed herein, based on the selective inhibition of tumor-associated isoform CA IX may be particularly preferred CA IX specific inhibitors, that could be used in new anticancer therapies and in the diagnostic/prognostic methods of this invention.  
           [0032]    The inventors have shown that CA IX is capable of reducing E-cadherin-mediated cell-cell adhesion that may be important for increased invasion capacity of the cells [103]. CA IX was found by the inventors also to contribute to acidification of extracellular pH in hypoxia but not in normoxia (unpublished data). The latter result indicates that hypoxia up-regulates both expression level and enzyme activity of CA IX, that is, hypoxia activates the CA catalytic activity of CA IX. That is a very important finding because intratumoral hypoxia is a clinically relevant factor increasing aggressiveness of tumor cells and reducing success of therapy. Hypoxia is usually accompanied by acidification of extracellular microenvironment, which facilitates tumor invasion and metastasis. CA IX appears to participate in this phenomenon by catalyzing hydration of carbon dioxide to generate bicarbonate ions that are then transported into cell interior and protons that acidify extracellular pH. Therefore, inhibition of the CA IX catalytic activity resulting in reduced extracellular acidification may have direct anticancer effects or may modulate efficiency of those conventional chemotherapeutic drugs whose uptake is pH-dependent.  
           [0033]    The instant invention is related to (1) the recognition that certain carbonic anhydrase inhibitors (CAIs), preferably sulfonamides, selectively target the cancer-related, hypoxia-induced MN/CA IX; (2) the use of such CAIs, preferably sulfonamides, as lead compounds for the design and synthesis of MN/CA IX-specific inhibitors; (3) the employment of said MN/CA IX-specific inhibitors for anticancer therapy based upon the inhibition of MN/CA IX-mediated acidification of tumor microenvironments; and (4) the use of the specificity of potent MN/CA IX-specific inhibitors for diagnostic/prognostic methods including imaging methods, such as scintigraphy, and for gene therapy. The invention is particularly directed to the use of CA IX-specific inhibitors for the development of drugs possessing anticancer properties and to modulate conventional chemotherapy for preneoplastic and neoplastic disease characterized by CA IX expression, particularly CA IX overexpression.  
           [0034]    In one aspect, the invention concerns methods of treating a mammal for a pre-cancerous or cancerous disease, wherein said disease is characterized by overexpression of MN/CA IX protein, comprising administering to said mammal a therapeutically effective amount of a composition comprising a compound, wherein said compound is selected from the group consisting of organic and inorganic molecules, and wherein said compound is determined to be a potent inhibitor of MN/CA IX enzymatic activity in a screening assay comprising:  
           [0035]    a) preparing serial dilutions of said compound and serial dilutions of MN/CA IX protein or a fragment of the MN/CA IX protein that comprises the carbonic anhydrase domain;  
           [0036]    b) preincubating a dilution of said compound with a dilution of said MN/CA IX protein or said MN/CA IX protein fragment for ten minutes at 20° C.;  
           [0037]    c) combining said preincubated mixture of said diluted compound and said diluted MN/CA IX protein or protein fragment with a substrate, consisting essentially of a saturated CO 2  solution, phenol red to 0.2 mM, Na 2 SO 4  to 0.1M, and Hepes buffer (pH 7.5) to 10 mM, in a reaction vessel for a period of 10 to 100 seconds at 20° C.;  
           [0038]    d) concurrently measuring the optical density, at the absorbance maximum of 557 nm, of the contents of said reaction vessel, using a stopped flow spectrophotometer; and  
           [0039]    e) determining the inhibition constant K I  of said compound;  
           [0040]    wherein if said inhibition constant K I  is determined to be less than about 50 nanomolar, said compound is determined be a potent inhibitor of MN/CA IX enzymatic activity; and wherein said compound is not selected from the group consisting of acetazolamide, ethoxzolamide, methazolamide and cyanate. Said mammal is preferably human, and said K I  is preferably less than about 35 nanomolar, more preferably less than about 25 nanomolar, and still more preferably less than about 10 nanomolar.  
           [0041]    Such methods can also be framed as methods of treating precancer and/or cancer, or inhibiting the growth of precancerous and/or cancerous cells in a mammalian subject, wherein said precancer and cancer are characterized by the overexpression of MN/CA IX. Said methods can also be framed as inhibiting the growth of such precancerous or cancerous mammalian cells overexpressing MN/CA IX comprising contacting said cells with a CA IX-specific inhibitor of this invention.  
           [0042]    The CA IX-specific inhibitors of this invention can be administered in a therapeutically effective amount, preferably dispersed in a physiologically acceptable nontoxic liquid vehicle. Different routes of administration may be preferred depending on the site or type of preneoplastic/neoplastic disease, for example, solid or non-solid tumor or metastasis. In general, parenteral administration would be preferred to avoid undesired effects of systemic treatment, for example, those that could be occasioned by binding of the inhibitors to the gastrointestinal mucosa. Injection into or into the vicinity of the preneoplastic/neoplastic disease would be generally preferred. For example, such injections could be intravenous, intraperitoneal, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intramedullary, intralesional, intradermal, among other routes of injection. Also, other modes of administration, for example, by suppository or topically, can be used as would be appropriate to the target disease. The pharmaceutical formulation would be designed in accordance with known standards as suitable for the route of administration.  
           [0043]    Said CA IX-specific inhibitors are preferably organic, more preferably aromatic or heterocyclic, and still more preferably an aromatic sulfonamide or a heterocyclic sulfonamide. Said aromatic sulfonamide may be a substituted aromatic sulfonamide, wherein said aromatic sulfonamide comprises an aromatic ring structure bearing a sulfonamide moiety bonded to said ring structure and optionally bearing one or more substituents independently selected from the group consisting of halogeno, nitro, and an alkylamino group, wherein the alkyl radical of said alkylamino group comprises 1 to 4 carbon atoms.  
           [0044]    Preferably the CA IX-specific inhibitors of this invention are more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of a carbonic anhydrase selected from the group consisting of CA I, CA II and CA IV. More preferably, the CA IX-specific inhibitors are more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of at least two carbonic anhydrases selected from the group consisting of CA I, CA II and CA IV. Still more preferably, the CA IX-specific inhibitors are more potent inhibitor of MN/CA IX enzymatic activity than of the enzymatic activity of each of the carbonic anhydrases in the group consisting of CA I, CA II and CA IV.  
           [0045]    However, since CA II is a particularly abundant and significant CA, that is cytosolic, it is important when the CA IX-specific inhibitors of this invention are not membrane-impermeant, that they may be more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of CA II. A method comprising the following steps provides an exemplary screening assay that can be used to determine the K I  of a compound in inhibiting the enzymatic activity of CA II:  
           [0046]    a) preparing serial dilutions of said compound and serial dilutions of CA II;  
           [0047]    b) preincubating a dilution of said compound with a dilution of CA II for ten minutes at 20° C.;  
           [0048]    c) combining said preincubated mixture of said compound and said CA II with a substrate solution, consisting essentially of 4-nitrophenylacetate in anhydrous acetonitrile, in a reaction vessel for a period of 1 to 3 minutes at 25° C.;  
           [0049]    d) concurrently measuring the optical density, at the absorbance maximum of 400 nm, of the contents of said reaction vessel, using a spectrophotometer; and  
           [0050]    e) determining the inhibition constant K I  of said compound.  
           [0051]    Exemplary and preferred aromatic sulfonamide or heterocyclic sulfonamide CA IX-specific inhibitors of this invention are selected from the group consisting of:  
                         

                         

                         

                         
 
           [0052]    Exemplary preferred aromatic sulfonamide CA IX-specific inhibitors are selected from the group consisting of:  
                         
 
           [0053]    A preferred aromatic sulfonamide CA IX-specific inhibitor can be that wherein a halogen atom is bonded to at least one carbon atom in the aromatic ring of said aromatic sulfonamide.  
           [0054]    Preferred heterocyclic sulfonamide CA IX-specific inhibitors can be substituted heterocyclic sulfonamides, wherein said substituted heterocyclic sulfonamide comprises a heterocyclic ring structure bearing a sulfonamide moiety bonded to said ring structure and optionally bearing one or more substituents independently selected from a group consisting of halogeno, nitro, and an alkylamino group, wherein the alkyl radical of said alkylamino group comprises 1 to 4 carbon atoms. Preferred heterocyclic sulfonamide CA IX-specific inhibitors may be halogenated.  
           [0055]    Further preferred heterocyclic sulfonamide CA IX-specific inhibitors are selected from the group consisting of:  
                         
 
           [0056]    Further preferred methods of treating mammals for pre-cancerous or cancerous disease, wherein said disease is characterized by overexpression of MN/CA IX protein, comprise administering to said mammal membrane-impermeant CA IX-specific inhibitors. A therapeutically effective amount of such a membrane-impermeant CA IX-specific inhibitor can be administered in a composition comprising the membrane-impermeant compound, wherein said membrane-impermeant inhibitor compound is selected from the group consisting of organic and inorganic molecules, and wherein said membrane-impermeant compound is determined to be a potent inhibitor of MN/CA IX enzymatic activity in a screening assay comprising:  
           [0057]    a) preparing serial dilutions of said membrane-impermeant compound and serial dilutions of MN/CA IX protein or a fragment of the MN/CA IX protein that comprises the carbonic anhydrase domain;  
           [0058]    b) preincubating a dilution of said membrane-impermeant compound with a dilution of said MN/CA IX protein or said MN/CA IX protein fragment for ten minutes at 20° C.;  
           [0059]    c) combining said preincubated mixture of said diluted compound and said diluted MN/CA IX protein or protein fragment with a substrate, consisting essentially of a saturated CO 2  solution, phenol red to 0.2 mM, Na 2 SO 4  to 0.1M, and Hepes buffer (pH 7.5) to 10 mM, in a reaction vessel for a period of 10 to 100 seconds at 20° C.;  
           [0060]    d) concurrently measuring the optical density, at the absorbance maximum of 557 nm, of the contents of said reaction vessel, using a stopped flow spectrophotometer; and  
           [0061]    e) determining the inhibition constant K I  of said membrane-impermeant compound,  
           [0062]    wherein if said inhibition constant K I  is determined to be less than about 50 nanomolar, said membrane-impermeant compound is determined be a potent inhibitor of MN/CA IX enzymatic activity. The mammal is preferably a human, and the K I  is preferably less than 35 nM, more preferably less than about 25 nM, and still more preferably less than about 10 nanomolar.  
           [0063]    Such a membrane-impermeant CA IX specific inhibitor compound is preferably organic, and more preferably a pyridinium derivative of an aromatic sulfonamide or a pyridinium derivative of a heterocyclic sulfonamide. Such membrane-impermeant CA IX-specific inhibitor compounds are preferably more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of a carbonic anhydrase selected from the group consisting of CA I, CA II and CA IV, and still more preferably more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of at least two carbonic anhydrases selected from the group consisting of CA I, CA II and CA IV. Further more preferably, said membrane-impermeant CA IX-specific inhibitor compounds are more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of each of the carbonic anhydrases in the group consisting of CA I, CA II and CA IV. Since both CA IX and CA IV are membrane bound CAs, it is particularly important that the membrane-impermeant CA IX-specific inhibitor compounds are more potent inhibitors of MN/CA IX enzymatic activity than of the enzymatic activity of CA IV.  
           [0064]    A method comprising the following steps provides an exemplary screening assay that can be used to determine the K I  of a compound inhibiting the enzymatic activity of CA IV:  
           [0065]    a) preparing serial dilutions of said membrane-impermeant compound and serial dilutions of CA IV;  
           [0066]    b) preincubating a dilution of said membrane-impermeant compound with a dilution of CA IV for ten minutes at 20° C.;  
           [0067]    c) combining said preincubated mixture of said compound and said CA IV with a substrate solution, consisting essentially of 4-nitrophenylacetate in anhydrous acetonitrile, in a reaction vessel for a period of 1 to 3 minutes at 25° C.;  
           [0068]    d) concurrently measuring the optical density, at the absorbance maximum of 400 nm, of the contents of said reaction vessel using a spectrophotometer; and  
           [0069]    e) determining the inhibition constant K I  of said membrane-impermeant compound.  
           [0070]    Preferred membrane-impermeant CA IX-specific inhibitor compounds that are pyridinium derivatives of aromatic sulfonamides are selected from the group consisting of sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide. Preferred pyridinium derivatives of aromatic sulfonamides can have the general formula of:  
                         
 
           [0071]    wherein  
           [0072]    n is 0, 1, or 2;  
           [0073]    R2, R3, R4 and R6 are each independently selected from the group consisting of hydrogen, alkyl moieties comprising from 1 to 12 carbon atoms, and aryl moieties. Further preferred are such compounds wherein  
           [0074]    R2 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl and phenyl;  
           [0075]    R3 is selected from the group consisting of hydrogen and methyl;  
           [0076]    R4 is selected from the group consisting of hydrogen, methyl and phenyl; and  
           [0077]    R6 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, and phenyl. Still further preferred are such compounds wherein  
           [0078]    R3 is hydrogen;  
           [0079]    R4 and R6 are phenyl;  
           [0080]    when n is 0, R2 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, and phenyl; and  
           [0081]    when n is 1 or 2, R2 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, and phenyl. Other preferred such compounds include those wherein  
           [0082]    R3 is hydrogen;  
           [0083]    R4 is phenyl; and  
           [0084]    when n is 0, R2 and R6 are the same and are selected from the group consisting of methyl, ethyl, n-propyl, and iso-propyl; and  
           [0085]    when n is 1 or 2, R2 and R6 are the same and are selected from the group consisting of methyl, ethyl, n-propyl and iso-propyl. Other preferred compounds include those wherein R2, R3, R4 and R6 are methyl. Still further preferred are such CA IX-specific inhibitor compounds wherein  
           [0086]    when n is 0, 1 or 2, R2, R4 and R6 are methyl, and R3 is hydrogen; or  
           [0087]    when n is 1 or 2, R2 is iso-propyl, R3 is hydrogen, R4 is methyl, and R6 is methyl or iso-propyl; or  
           [0088]    when n is 1 or 2, R2 and R6 are phenyl, and R3 and R4 are hydrogen.  
           [0089]    Still more preferred such compounds are those wherein  
           [0090]    when n is 2, R2 and R6 are methyl, R3 is hydrogen, and R4 is phenyl; or  
           [0091]    when n is 2, R2 and R6 are ethyl, R3 is hydrogen, and R4 is phenyl; or  
           [0092]    when n is 2, R2, R3, R4 and R6 are methyl.  
           [0093]    When said CA IX-specific inhibitors are membrane-impermeant pyridinium derivatives of a heterocyclic sulfonamides, a preferred compound is a pyridinium derivative of aminobenzolamide.  
           [0094]    Preferred CA IX-specific inhibitor compounds that are pyridinium derivatives of heterocyclic sulfonamides may have the general formula of:  
                         
 
           [0095]    wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, alkyl moieties comprising from 1 to 12 carbon atoms, and aryl moieties. Further preferred are such compounds wherein  
           [0096]    R1 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, tert-butyl and phenyl;  
           [0097]    R2 is selected from the group consisting of hydrogen and methyl;  
           [0098]    R3 is selected from the group consisting of hydrogen, methyl, n-nonyl, and phenyl;  
           [0099]    R4 is selected from the group consisting of hydrogen and methyl; and  
           [0100]    R5 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, tert-butyl, n-nonyl and phenyl. Further preferred are such compounds wherein  
           [0101]    R2 and R4 are hydrogen;  
           [0102]    R3 is methyl; and  
           [0103]    R1 and R5 are the same and selected from the group consisting of methyl, iso-propyl, and tert-butyl. Still further preferred are such compounds wherein  
           [0104]    R2 and R4 are hydrogen;  
           [0105]    R3 is phenyl; and  
           [0106]    R1 and R5 are the same and selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, and phenyl. Additionally are preferred such compounds wherein  
           [0107]    R1 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, and n-butyl;  
           [0108]    R2 and R4 are hydrogen; and  
           [0109]    R3 and R5 are phenyl. Other preferred such compounds are those wherein  
           [0110]    R2 and R4 are hydrogen, R3 is hydrogen or methyl, and R1 and R5 are phenyl; or  
           [0111]    R1, R2, and R5 are methyl, R3 is phenyl, and R4 is hydrogen; or  
           [0112]    R1 and R4 are methyl, R2-is hydrogen, and R3 and R5 are n-nonyl.  
           [0113]    Also preferred such compounds are those wherein  
           [0114]    R1 is methyl or iso-propyl, R3 and R5 are methyl, and R2 and R4 are hydrogen; or  
           [0115]    R1 and R5 are the same and are methyl or ethyl, R2 and R4 are hydrogen, and R3 is phenyl; or  
           [0116]    R1, R2, R3 and R5 are methyl, and R4 is hydrogen.  
           [0117]    In another aspect, this invention concerns methods of inhibiting tumor growth in a patient having a tumor, the cells of which tumor are characterized by overexpression of MN/CA IX protein, comprising administering to said patient a therapeutically effective amount of a composition comprising a compound, wherein said compound is selected from the group consisting of organic and inorganic molecules, and wherein said compound is determined to be a potent inhibitor of MN/CA IX enzymatic activity in a screening assay as outlined above for MN/CA IX using a saturated C0 2  solution.  
           [0118]    Still further, this invention concerns novel compounds that are useful as CA IX-specific inhibitors in a variety of methods disclosed herein. Such novel compounds include pyridinium derivatives of heterocyclic sulfonamides with the general formula of:  
                         
 
           [0119]    wherein  
           [0120]    R1 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, tert-butyl and phenyl;  
           [0121]    R2 is selected from the group consisting of hydrogen and methyl;  
           [0122]    R3 is selected from the group consisting of hydrogen, methyl, n-nonyl and phenyl;  
           [0123]    R4 is selected from the group consisting of hydrogen and methyl; and  
           [0124]    R5 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, tert-butyl, n-nonyl and phenyl, except that  
           [0125]    R1 cannot be methyl when R2 and R4 are hydrogen and R3 and R5 are methyl; and  
           [0126]    R1 cannot be methyl when R2 and R4 are hydrogen, R3 is phenyl and R5 is methyl; and  
           [0127]    R1 cannot be phenyl when R2 and R4 are hydrogen and R3 and R5 are phenyl. Preferred such pyridinium derivatives of heterocyclic sulfonamides include those wherein  
           [0128]    R2 and R4 are hydrogen;  
           [0129]    R3 is methyl; and  
           [0130]    R1 and R5 are the same and selected from the group consisting of iso-propyl and tert-butyl, and those wherein  
           [0131]    R2 and R4 are hydrogen;  
           [0132]    R3 is phenyl; and  
           [0133]    R1 and R5 are the same and selected from the group consisting of ethyl, iso-propyl, n-propyl, and n-butyl, and further preferably those wherein  
           [0134]    R1 is selected from the group consisting of methyl, ethyl, iso-propyl, n-propyl, n-butyl, and tert-butyl;  
           [0135]    R2 and R4 are hydrogen; and  
           [0136]    R3 and R5 are phenyl. Still further preferred are those pyridinium derivatives of heterocyclic sulfonamides, wherein  
           [0137]    R1 is iso-propyl, R3 and R5 are methyl, and R2 and R4 are hydrogen; or  
           [0138]    R2 and R4 are hydrogen, R3 is hydrogen or methyl, and R1 and R5 are phenyl; or  
           [0139]    R1, R2, and R5 are methyl, R3 is phenyl, and R4 is hydrogen; or  
           [0140]    R1, R2, R3 and R5 are methyl and R4 is hydrogen; or  
           [0141]    R1 and R4 are methyl, R2 is hydrogen and R3 and R5 are n-nonyl.  
           [0142]    In another therapeutic aspect of the invention, the CA IX-specific inhibitors can be conjugated to radioisotopes for administration. Also, the CA IX-specific inhibitors can be administred concurrently and/or sequentially with radiation and/or with a therapeutically effective amount in a physiologically acceptable formulation of one or more of the following compounds selected from the group consisting of: conventional anticancer drugs, chemotherapeutic agents, different inhibitors of cancer-related pathways, bioreductive drugs, CA IX-specific antibodies and CA IX-specific antibody fragments that are biologically active. Preferably said CA IX-specific antibodies and/or CA IX-specific antibody fragments are humanized or fully human, and may be attached to a cytotoxic entity.  
           [0143]    In another therapeutic aspect, this invention concerns methods of treating a mammal for a precancerous or cancerous disease, wherein said disease is characterized by overexpression of MN/CA IX protein, comprising administering to said mammal a therapeutically effective amount in a physiologically acceptable formulation of a vector conjugated to a potent CA IX-specific inhibitor, wherein said vector expresses a wild-type gene that is absent from or mutated in a CA IX expressing cell, that is precancerous or cancerous, and wherein the wild type gene product has an anticancer effect in said cell; or wherein said vector comprises a gene that expresses a cytotoxic protein. An exemplary wild-type gene would be the von Hippel-Lindau gene known to be directly involved in the constitutive expression of CA IX in renal cell carcinoma.  
           [0144]    Preferably said vector comprises a MN/CA IX promoter or a MN/CA IX promoter fragment, wherein said promoter or promoter fragment comprises one or more hypoxia response elements (HREs), and wherein said promoter or promoter fragment is operably linked to said wild-type gene or to said gene that expresses a cytotoxic protein. Preferably the CA IX-specific inhibitor conjugated to the vector has a K I  as determined above in the C0 2  saturation assay to be less than about 50 nM, more preferably less than about 35 nM, still more preferably less than about 25 nM and still further more preferably less than about 10 nM. Preferably, said potent MN/CA IX inhibitor is not selected from the group consisting of acetazolamide, ethoxzolamide, methazolamide and cyanate.  
           [0145]    Still in another aspect, this invention concerns methods that are diagnostic or diagnostic and prognostic for precancer or cancer. For example, such methods may comprise contacting a mammalian sample with a CA IX-specific inhibitor conjugated to a label or a visualizing means, and detecting or detecting and quantifying binding of said CA IX-specific inhibitor to cells in said sample by detecting or detecting and quantifying said label or said visualizing means on cells in said sample, wherein said detection or said detection and quantitation at a level above that for a control sample is indicative of precancerous or cancerous cells that overexpress CA IX in said sample.  
           [0146]    Such methods can be of particular diagnostic and prognostic importance by detecting or detecting and quantitating CA IX activated by hypoxic conditions. Hypoxia combined with CA IX overepression indicates that the mammal from whom the sample was taken is considered to have a poorer prognosis, and decisions on treatment for said mammal are made in view of the presence of said hypoxic conditions. MN/CA IX as a hypoxia marker is useful in general in making therapeutic decisions. For example, a cancer patient whose tumor is known to express MN/CA IX at an abnormally high level would not be a candidate for certain kinds of chemotherapy and radiotherapy, but would be a candidate for hypoxia-selective chemotherapy.  
           [0147]    Brown, J. M. [16] points out at page 157 that “solid tumours are considerably less well oxygenated than normal tissues. This leads to resistance to radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumour metastases.” Brown explains how tumor hypoxia can be exploited in cancer treatment. One strategy to exploit tumor hypoxia for cancer treatment proposed by Brown [16] is to use drugs that are toxic only under hypoxic conditions. Exemplary and preferred drugs that could be used under that strategy include tirapazamine and AQ4N, a di-N-oxide analogue of mitozantrome.  
           [0148]    A second mode of exploiting hypoxia proposed by Brown [16] is by gene therapy strategies developed to take advantage of the selective induction of HIF-1. Brown notes that a tumor-specific delivery system can be developed wherein a promoter that is highly responsive to HIF-1 would drive the expression of a conditionally lethal gene under hypoxic but not normoxic conditions. The MN/CA IX promoter is just such a promoter highly responsive to hypoxia, as well as MN/CA IX promoter fragments comprising one or more HREs. “Expression of an enzyme not normally found in the human body could, under the control of a hypoxia-responsive promoter [the MN/Ca IX promoter], convert a nontoxic pro-drug into a toxic drug in the tumour.” [Brown [16], page 160.] Exemplary is the use of the bacterial cytosine deaminase, which converts the nontoxic 5-fluorocytosine to the anticancer drug 5-fluorouracil (5FU) cited by Brown to Trinh et al. [109].  
           [0149]    Ratcliffe et al., U.S. Pat. Nos. 5,942,434 and 6,265,390 explain how anti-cancer drugs become activated under hypoxia [119], but that the use of a drug activation system, wherein the enzyme that activates the drug is significantly increased under hypoxia, results in much enhanced therapeutic effect.  
           [0150]    This invention further concerns methods for imaging tumors and/or metastases that express CA IX in a patient comprising the administration of a CA IX-specific inhibitor linked to an imaging agent to said patient. A preferred imaging method would encompass scintigraphy.  
           [0151]    The assays of this invention are both diagnostic and/or prognostic, i.e., diagnostic/prognostic. The term “diagnostic/ prognostic” is herein defined to encompass the following processes either individually or cumulatively depending upon the clinical context: determining the presence of disease, determining the nature of a disease, distinguishing one disease from another, forecasting as to the probable outcome of a disease state, determining the prospect as to recovery from a disease as indicated by the nature and symptoms of a case, monitoring the disease status of a patient, monitoring a patient for recurrence of disease, and/or determining the preferred therapeutic regimen for a patient. The diagnostic/prognostic methods of this invention are useful, for example, for screening populations for the presence of neoplastic or pre-neoplastic disease, determining the risk of developing neoplastic disease, diagnosing the presence of neoplastic and/or pre-neoplastic disease, monitoring the disease status of patients with neoplastic disease, and/or determining the prognosis for the course of neoplastic disease.  
           [0152]    The present invention is useful for treating and for screening the presence of a wide variety of preneoplastic/neoplastic diseases including carcinomas, such as, mammary, colorectal, urinary tract, ovarian, uterine, cervical, endometrial, squamous cell and adenosquamous carcinomas; head and neck cancers; mesodermal tumors, such as, neuroblastomas and retinoblastomas; sarcomas, such as osteosarcomas and Ewing&#39;s sarcoma; and melanomas. Of particular interest are gynecological cancers including ovarian, uterine, cervical, vaginal, vulval and endometrial cancers, particularly ovarian, uterine cervical and endometrial cancers. Also of particular interest are cancers of the breast, of gastrointestinal tract, of the stomach including esophagus, of the colon, of the kidney, of the prostate, of the liver, of the urinary tract including bladder, of the lung, and of the head and neck. Gynecologic cancers of particular interest are carcinomas of the uterine cervix, endometrium and ovaries; more particularly such gynecologic cancers include cervical squamous cell carcinomas, adenosquamous carcinomas, adenocarcinomas as well as gynecologic precancerous conditions, such as metaplastic cervical tissues and condylomas.  
           [0153]    The invention provides methods and compositions for evaluating the probability of the presence of malignant or pre-malignant cells, for example, in a group of cells freshly removed from a host. Such an assay can be used to detect tumors, quantitate their growth, and help in the diagnosis and prognosis of disease. The assays can also be used to detect the presence of cancer metastasis, as well as confirm the absence or removal of all tumor tissue following surgery, cancer chemotherapy and/or radiation therapy. It can further be used to monitor cancer chemotherapy and tumor reappearance.  
           [0154]    The presence of MN antigen can be detected and/or quantitated using a number of well-defined diagnostic assays. Those in the art can adapt any of the conventional immunoassay formats to detect and/or quantitate MN antigen as herein disclosed. The immunoassays of this invention can be embodied in test kits which comprise the potent CA IX-specific inhibitors of this invention, appropriately labeled and/or linked to a visualizing means, as known in the art. Such test kits can be in solid phase formats, but are not limited thereto, and can also be in liquid phase format, and can be based on immunohistochemical assays, ELISAS, particle assays, radiometric or fluorometric assays either unamplified or amplified, using, for example, avidin/biotin technology, among other assay formats.  
           [0155]    Exemplary CA IX-specific inhibitors of the invention are shown herein to treat transfected cells that constitutively express MN/CA IX compared to non-transfected cells with no MN/CA IX expression. The exemplary CA IX-specific inhibitors are shown to inhibit acidification of extracellular pH induced by MN/CA IX in cell cultures exposed to hypoxia.  
           [0156]    Further, labeled exemplary CA IX-specific inhibitors, such as labeled sulfonamides, for example, conjugated to fluorescein isothiocyanate (FITC), are shown to bind to the surface of MN/CA IX transfected cells, and not to control cells, only in hypoxia but not in normoxia. Those experiments confirm that CA IX-specific inhibitors, such as the sulfonamide compounds described herein, can specifically target MN/CA IX under conditions characteristic of intratumoral microenvironments.  
           [0157]    The CA IX-specific inhibitors of this invention can be used diagnostically and prognostically for precancer and cancer, and to determine the status of a patient, and therapeutically, individually or in different combinations with conventional therapeutic regimens to treat precancers and/or cancer. The CA IX-specific inhibitors may also be used in cancer research.  
           [0158]    More particularly for treating precancer and/or cancer, the CA IX-specific inhibitors of this invention can be used to hinder cancer expansion and/or progression by blocking CA IX activity. The CA IX-specific inhibitors can be conjugated to radioisotopes for radiotherapy. The CA IX-specific inhibitors can be combined with CA IX-specific antibodies and a variety of conventional therapeutic drugs, different inhibitors of cancer-related pathways, bioreductive drugs, and/or radiotherapy, wherein different combinations of treatment regimens with the CA IX-specific inhibitors of this invention may increase overall treatment efficacy. Particularly, the CA IX-specific inhibitors of this invention may be combined with therapy using MN/CA IX-specific antibodies and/or CA IX-specific antibody fragments, preferably humanized CA IX-specific antibodies and/or biologically active fragments thereof, and more preferably fully human CA IX-specific antibodies and/or fully human CA IX-specific biologically active antibody fragments. Said CA IX-specific antibodies and biologically active CA IX-specific antibody fragments, preferably humanized and more preferably fully human, may be conjugated to a cytotoxic entity, for example, a cytotoxic protein, such as ricin A, among many other cytotoxic entities.  
           [0159]    Still further, a CA IX-specific inhibitor of this invention could be coupled to a vector for targeted delivery to CA IX-specific expressing cells for gene therapy (for example, with the wild-type von Hippel-Lindau gene), or for effecting the expression of cytotoxic proteins, preferably wherein said vector comprises a MN/CA IX promoter or MN/CA IX promoter fragment comprising the MN/CA IX hypoxia response element (HRE) or a HRE of another gene, and more preferably wherein the CA IX promoter or CA IX promotor fragment comprises more than one HRE, wherein said HRE or HREs is or are either of MN/CA IX, and/or of other genes and/or of genetically engineered HRE consensus sequences in a preferred context.  
           [0160]    Particularly, the CA IX-specific inhibitors of this invention can be used diagnostically/prognostically to detect precancerous and/or cancerous cells by binding to CA IX, preferably to CA IX activated by hypoxic conditions, wherein said CA IX specific inhibitors are coupled to a label or to some visualizing means. Such detection, particularly of hypoxic conditions, and CA IX overexpression, can be helpful in determining effective treatment options, and in predicting treatment outcome and the prognosis of disease development. Further the CA IX-specific inhibitors when labeled or linked to an appropriate visualizing means can be used for imaging tumors and/or metastases that express CA IX.  
           [0161]    The CA IX-specific inhibitors of this invention can also be used in basic and pre-clinical research. For example, the CA IX-specific inhibitors can be used to study the regulation of CA IX enzyme activity, to study the role of CA IX in tumor growth and metabolism, and to study the role of CA IX in response to treatment by drugs, radiation, inhibitors and other therapeutic regimens.  
           [0162]    Further methods are disclosed for the preparation of positively-charged, membrane-impermeant heterocyclic sulfonamide CA inhibitors with high affinity for the membrane-bound carbonic anhydrase CA IX. Particularly preferred CA IX-specific inhibitors are pyridinium derivatives of such aromatic and heterocyclic sulfonamides. The general structure of the preferred pyridinium derivatives of sulfonamides can be described as a pyridinium portion attached to the “tail” of an aromatic or heterocyclic sulphonamide portion of the compound.  
           [0163]    Further provided are screening assays for compounds that are useful for inhibiting the growth of a vertebrate, preferably mammalian, more preferably human, preneoplastic or neoplastic cell that abnormally expresses MN protein. Said screening assays comprise tests for the inhibition of the enzymatic activity of MN by said compounds. Additional assays provided herein test said compounds for their cell membrane impermeance.  
           [0164]    Aspects of the instant invention disclosed herein are described in more detail below.  
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         Abbreviations  
         [0290]    The following abbreviations are used herein:  
           [0291]    aa—amino acid  
           [0292]    AAZ—acetazolamide  
           [0293]    ATCC—American Type Culture Collection  
           [0294]    bp—base pairs  
           [0295]    BRL—Bethesda Research Laboratories  
           [0296]    BRZ—brinzolamide  
           [0297]    BSA—bovine serum albumin  
           [0298]    CA—carbonic anhydrase  
           [0299]    CAI—carbonic anhydrase inhibitor  
           [0300]    CAM—cell adhesion molecule  
           [0301]    CARP—carbonic anhydrase related protein  
           [0302]    Ci—curie  
           [0303]    cm—centimeter  
           [0304]    CNS—central nervous system  
           [0305]    cpm—counts per minute  
           [0306]    C-terminus—carboxyl-terminus  
           [0307]    ° C.—degrees centigrade  
           [0308]    DCP—dichlorophenamide  
           [0309]    DEAE—diethylaminoethyl  
           [0310]    DMEM—Dulbecco modified Eagle medium  
           [0311]    ds—double-stranded  
           [0312]    DZA—dorzolamide  
           [0313]    EDTA—ethylenediaminetetraacetate  
           [0314]    EZA—ethoxzolamide  
           [0315]    F—fibroblasts  
           [0316]    FCS—fetal calf serum  
           [0317]    FITC—fluorescein isothiocyanate  
           [0318]    H—HeLa cells  
           [0319]    IC—intracellular  
           [0320]    kb—kilobase  
           [0321]    kbp—kilobase pairs  
           [0322]    kd or kDa—kilodaltons  
           [0323]    K I —inhibition constant  
           [0324]    KS—keratan sulphate  
           [0325]    LTR—long terminal repeat  
           [0326]    M—molar  
           [0327]    mA—milliampere  
           [0328]    MAb—monoclonal antibody  
           [0329]    ME—mercaptoethanol  
           [0330]    MEM—minimal essential medium  
           [0331]    min.—minute(s)  
           [0332]    mg—milligram  
           [0333]    ml—milliliter  
           [0334]    mM—millimolar  
           [0335]    MMC—mitomycin C  
           [0336]    mmol—millimole  
           [0337]    MZA—methazolamide  
           [0338]    N—normal concentration  
           [0339]    NEG—negative  
           [0340]    ng—nanogram  
           [0341]    nm—nanometer  
           [0342]    nM—nanomolar  
           [0343]    nt—nucleotide  
           [0344]    N-terminus—amino-terminus  
           [0345]    ODN—oligodeoxynucleotide  
           [0346]    ORF—open reading frame  
           [0347]    PA—Protein A  
           [0348]    PBS—phosphate buffered saline  
           [0349]    PCR—polymerase chain reaction  
           [0350]    PG—proteoglycan  
           [0351]    pl—isoelectric point  
           [0352]    PMA—phorbol 12-myristate 13-acetate  
           [0353]    POS—positive  
           [0354]    Py—pyrimidine  
           [0355]    QAS—quaternary ammonian sulfonilamide  
           [0356]    QSAR—quantitative structure-activity relationship(s)  
           [0357]    RACE—rapid amplification of cDNA ends  
           [0358]    RCC—renal cell carcinoma  
           [0359]    RIA—radioimmunoassay  
           [0360]    RIP—radioimmunoprecipitation  
           [0361]    RIPA—radioimmunoprecipitation assay  
           [0362]    RNP—RNase protection assay  
           [0363]    RT-PCT—reverse transcription polymerase chain reaction  
           [0364]    SAC— Staphylococcus aureus  cells  
           [0365]    SAR—structure-activity relationship  
           [0366]    sc—subcutaneous  
           [0367]    SDS—sodium dodecyl sulfate  
           [0368]    SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel electrophoresis  
           [0369]    SINE—short interspersed repeated sequence  
           [0370]    SP—signal peptide  
           [0371]    SP-RIA—solid-phase radioimmunoassay  
           [0372]    TBE—Tris-borate/EDTA electrophoresis buffer  
           [0373]    TC—tissue culture  
           [0374]    TCA—trichloroacetic acid  
           [0375]    TC media—tissue culture media  
           [0376]    TC—tissue culture  
           [0377]    tk—thymidine kinase  
           [0378]    TM—transmembrane  
           [0379]    Tris—tris (hydroxymethyl) aminomethane  
           [0380]    μCi—microcurie  
           [0381]    μg—microgram  
           [0382]    μl—microliter  
           [0383]    μM—micromolar  
         Cell Lines  
         [0384]    BL21 (DE3)— Escherichia coli  strain described by Lindskog&#39;s group (for CA I, II expression)[Lindskog et al., “Structure-function relations in human carbonic anhydrase II as studied by site-directed mutagenesis,” in  Carbonic anhydrase—From biochemistry and genetics to physiology and clinical medicine,  Botre et al., Eds., VCH, Weinheim, pp. 1-13 (1991)] 
           [0385]    BL21-GOLD— Escherichia coli  strain (from Stratagene) used for CA IX  
           [0386]    (DE3) expression)  
         Nucleotide and Amino Acid Sequence Symbols  
         [0387]    The following symbols are used to represent nucleotides herein:  
                                                   Base               Symbol   Meaning                           A   adenine           C   cytosine           G   guanine           T   thymine           U   uracil           I   inosine           M   A or C           R   A or G           W   A or T/U           S   C or G           Y   C or T/U           K   G or T/U           V   A or C or G           H   A or C or T/U           D   A or G or T/U           B   C or G or T/U           N/X   A or C or G or T/U                      
 
           [0388]    There are twenty main amino acids, each of which is specified by a different arrangement of three adjacent nucleotides (triplet code or codon), and which are linked together in a specific order to form a characteristic protein. A three-letter or one-letter convention is used herein to identify said amino acids, as, for example, in FIG. 1 as follows:  
                                                           3 Ltr.   1 Ltr.           Amino acid name   Abbrev.   Abbrev.                           Alanine   Ala   A           Arginine   Arg   R           Asparagine   Asn   N           Aspartic Acid   Asp   D           Cysteine   Cys   C           Glutamic Acid   Glu   E           Glutamine   Gln   Q           Glycine   Gly   G           Histidine   His   H           Isoleucine   Ile   I           Leucine   Leu   L           Lysine   Lys   K           Methionine   Met   M           Phenylalanine   Phe   F           Proline   Pro   P           Serine   Ser   S           Threonine   Thr   T           Tryptophan   Trp   W           Tyrosine   Tyr   Y           Valine   Val   V           Unknown or other       X                      
 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0389]    FIGS.  1 A-C provides the nucleotide sequence for MN/CA IX full-length cDNA [SEQ ID NO: 1]. FIGS.  1 A-C also sets forth the predicted amino acid sequence [SEQ ID NO: 2] encoded by the cDNA.  
         [0390]    FIGS.  2 A-F provides a 10,898 bp complete genomic sequence of MN/CA9 [SEQ ID NO: 3]. The base count is as follows: 2654 A; 2739 C; 2645 G; and 2859 T. The 11 exons are in general shown in capital letters, but exon 1 is considered to begin at position 3507 as determined by RNase protection assay.  
         [0391]    [0391]FIG. 3 provides an exon-intron map of the human MN/CA9 gene. The positions and sizes of the exons (numbered, cross-hatched boxes), Alu repeat elements (open boxes) and an LTR-related sequence (first unnumbered stippled box) are adjusted to the indicated scale. The exons corresponding to individual MN/CA IX protein domains are enclosed in dashed frames designated PG (proteoglycan-like domain), CA (carbonic anhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmic tail). Below the map, the alignment of amino acid sequences illustrates the extent of homology between the MN/CA IX protein PG region (aa 53-111) [SEQ ID NO: 4] and the human aggrecan (aa 781-839) [SEQ ID NO: 5].  
         [0392]    FIGS.  4  A-B shows the chemical structures of the 26 different sulfonamide compounds tested in Example 1.  
         [0393]    [0393]FIG. 5 shows the scheme for the general synthesis of compounds 71-91 of Example 3 (Scheme 1).  
         [0394]    [0394]FIG. 6 shows the scheme for the reaction between a pyrylium salt and an amine (Scheme 2), as described in Example 3. 
     
    
     DETAILED DESCRIPTION  
       [0395]    The novel methods of the present invention comprise inhibiting the growth of tumor cells which overexpress MN protein with compounds that inhibit the enzymatic activity of MN protein. Said compounds are organic or inorganic, preferably organic, more preferably sulfonamides. Still more preferably, said compounds are pyridinium derivatives of aromatic or heterocyclic sulfonamides. These preferred pyridinium derivatives of sulfonamides are likely to have fewer side effects than other compounds in three respects: they are small molecules, they are membrane-impermeant, and they are specific potent inhibitors of the enzymatic activity of the tumor-associated MN/CA IX protein.  
         [0396]    The use of oncoproteins as targets for developing new cancer therapeutics is considered conventional by those of skill in the art. [See, e.g., Mendelsohn and Lippman [61]. However, the application of such approaches to MN is new. In comparison to other tumor-related molecules (e.g. growth factors and their receptors), MN has the unique property of being differentially expressed in preneoplastic/neoplastic and normal tissues, which are separated by an anatomic barrier.  
         [0397]    The pyridinium derivatives of sulfonamides of the present invention can be formed, for example, by creating bonds between pyrylium salts and aromatic or heterocyclic sulfonamide reagents, as described below. The aromatic or heterocyclic sulfonamide portion of a pyridinium salt of a sulfonamide compound can be called the “head,” and the pyridinium portion can be called the “tail.” 
         [0398]    It can be appreciated by those of skill in the art that various other types of linkages can couple the pyridinium portion with the sulfonamide portion. It can further be appreciated that alternate methods, in addition to those disclosed herein, can be used to make the pyridinium derivatives of the present invention.  
         [0399]    As used herein, “cancerous” and “neoplastic” have equivalent meanings, and “precancerous” and “preneoplastic” have equivalent meanings.  
         [0400]    As used herein, the term “aromatic” when applied to sulphonamide structures means “comprising an aromatic ring, without an additional heterocyclic ring.” The term “heterocyclic” when applied to sulphonamide structures means “comprising a heterocyclic ring, with or without an additional aromatic ring.” 
         [0401]    As used herein, the term “alkyl”, alone or in combination, refers to a straight-chain or branched-chain alkyl radical containing from 1 to 12, preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms. Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the like.  
         [0402]    The term “aryl”, alone or in combination, means a phenyl or naphthyl radical which optionally carries one or more substituents selected from alkyl, alkoxy, halogen, hydroxy, amino, nitro, cyano, haloalkyl, carboxy, alkoxycarbonyl, cycloalkyl, heterocycloalkyl, amido, mono and dialkyl substituted amino, mono and dialkyl substituted amido and the like, such as phenyl, p-tolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl, 3-methyl-4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl, 3-aminophenyl, 3-acetamidophenyl, 4-acetamidophenyl, 2-methyl-3-acetamidophenyl, 2-methyl-3-aminophenyl, 3-methyl-4-aminophenyl, 2-amino-3-methylphenyl, 2,4-dimethyl-3-aminophenyl, 4-hydroxyphenyl, 3-methyl-4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, 3-amino-I-naphthyl, 2-methyl-3-amino-1-naphthyl, 6-amino-2-naphthyl, 4,6-dimethoxy-2-naphthyl and the like.  
         [0403]    Preferred sulfonamides of the present invention are aromatic and heterocyclic sulfonamides. The structures of representative sulfonamides of this group, designated 1-26, are shown in FIG. 4.  
         [0404]    More preferred sulfonamides of the present invention are pyridinium derivatives of aromatic sulfonamides and have the general formula (A) below,  
                         
 
         [0405]    wherein n is 0, 1, or 2; and R2, R3, R4 and R6 are each independently selected from the group consisting of hydrogen, alkyls and aryls. The structures of representative sulfonamides of this group, designated 27 through 70, are shown as derivatives of the general structure (A), in Table 2.  
         [0406]    Alternatively, more preferred sulfonamides of the present invention are pyridinium derivatives of heterocyclic sulfonamides and have the general formula (B) below, wherein said pyridinium derivative of a heterocyclic sulfonamide has the general formula of:  
                         
 
         [0407]    wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, akyls and aryls. The structures of representative sulfonamides of this group, designated 71 through 91, are shown as derivatives of the general structure (B), in Table 3.  
         [0408]    Representative sulfonamide derivatives of the group of compounds represented by the general formulas (A) and (B) have CA IX inhibitory activity, and are potentially useful therapeutically as anticancer agents in treating MN-associated tumors.  
         [0409]    Further, biologic activity of the identified sulfonamides will be tested in vitro by inhibition of the carbonic anhydrase enzymatic activity of the MN protein, by effects on cell morphology and growth characteristics of MN-related tumor cells (HeLa) and of control cells [104]. In vivo screening will be carried out in nude mice that have been injected with HeLa cells.  
       Representative Sulfonamide Inhibitors of CA IX  
       [0410]    The sulfonamides investigated in Example 1 for the inhibition of the tumor-associated isozyme CA IX, of types 1-26 are shown in FIGS.  4 A-B. Compounds 1-6, 11-12, 20 and 26 are commercially available, whereas 7-10 [43], 13-19 [24, 90, 97] and 21-25 [79] were prepared as reported earlier. The six clinically used compounds were also assayed. For Example 2 compounds (pyridinium derivatives of aromatic sulfonamides), reaction of sulfanilamide, homosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide with 2,6-di-, 2,4,6-tri- or 2,3,4,6-tetrasubstituted pyrylium salts afforded the pyridinium salts 27-70 investigated here, by the general Bayer-Piccard synthesis [9,10, 97].  
         [0411]    As described in Example 3, a series of positively-charged sulfonamides, designated here as compounds 71-91, were obtained by reaction of aminobenzolamide (5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) with tri-/tetra-substituted pyrilium salts possessing alkyl-, aryl- or combinations of alkyl and aryl groups at the pyridinium ring (described below). Three of these compounds (71, 75, and 87) have been described elsewhere [25, 85]; all other compounds of this series are new.  
       Heterocyclic Sulfonamide Inhibitors of CA IX: Synthesis of Pyridinium Derivatives of Aminobenzolamide  
       [0412]    Chemistry: Reaction of aminobenzolamide (5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) [97] with 2,6-di-, 2,4,6-tri- or 2,3,4,6-tetrasubstituted pyrylium salts afforded the pyridinium salts 71-91 investigated here, by the general synthesis of such derivatives with nucleophiles (Scheme 1 as shown in FIG. 5) [6, 26, 108].  
         [0413]    Preparation of compounds: A large number of positively-charged sulfonamides, prepared by reaction of amino-sulfonamides with pyrylium salts [23, 88, 89] were recently reported by this group, and generally tested as inhibitors of the “classical” isozymes CA I, II and IV [81, 96, 97, 98]. Based on QSAR studies on several series of CA inhibitors, including some positively-charged derivatives [23, 88, 89], it emerged that the enhancement of CA inhibitory activity is correlated with increased positive charges on the heterocyclic/aromatic ring incorporated in such molecules, as well as with “long” inhibitor molecules per se (i.e., molecules extending on the direction passing through the Zn(II) ion of the enzyme, the sulfonamide nitrogen atom and the long axis of the inhibitor) [23, 88, 89]. It appeared thus of interest to try to explore this result, designing positively-charged, long sulfonamide CAIs. Thus, we thought of attaching substituted-pyridinium moieties to an already potent and long-molecule CAI suitable for reaction with pyrylium salts, i.e., aminobenzolamide [97]. Indeed, this compound acts as a very potent CAI against isozymes I, II and IV (with inhibition constants in the low nanomolar range—see later in the text). The substitution pattern of the pyridinium ring was previously shown [81, 96, 97, 98] to be critical for the biological activity of this type of sulfonamide CAIs. Thus, a large series of of 2,4,6-trialkylpyridinium-; 2,6-dialkyl-4-phenylpyridinium-; 2-alkyl-4,6-diphenylpyridinium-; 2,4,6-triphenylpyridinium-, together with various 2,6-disubstituted-pyridinium and 2,3,5,6-tetrasubstituted-pyridinium aminobenzolamide derivatives have been prepared by the reaction described in Scheme 1 (Shown in FIG. 5).  
         [0414]    Although apparently simple, the reaction between a pyrylium salt and an amine, leading to pyridinium salts, is in reality a complicated process (Scheme 2, shown in FIG. 6), as established by detailed spectroscopic and kinetic data from Balaban&#39;s and Katritzky&#39;s groups [6, 26, 108]. Thus, the nucleophilic attack of a primary amine RNH 2  on pyrylium cations generally occurs in the α position, with the formation of intermediates of type IV (depicted in FIG. 6), which by deprotonation in the presence of bases lead to the 2-amino-tetradehydropyran derivatives V. In many cases the deprotonation reaction is promoted by the amine itself, when this is basic enough (this being the reason why in many cases one works at molar ratios pyrylium:amine of 1:2 when pyridinium salts are prepared by this method), or by external catalysts added to the reaction mixture, such as triethylamine [6, 26, 108]. The derivatives V are generally unstable, being tautomers with the ketodieneamines VI which are the key intermediates for the conversion of pyryliums into pyridiniums [6, 26, 108]. In acidic media, in the rate-determining step of the whole process, ketodieneamines VI may be converted to the corresponding pyridinium salts VII, although other products, such as vinylogous amides with diverse structures have also been isolated in such reactions [6, 26, 108]. A supplementary complication appears when the moiety substituting the 2- and/or 6-position(s) of the pyrylium ring is methyl, cases in which a concurrent cyclisation with formation of the anilines VIII in addition to the pyridinium salts VII, may take place too [6, 26, 108]. These concurrent reactions mentioned above are generally important when the amine to be converted into the pyridinium salt possesses weak nucleophilicity or basicity. This happens to be the case of aminobenzolamide. In fact, reaction of aminobenzolamide with several pyrylium salts, performed in a variety of conditions (different solvents, such as low molecular weight alcohols (MeOH, EtOH, i-PrOH); DMF; methylene chloride; acetonitrile; diverse molar ratios of the reagents; temperatures from 25 to 150° C.; reaction times between 15 min and 48 hours, etc) led only to the isolation of the unreacted raw materials. The only conditions which led to the formation of the pyridinium salts III (depicted in FIG. 5) were the following: anhydrous methanol in the presence of acetic anhydride as solvent and triethylamine as catalysts for the deprotonation of the intermediates IV. Acetic anhydride had the role of reacting with the water formed in the condensation reaction. This water may in fact act as a competitive nucleophile with aminobenzolamide when reacting with the pyrylium cation, and as a consequence the yields in pyridinium salts would dramatically be decreased. After the rapid formation of the ketodieneamine, catalyzed by triethylamine (and in the presence of the acetic anhydride as water scavenging agent), the cyclisation to the pyridinium ring (the rate-determining step) has been achieved by refluxation in the presence of acetic acid (2-5 hours). Still the yields were not always good, especially for the 2-methyl-containing derivatives.  
       Preparation of MN Proteins and/or Polypeptides  
       [0415]    The terms “MN/CA IX” and “MN/CA9” are herein considered to be synonyms for MN. Also, the G250 antigen is considered to refer to MN protein/polypeptide [112].  
         [0416]    Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNA sequence shown herein in FIGS.  1 A- 1 C [SEQ ID NO: 1], the MN amino acid sequence [SEQ ID NO: 2] also shown in FIGS.  1 A- 1 C, and the MN genomic sequence [SEQ ID NO: 3] shown herein in FIGS.  2 A- 2 F. The MN gene is organized into 11 exons and 10 introns.  
         [0417]    The first thirty seven amino acids of the MN protein shown in FIGS.  1 A- 1 C is the putative MN signal peptide [SEQ ID NO: 6]. The MN protein has an extracellular domain [amino acids (aa) 38-414 of FIGS.  1 A- 1 C [SEQ ID NO: 7], a transmembrane domain [aa 415-434; SEQ ID NO: 8] and an intracellular domain [aa 435-459; SEQ ID NO: 9]. The extracellular domain contains the proteoglycan-like domain [aa 53-111: SEQ ID NO: 4] and the carbonic anhydrase (CA) domain [aa 135-391; SEQ ID NO: 5].  
         [0418]    The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides) is herein defined to mean proteins and/or polypeptides encoded by an MN gene or fragments thereof. An exemplary and preferred MN protein according to this invention has the deduced amino acid sequence shown in FIG. 1. Preferred MN proteins/polypeptides are those proteins and/or polypeptides that have substantial homology with the MN protein shown in FIG. 1. For example, such substantially homologous MN proteins/polypeptides are those that are reactive with the MN-specific antibodies, preferably the Mab M75 or its equivalent. The VU-M75 hybridoma that secretes the M75 Mab was deposited at the ATCC under HB 11128 on Sep. 17, 1992.  
         [0419]    A “polypeptide” or “peptide” is a chain of amino acids covalently bound by peptide linkages and is herein considered to be composed of 50 or less amino acids. A “protein” is herein defined to be a polypeptide composed of more than 50 amino acids. The term polypeptide encompasses the terms peptide and oligopeptide.  
         [0420]    It can be appreciated that a protein or polypeptide produced by a neoplastic cell in vivo could be altered in sequence from that produced by a tumor cell in cell culture or by a transformed cell. Thus, MN proteins and/or polypeptides which have varying amino acid sequences including without limitation, amino acid substitutions, extensions, deletions, truncations and combinations thereof, fall within the scope of this invention. It can also be appreciated that a protein extant within body fluids is subject to degradative processes, such as, proteolytic processes; thus, MN proteins that are significantly truncated and MN polypeptides may be found in body fluids, such as, sera. The phrase “MN antigen” is used herein to encompass MN proteins and/or polypeptides.  
         [0421]    It will further be appreciated that the amino acid sequence of MN proteins and polypeptides can be modified by genetic techniques. One or more amino acids can be deleted or substituted. Such amino acid changes may not cause any measurable change in the biological activity of the protein or polypeptide and result in proteins or polypeptides which are within the scope of this invention, as well as, MN muteins.  
         [0422]    The MN proteins and polypeptides of this invention can be prepared in a variety of ways according to this invention, for example, recombinantly, synthetically or otherwise biologically, that is, by cleaving longer proteins and polypeptides enzymatically and/or chemically. A preferred method to prepare MN proteins is by a recombinant means. Particularly preferred methods of recombinantly producing MN proteins are described below. A representative method to prepare the MN proteins shown in FIG. 1 or fragments thereof would be to insert the full-length or an appropriate fragment of MN cDNA into an appropriate expression vector as exemplified in the Materials and Methods section.  
       MN Gene  
       [0423]    FIGS.  1 A-C provides the nucleotide sequence for a full-length MN cDNA clone [SEQ ID NO: 1] isolated as described in Zavada et al., WO 95/34650. FIGS.  2 A-F provides a complete MN genomic sequence [SEQ ID NO: 3].  
         [0424]    The ORF of the MN cDNA shown in FIG. 1 has the coding capacity for a 459 amino acid protein with a calculated molecular weight of 49.7 kd. The overall amino acid composition of the MN/CA IX protein is rather acidic, and predicted to have a pl of 4.3. Analysis of native MN/CA IX protein from CGL3 cells by two-dimensional electrophoresis followed by immunoblotting has shown that in agreement with computer prediction, the MN/CA IX is an acidic protein existing in several isoelectric forms with pis ranging from 4.7 to 6.3.  
         [0425]    The CA domain is essential for induction of anchorage independence, whereas the TM anchor and IC tail are dispensable for that biological effect. The MN protein is also capable of causing plasma membrane ruffling in the transfected cells and appears to participate in their attachment to the solid support. The data evince the involvement of MN in the regulation of cell proliferation, adhesion and intercellular communication.  
       Enzymatic Screening Assays  
       [0426]    Assays are provided herein for the screening of compounds for inhibition of the enzymatic activity of the MN protein. Such assays comprise the incubation of said compound with said MN protein and with a substrate selected from the group consisting of saturated CO 2  and 4-nitrophenylacetate, preferably saturated CO 2 , and determination of the inhibition constant K I  of said compound, wherein said enzymatic activity of the MN protein is measured by the pH change of an indicator by stopped flow spectrophotometer.  
         [0427]    Screening of representative heterocyclic and aromatic sulfonamides for inhibition of MN protein: From Example 1, it was found that the inhibition profile of isozyme CA IX is very different from that of the classical isozymes CA I and II (cytosolic) as well as CA IV (membrane-bound). The following particular features may be noted: (i) all the 32 sulfonamides investigated in Example 1 act as CA IX inhibitors, with inhibition constants in the range of 14-285 nM (the corresponding affinities for the other three isozymes vary in a much wider range, as seen from data of Table 1). Based on these data, it can be noted that CA IX is a sulfonamide avid CA, similarly to CA II, the isozyme considered up to now to be responsible for the majority of pharmacological effect of sulfonamides [22, 29, 83,93, 94, 95, 102]. Still, many other differences are observed between CA IX and other isozymes for which inhibitors were developed for clinical use; (ii) for CA I, II and IV, generally, aromatic sulfonamides behave as weaker inhibitors as compared to heterocyclic derivatives (compare 1-6, or DCP), as aromatic compounds, with 15,21, AAZ, MZA, EZA, DZA or BRZ among others (as heterocyclic sulfonamides). In the case of CA IX, such a fine distinction is rather difficult to be made, since both aromatic (such as 1, 6, 11, 12, 17, 18, 22-26) derivatives, as well as heterocyclic compounds (such as 14, 15, 21, and the clinically used sulfonamides—except dichlorophenamide) possess rather similar inhibition constants, in the range of 14-50 nM; (iii) orthanilamide derivatives (such as 1, 17 and 22) behave as very potent CA IX inhibitors (K I -s in the range of 20-33 nM), although they are weak or medium-weak inhibitors of CA I, II and IV; (iv) 1,3-benzene-disulfonamide derivatives (such as 11, 12 and DCP) are again strong CA IX inhibitors, with K I -s in the range of 24-50 nM, although their CA II, I and IV inhibition profile is not particularly strong; (v) metanilamide 2, sulfanilamide 3, and 4-hydrazino-benzenesulfonamide 4 show CA IX inhibition data quite similar with those against CA II, whereas homosulfanilamide 5 and 4-aminoethyl-benzensulfonamide 6 act as better CA IX inhibitors as compared to CA II inhibition; (vi) the halogenosulfanilamides 7-10 are much weaker inhibitors of CA IX than of CA II, a finding difficult to interpret at this moment; (vii) the strongest CA II inhibitor among the investigated compounds, 4-aminobenzolamide 15 (K I  of 2 nM) is not the strongest CA IX inhibitor (K I  of 38 nM). Instead, the best CA IX inhibitor detected so far is the ethoxzolamide phenol 21 (K I  of 14 nM). It is interesting to note that 21 and EZA have the same affinity for CA II, whereas their affinity for CA IX is rather different, with the phenol more active than the ethoxy-derivative; (viii) among the clinically used compounds, the best inhibitor is acetazolamide, followed by methazolamide, ethoxzolamide and brinzolamide. The most ineffective (but appreciably inhibiting the isozyme IX) are dichlorophenamide and dorzolamide; (ix) sulfonamides 20 and 22-26 behave as very good CA IX inhibitors, with K I -s in the range of 16-32 nM, being slightly more effective than the clinically used CAIs mentioned above, and among the best CA IX inhibitors detected so far. It is thus envisageable that such compounds may be used as lead molecules for obtaining more potent and eventually specific CA IX inhibitors, with applications as antitumor agents.  
         [0428]    Screening of representative pyridinium derivatives of aromatic sulfonamides for inhibition of MN protein: From Example 2, wherein membrane-impermeant pyridinium derivatives of sulfonamides were tested for their ability to inhibit the enzymatic activity of CA IX, the following conclusions were drawn from data of Table 2: (i) for a given substitution pattern of the pyridinium ring, the 4-aminoethyl-benzenesulfonamide derivatives 55-70 were more active than the corresponding homosulfanilamide derivatives 39-54, which in turn were more active than the corresponding sulfanilamides 27-38. This behavior has also been observed for the other three investigated isozymes [96]; (ii) some of the derivatives possessing bulky substitutents at the pyridinium ring (mainly phenyls, tert-butyls; n-butyl, n-propyl or iso-propyl), such as 34-37, 51 and 67, were very ineffective CA IX inhibitors, showing inhibition constants &gt;500 nM; (iii) another group of compounds, including 27, 30-33, 44, and 60 showed a moderate inhibitory power towards the tumor-associated isozyme IX, showing K I  values in the range of 160-450 nM. Most of these compounds are sulfanilamide derivatives (except 44 and 60), and the substitution pattern at the pyridinium ring includes (with one exception, 27) at least one phenyl group in 4, or two phenyls in the 2 and 4 positions. It should be noted that the corresponding homosulfanilamides and 4-aminoethylbenzene-sulfonamides incorporating the same substitution pattern as the compounds mentioned above (sulfanilamides), lead to much better CA IX inhibitors (see later in the text); (iv) a third group of derivatives, including 38, 45-50, 52, 53, 61, 63-66, 68 and 69, showed good CA IX inhibitory properties, with K I  values in the range of 64-135 nM. As mentioned above, except for the tetramethyl-pyridinium-substituted derivative 38, most of these compounds incorporate 4-phenyl-pyridinium or 2,4-diphenylpyridinium moieties, whereas the group in position 6 is generally quite variable (alkyls or phenyl are tolerated). The most interesting observation regarding this subtype of CA IX inhibitors is constituted by the fact that the 2,4,6-triphenyl-pyridinium- and 2,6-diphenyl-pyridinium derivatives of homosulfanilamide and 4-aminoethylbenzenesulfonamide (52-53 and 68-69) efficiently inhibit isozyme IX, although they act as very weak inhibitors for isozymes I, II and IV (Table 2). As it will be discussed shortly, this may be due to the fact that the hCA IX active site is larger than that of the other investigated isozymes, notably CA II, I and IV; (v) a last group of derivatives (28-29; 39-43; 54; 55-59; 62 and 70) showed very good CA IX inhibitory properties, these compounds possessing K I  values in the range of 6-54 nM, similarly to the clinically used inhibitors acetazolamide, methazolamide, dichlorophenamide and indisulam, for which the inhibition data are provided for comparison. It should be noted that three derivatives 58, 59 and 70 showed inhibition constants &lt;10 nM, these being the most potent CA IX inhibitors ever reported up to now. Correlated with their membrane-impermeability [96, 85], it may be assumed that in vivo such compounds may lead for the first time to a selective CA IX inhibition. Thus, the best substitution pattern at the pyridinium ring includes either only compact alkyls (39-41, 54, 55 and 70), or 2,6-dialkyl-4-phenyl-pyridinium moieties (all compounds mentioned above except 62, which incorporates a 2-methyl-4,6-diphenylpyridinium ring); (vi) the number of the substitutents at the pyridinium ring seems to be less important for the activity of this series of CAIs, since both di-, tri- or tetrasubstituted derivatives showed good inhibitory potency. The nature of these groups on the other hand—as discussed in detail above—is the most important parameter influencing CA inhibitory properties (together with the linker between the benzenesulfonamide moiety and the substituted pyridinium ring); (vii) the isozyme most similar to hCA IX regarding the affinity for these inhibitors was hCA II (which has 33% homology with hCA IX) [Pastorek et al. (1994), supra] whereas the affinities of isozymes I and IV were rather different.  
         [0429]    Screening of representative Dyridinium derivatives of heterocyclic sulfonamides for inhibition of MN protein, and comparison with inhibition of other CA isozymes: Isozyme I. As seen from data of Table 3, all derivatives 71-91 reported here act as very efficient CAIs against this isozyme which is generally the most “resistant” to inhibitors of this type [30, 31, 100, 102]. Indeed, aminobenzolamide is already a highly potent CA I inhibitor (K I  of 6 nM), whereas inhibitors 71-91 show inhibition constants in the range of 3-12 nM, in contrast to the clinically used sulfonamide CAIs which are much less effective inhibitors, with K I  values in the range of 30-1200 nM (Table 3). Thus, derivatives possessing several bulky groups (i-Pr; t-Bu; n-Pr; n-Bu; Ph, etc) substituting the pyridinium moiety, such as 73, 74, 77, 78, 82, 84, 85 showed a decreased inhibitory activity as compared to aminobenzolamide, with K I  values in the range of 7-12 nM (aminobenzolamide has a K I  of 6 nM against hCA I). The rest of the compounds were more efficient as compared to aminobenzolamide in inhibiting this isozyme, with K I  values in the range of 3-5 nM. Best CA I inhibitors were 75, and 89-91 (K I  of 3 nM), all of which containing either only alkyl moieties or 4-Ph and other alkyl moieties substituting the pyridinium ring. These are probably the best CA I inhibitors ever reported up to now, since the clinically used CAIs show much higher inhibition constants against isozyme I (Table 3).  
         [0430]    Isozyme II. Aminobenzolamide is already a very potent CA II inhibitor, with an inhibition constant around 2 nM. Several of the new inhibitors, such as 74, 77, 78, 82-88 act as weaker CA II inhibitors as compared to aminobenzolamide, with K I  values in the range of 3.13-5.96 nM (but all these compounds act as potent inhibitors, being much more effective than the clinically used CAIs acetazolamide, methazolamide, dichlorophenamide or indisulam—see Table 3). Again the substitution pattern at the pyridinium ring is the main discriminator of activity for these compounds: all the less active derivatives mentioned above incorporate at least two bulky/long aliphatic groups, mainly in positions 2- and 6- of the pyridinium ring (n-Pr; t-Bu; n-Bu; and Ph). The best CA II inhibitors among derivatives 71-91 were those incorporating more compact 2,6-substituents at the pyridinium ring (such as Me, Et) together with a 4-Me or 4-Phe moiety, or those incorporating only aliphatic such groups, such as 71-73, 75, 76, 79-81, 89-91, which showed K I  values in the range of 0.20-1.61 nM (thus, for the best inhibitors a factor of 10 increase in inhibitory power as compared to aminobenzolamide). It should be mentioned that iso-propyl-substituted compounds (73, 79) are active as CA II inhibitors, although their activity against CA I was not so good.  
         [0431]    Isozyme IV. Most sulfonamides show inhibitory activity against CA IV intermediate between those towards CA I (less susceptible) and CA II (very high affinity for sulfonamides). This is also the trend observed with the sulfonamides investigated here, derivatives of aminobenzolamide. Thus, the parent sulfonamide (shown in FIG. 5) is a potent CA IV inhibitor, with a K I  value around 5 nM. The new derivatives of general formula (B) incorporating bulky pyridinium-ring substituents (such as 74, 77, 78, 82, 84-88, 90) were less effective than aminobenzolamide, showing K I  values in the range of 5.2-10.3 nM, whereas the compounds showing the other substitution pattern mentioned above were better CA IV inhibitors, showing K I  values in the range of 2.0-4.7 nM.  
         [0432]    Isozyme IX. Aminobenzolamide is less inhibitory against this isozyme (K I  of 38 nM) as compared to other isozymes discussed above. This behavior is difficult to explain at this point, since no X-ray crystal structure of this isozyme has been reported. A very encouraging result obtained with the new derivatives of general formula (B) reported here, was the observation that several of them show very high affinity for CA IX, with K I  values in the range of 3-9 nM (derivatives 71, 72, 75, 76, and 89). It may be seen that all of them incorporate aliphatic moieties (Me, Et and i-Pr) in positions 2- and 6- of the pyridinium ring, and either 4-Me or 4-Ph moieties. Only one compound is tetrasubstituted (89), again possessing only methyl moieties. The best CA IX inhibitor (and the best ever reported up to now) was 71, which is almost 13 times more effective than benzolamide in inhibiting this isozyme. Another group of new derivatives, such as 73, 74, 77, 79, 80, 81, 83, 86-88, 90, 91, showed effective CA IX inhibition, with K I  values in the range of 12-35 nM, being thus more effective than aminobenzolamide. They incorporate slightly bulkier groups as compared to the previously discussed ones. Again the less effective inhibitors (K I  values in the range of 40-43 nM) were those incorporating several bulky pyridinium substituents, such as 78, 84, 85 which contained either two n-Bu or one Ph and n-Bu/t-Bu in positions 2- and 6- of the pyridinium ring. Thus, SAR is now rather clear for this type of CAIs: best CA IX inhibitors should contain either only small, compact aliphatic moieties substituting the pyridinium ring, or they tolerate a 4-Ph moiety, but the 2,6-substituents should again be small, compact aliphatic moieties. In this particular case, 2,4,6-trisubstituted-pyridinium derivatives were more effective CA IX inhibitors as compared to the tetrasubstituted derivatives.  
         [0433]    Membrane impermeability of Heterocyclic Sulfonamide Inhibitors of CA IX. As seen from data of Table 4 of Example 3, incubation of human red cells (which contain high concentrations of isozymes I and II, i.e., 150 μM hCA I and 20 μM hCA II, but not the membrane-bound CA IV or CA IX) [118] with millimolar concentrations of different sulfonamide inhibitors, such as acetazolamide, or methazolamide, led to saturation of the two isozymes present in erythrocytes with inhibitor, already after short periods of incubation (30 min), whereas for benzolamide or aminobenzolamide, a similar effect is achieved after somehow longer periods (60 min) (Table 4). This is obviously due to the high diffusibility through membranes of the first three inhibitors, whereas benzolamide/aminobenzolamide with a pK a  of 3.2 for the second sulfonamido group [58] being present mainly as an (di)anion at the pH at which the experiment has been done (7.4), is already less diffusible and penetrates membranes in a longer time. Different cationic sulfonamides synthesized by us here, such as 71, 76, 89, 91, in the same conditions, were detected only in very small amounts within the blood red cells, proving that they were unable to penetrate through the membranes, obviously due to their cationic nature. Even after incubation times as long as one hour (and longer, data not shown), only traces of such cationic sulfonamides were present inside the blood red cells, as proved by the three assay methods used for their identification in the cell lysate, which were in good agreement with each other (Table 4). This demonstrates that the proposed approach for achieving membrane impermeability works well for the designed positively-charged sulfonamide CAIs of the general formula (B) (shown above), since the very small amount of sulfonamide detected may be due to contamination of the lysates with very small amount of membranes.  
       Design of Membrane-Impermeant Sulfonamide Inhibitors of CA IX  
       [0434]    No X-ray crystal structure of isozyme IX is available up to now, in strong contrast with hCA II, for which many X-ray crystal structures are available (alone or in complexes with inhibitors and activators) [1, 2, 14, 15, 19a, 19b, 37, 38]. Examining the active site residues of these two isozymes and the architecture of hCA II, may help explain the above inhibition data and their relevance for CA IX specific inhibitors.  
         [0435]    First of all, the zinc ligands and the proton shuttle residue of these two isozymes are identical [33, 43, 72, 100, 101, 102, 114, 115, 117]. An important difference is constituted by the amino acid in position 131, which is Phe for hCA II and Val for hCA IX. Phe 131 is known to be very important for the binding of sulfonamide inhibitors to hCA II [2, 46, 47]: in many cases this bulky side chain limits the space available for the inhibitor aromatic moieties, or it may participate in stacking interactions with groups present in it (for recent examples see refs. [2, 46, 47]. Thus, the presence of a less bulky such residue in hCA IX (i.e., a valine) which is also unavailable for participation to stacking interactions has as a consequence the fact that the hCA IX active site is larger than that of hCA II. A second potentially important residue is 132, which is Gly in hCA II and Asp in hCA IX. This residue is situated on the rim of the hydrophilic half of the entrance to the active site of hCA II (and presumably also of hCA IX) and it is critical for the interaction with inhibitors possessing elongated molecules, as recenly shown by us [19b]. Strong hydrogen bonds involving the CONH moiety of Gly 132 were shown to stabilize the complex of this isozyme with a p-aminoethylbenzenesulfonamide derived inhibitor [19b]. In the case of hCA IX, the presence of aspartic acid in this position at the entrance of the active site may signify that: (i) stronger interactions with polar moieties of the inhibitor bound within the active site should be possible, since the COOH moiety possesses more donor atoms; (ii) this residue may have flexible conformations, fine-tuning in this way the interaction with inhibitors. Thus, the stronger hCA IX inhibition with some of these inhibitors (as compared to their affinity for isozyme II), such as for example 46-50, 52, 53, 55, 58, 62 and 68-70, might be explained just by the different interactions with the two active site residues mentioned above.  
       Therapeutic Use of MN-Specific Inhibitors  
       [0436]    The MN-specific inhibitors of this invention, organic and/or inorganic, preferably organic, and as outlined above, may be used therapeutically in the treatment of neoplastic and/or pre-neoplastic disease, either alone or in combination with other chemotherapeutic drugs.  
         [0437]    The MN-specific inhibitors can be administered in a therapeutically effective amount, preferably dispersed in a physiologically acceptable, non-toxic liquid vehicle.  
       Materials and Methods  
       [0438]    General. Melting points: heating plate microscope (not corrected); IR spectra: KBr pellets, 400-4000 cm −1  Perkin-Elmer 16PC FTIR spectrometer;  1 H-NMR spectra: Varian 300CXP apparatus (chemical shifts are expressed as δ values relative to Me 4 Si as standard); Elemental analysis: Carlo Erba Instrument CHNS Elemental Analyzer, Model 1106. All reactions were monitored by thin-layer chromatography (TLC) using 0.25-mm precoated silica gel plates (E. Merck). Pyrylium salts were prepared by literature procedures, generally by olefin (or their precursors) bisacylation, as described in the literature [6, 26, 108], whereas aminobenzolamide as described earlier [97]. Other sulfonamides used as standards were commercially available.  
       General Procedure for the Preparation of Compounds 71-91 (Pyridinium Derivatives of aminobenzolamide)  
       [0439]    An amount of 2.9 mM of aminobenzolamide [97] and 2.9 mM of pyrylium salt II (depicted in FIG. 5) were suspended in 5 mL of anhydrous methanol and poured into a stirred mixture of 14.5 mM of triethylamine and 5.8 mM of acetic anhydride. After five minutes of stirring, another 10 mL of methanol were added to the reaction mixture, which was heated to reflux for 15 min. Then 14.5 mM of acetic acid was added and heating was continued for 2-5 hours. The role of the acetic anhydride is to react with the water formed during the condensation reaction between the pyrylium salt and the aromatic amine, in order to shift the equilibrium towards the formation of the pyridinium salts of the general formula (B) (shown above). In the case of aminobenzolamide, this procedure is the only one which gave acceptable yields in pyridinium salts, probably due to the deactivating effect of the sulfamoylaminothiadiazole moiety on the amine group, which becomes poorly nucleophilic and unreactive towards these reagents. The precipitated pyridinium salts obtained were purified by treatment with concentrated ammonia solution (which also converts the eventually unreacted pyrylium salt to the corresponding pyridine which is soluble in acidic medium), reprecipitation with perchloric acid and recrystallization from water with 2-5% HClO 4 .  
         [0440]    Purification of Catalytic Domain of CA IX  
         [0441]    The cDNA of the catalytic domain of hCA IX (isolated as described by Pastorek et al. [72]) was amplified by using PCR and specific primers for the vector pCAL-n-FLAG (from Stratagene). The obtained construct was inserted in the PCAL-n-FLAG vector and then cloned and expressed in  Escherichia coli  strain BL21-GOLD(DE3) (from Stratagene). The bacterial cells were lysed and homogenated in a buffered solution (pH 8) of 4 M urea and 2% Triton X-100, as described by Wingo et al. [116]. The homogenate thus obtained was extensively centrifuged in order to remove soluble and membrane associated proteins as well as other cellular debris. The resulting pellet was washed by repeated homogenation and centrifugation in water, in order to remove the remaining urea and Triton X-100. Purified CA IX inclusion bodies were denaturated in 6 M guanidine hydrochloride and refolded into the active form by snap dilution into a solution of 100 mM MES (pH 6), 500 mM L-arginine, 2 mM ZnCl 2 , 2 mM EDTA, 2 mM reduced glutathione, 1 mM oxidized glutathione. Active hCA IX was extensively dialysed into a solution of 10 mM Hepes (pH 7.5), 10 mM Tris HCl, 100 mM Na 2 SO 4  and 1 mM ZnCl 2 . The amount of protein was determined by spectrophometric measurements and its activity by stopped-flow measurements, with CO 2  as substrate [44]. Optionally, the protein was further purified by sulfonamide affinity chromatography [44], the amount of enzyme was determined by spectrophometric measurements and its activity by stopped-flow measurements, with CO 2  as substrate [44].  
         [0442]    CA I, II and IV Purification  
         [0443]    Human CA I and CA II cDNAs were expressed in  Escherichia coli  strain BL21 (DE3) from the plasmids pACA/hCA I and pACA/hCA II described by Lindskog&#39;s group [54]. Cell growth conditions were those described in ref. [12], and enzymes were purified by affinity chromatography according to the method of Khalifah et al. [45]. Enzyme concentrations were determined spectrophotometrically at 280 nm, utilizing a molar absorptivity of 49 mM −1 .cm −1  for CA I and 54 mM −1 .cm −1  for CA II, respectively, based on M r =28.85 kDa for CA I, and 29.3 kDa for CA II, respectively [53, 84]. CA IV was isolated from bovine lung microsomes as described by Maren et al, and its concentration has been determined by titration with ethoxzolamide [59].  
       Enzyme Assays  
       [0444]    CA CO2 Hydrase Activity Assay  
         [0445]    An SX.18MV-R Applied Photophysics stopped-flow instrument has been used for assaying the CA CO 2  hydration activity assays [44]. A stopped flow variant of the Poker and Stone spectrophotometric method [76] has been employed, using an SX.18MV-R Applied Photophysics stopped flow instrument, as described previously [43]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na 2 SO 4  (for maintaining constant the ionic strength), following the CA-catalyzed CO 2  hydration reaction for a period of 10-100 s. Saturated CO 2  solutions in water at 20° C. were used as substrate [44]. Stock solutions of inhibitor (1 mM) were prepared in distilled-deionized water with 10-20% (v/v) DMSO (which is not inhibitory at these concentrations) and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results.  
         [0446]    CA Esterase Activity Assay  
         [0447]    Initial rates of 4-nitrophenylacetate hydrolysis catalysed by different CA isozymes were monitored spectrophotometrically, at 400 nm, with a Cary 3 instrument interfaced with an IBM compatible PC [76]. Solutions of substrate were prepared in anhydrous acetonitrile; the substrate concentrations varied between 2.10 −2  and 1.10 −6  M, working at 25° C. A molar absorption coefficient ε of 18,400 M −1 .cm −1  was used for the 4-nitrophenolate formed by hydrolysis, in the conditions of the experiments (pH 7.40), as reported in the literature [76]. Non-enzymatic hydrolysis rates were always subtracted from the observed rates. Triplicate experiments were done for each inhibitor concentration, and the values reported throughout the paper are the mean of such results. Stock solutions of inhibitor (1-3 mM) were prepared in distilled-deionized water with 10-20% (v/v) DMSO (which is not inhibitory at these concentrations) and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 10 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constant K I  was determined as described in references [44, 76].  
       Membrane Permeance Assay: Ex vivo Penetration through Red Blood Cells  
       [0448]    An amount of 10 mL of freshly isolated human red cells thoroughly washed several times with Tris buffer (pH 7.40, 5 mM) and centrifuged for 10 min were treated with 25 mL of a 2 mM solution of sulfonamide inhibitor. Incubation has been done at 37° C. with gentle stirring, for periods of 30-120 min. After the incubation times of 30, 60 and 120 min., respectively, the red cells were centrifuged again for 10 min, the supernatant discarded, and the cells washed three times with 10 mL of the above mentioned buffer, in order to eliminate all unbound inhibitor [81, 96, 98]. The cells were then lysed in 25 mL of distilled water, centrifuged for eliminating membranes and other insoluble impurities. The obtained solution was heated at 100° C. for 5 minutes (in order to denature CA-s) and sulfonamides possibly present have been assayed in each sample by three methods: a HPLC method [36]; spectrophotometrically [4] and enzymatically [76].  
         [0449]    HPLC: A variant of the methods of Gomaa [36] has been developed by us, as follows: a commercially available 5 μm Bondapak C-18 column was used for the separation, with a mobile phase made of acetonitrile-methanol-phosphate buffer (pH 7.4) 10:2:88 (v/v/v), at a flow rate of 3 mL/min, with 0.3 mg/mL sulphadiazine (Sigma) as internal standard. The retention times were: 12.69 min for acetazolamide; 4.55 min for sulphadiazine; 10.54 min for benzolamide; 12.32 min for aminobenzolamide; 3.15 min for 71; 4.41 min for 76; 3.54 min for 89; and 4.24 min for 91. The eluent was monitored continuously for absorbance (at 254 nm for acetazolamide, and wavelength in the range of 270-310 nm in the case of the other sulfonamides.  
         [0450]    Spectrophotometrically: A variant of the pH-induced spectrophotometric assay of Abdine et al. [4] has been used, working for instance at 260 and 292 nm, respectively, for acetazolamide; at 225 and 265 nm, respectively, for sulfanilamide, etc. Standardized solutions of each inhibitor have been prepared in the same buffer as the one used for the membrane penetrability experiments.  
         [0451]    Enzymatically: the amount of sulfonamide present in the lysate has been evaluated based on hCA II inhibition measured with the esterase method, as described above [76]. Standard inhibition curves have been obtained previously for each sulfonamide, using the pure compound, which were used thereafter for determining the amount of inhibitor present in the lysate. Mention should be made that the three methods presented above led to results in good agreement, within the limits of the experimental errors.  
         [0452]    Statistical analysis: Values are expressed±standard error of measurement. Statistical significance was determined using an unpaired t-test with p&lt;0.05 considered significant.  
         [0453]    The following examples are for purposes of illustration only and are not meant to limit the invention in any way.  
       EXAMPLE 1  
     Inhibition of the Tumor-Associated Isozyme IX with Aromatic and Heterocyclic Sulfonamides  
       [0454]    The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) Were also provided for comparison.  
         [0455]    Chemistry. Sulfonamides investigated for the inhibition of the tumor-associated isozyme CA IX, of types 1-26 are shown in FIGS.  4 A-B. Compounds 1-6, 11-12, 20 and 26 are commercially available, whereas 7-10 [43], 13-19 [24, 79, 90, 97] and 21-25 [79] were prepared as reported earlier. The six clinically used compounds were also assayed, since no such data are available in the literature.  
         [0456]    CA inhibition data. Inhibition data against four CA isozymes, CA I, II, IV and IX [44, 72, 116], with the above mentioned compounds 1-26 and the six clinically used inhibitors, are shown in Table 1.  
                                                                         TABLE 1                           CA I, II, IV and IX inhibition data with sulfonamides       1-26 and clinically used inhibitors.                K I * (nM)                Inhibitor   hAG I a     hCA II a     bCA IV b     hCA IX c                       1   45400   295   1310   33        2   25000   240   2200   238        3   28000   300   3000   294        4   78500   320   3215   305        5   25000   170   2800   103        6   21000   160   2450   33        7   8300   60   180   245        8   9800   110   320   264        9   6500   40   66   269       10   6000   70   125   285       11   5800   63   154   24       12   8400   75   160   39       13   8600   60   540   41       14   9300   19   355   30       15   6   2   5   38       16   164   46   129   34       17   185   50   144   20       18   109   33   72   31       19   95   30   72   24       20   690   12   154   16       21   55   8   17   14       22   21000   125   415   32       23   23000   133   438   30       24   24000   125   560   21       25   18000   110   450   22       26   135   40   86   26       AAZ   250   12   70   25       MZA   50   14   36   27       EZA   25   8   13   34       DCP   1200   38   380   50       DZA   50000   9   43   52       BRZ   —   3   45   37                                          
 
         [0457]    We report here the first inhibition study of the tumor-associated, transmembrane isozyme CA IX with a series of aromatic and heterocyclic sulfonamides, including also the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) are also provided for comparison. Very interesting inhibition profile against CA IX with these sulfonamides has been detected, which is a promising discovery for the potential design of CA IX-specific inhibitors, with applications as antitumor agents. Several nanomolar CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfanilamide, 4-carboxy-benzenesulfonamide, 1-naphthalene-sulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocyclic (such as 1,3,4-thiadiazole-2-sulfonamide, benzothiazole-2-sulfonamide, etc.) sulfonamides investigated.  
       EXAMPLE 2  
     The First Selective, Membrane-Impermeant Inhibitors Targeting the Tumor-Associated Isozyme IX  
       [0458]    Up to now no CA IX inhibition studies with this type of membrane-impermeant CAIs have been reported. Thus, we decided to explore some of the pyridinium derivatives of general formula (A) for their interaction with the catalytic domain of tumor-associated isozyme IX, recently cloned and purified by the inventors [33, 43, 114, 115, 117], as well as the cytosolic, physiologically relevant isozymes CA I, II and the membrane-anchored isozyme CA IV [88, 96].  
         [0459]    The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of positively-charged, pyridinium derivatives of sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. This is the first report of inhibitors that may selectively target CA IX, due to their membrane-impermeability and high affinity for this clinically relevant isozyme.  
         [0460]    CA Inhibition  
         [0461]    Data of Table 2 clearly show that most of the compounds 27-70 act as efficient CA IX inhibitors, and that their affinity for this isozyme differs considerably as compared to affinities for the cytosolic isozymes CA I and II, and the other membrane-associated isozyme investigated, CA IV.  
         [0462]    In a series of substituted-pyridinium derived sulfanilamides, homosulfanilamides and p-aminoethylbenzenesulfonamides, a large number of effective hCA IX inhibtors were detected. Some low nanomolar CA IX inhibitors were reported for the first time. Since these compounds are membrane-impermeant due to their salt-like character, and as hCA IX is present on the extracellular side of many tumors with poor clinical prognosis, compounds of this type target specifically this tumor-associate CA isozyme without affecting the cytosolic CAs known to play important physiological functions. Thus, compounds of this type may constitute the basis of new anticancer therapies based on CA inhibitors.  
                                                                                                                                                     TABLE 2                           Inhibition of isozymes hCA I, hCA II, bCA IV and hCA IX with the       pyridinium salts 27-70.                A                                                                          K I *                                hCA                   Com-                   I a     hCA II a     bCA IV b     hCA IX c         pound   R 2     R 3     R 4     R 6     (μM)   (nM)   (nM)   (nM)                    27   Me   H   Me   Me   10   150   290   165       28   Me   H   Ph   Me   7   60   211   48       29   Et   H   Ph   Et   6   60   182   43       30   n-Pr   H   Ph   n-Pr   10   120   194   178       31   i-Pr   H   Ph   i-Pr   5   50   90   160       32   Me   H   Ph   Ph   40   210   852   280       33   Et   H   Ph   Ph   43   400   1300   450       34   n-Pr   H   Ph   Ph   140   580   1483   &gt;500       35   i-Pr   H   Ph   Ph   125   440   2102   &gt;500       36   n-Bu   H   Ph   Ph   305   620   2155   &gt;500       37   Ph   H   Ph   Ph   290   510   2500   &gt;500       38   Me   Me   Me   Me   5   40   61   72       39   Me   H   Me   Me   7   50   92   38       40   i-Pr   H   Me   Me   6   50   80   42       41   i-Pr   H   Me   i-Pr   11   80   144   54       42   Me   H   Ph   Me   4   20   70   26       43   Et   H   Ph   Et   2   21   52   29       44   n-Pr   H   Ph   n-Pr   24   90   163   230       45   i-Pr   H   Ph   i-Pr   12   61   101   100       46   Me   H   Ph   Ph   32   121   161   64       47   Et   H   Ph   Ph   42   314   983   79       48   n-Pr   H   Ph   Ph   130   390   1260   85       49   i-Pr   H   Ph   Ph   112   370   1214   80       50   n-Bu   H   Ph   Ph   300   595   2104   135       51   t-Bu   H   Ph   Ph   110   321   1070   &gt;500       52   Ph   H   Ph   Ph   280   472   1956   120       53   Ph   H   H   Ph   280   493   1954   106       54   Me   Me   Me   Me   3   30   51   35       55   Me   H   Me   Me   4   21   60   14       56   i-Pr   H   Me   Me   2   15   32   31       57   i-Pr   H   Me   i-Pr   3   20   70   49       58   Me   H   Ph   Me   1   8   20   6       59   Et   H   Ph   Et   1   9   21   8       60   n-Pr   H   Ph   n-Pr   7   42   82   205       61   i-Pr   H   Ph   i-Pr   6   21   70   89       62   Me   H   Ph   Ph   18   103   144   37       63   Et   H   Ph   Ph   40   220   761   70       64   n-Pr   H   Ph   Ph   112   270   1055   84       65   i-Pr   H   Ph   Ph   94   350   864   78       66   n-Bu   H   Ph   Ph   290   544   2008   120       67   t-Bu   H   Ph   Ph   92   275   1000   &gt;500       68   Ph   H   Ph   Ph   270   419   1830   95       69   Ph   H   H   Ph   265   420   1905   81       70   Me   Me   Me   Me   2   10   21   8            acetazolamide   0.25   12   70   25       methazolamide   0.05   14   36   27       dichlorophenamide   1.2   38   380   50       indisulam   0.03   15   65   24                                                          
 
       EXAMPLE 3  
     Design of Selective, Membrane-Impermeant Heterocyclic Sulphonamide Inhibitors Targeting the Human Tumor-Associated Isozyme IX  
       [0463]    A series of positively-charged sulfonamides were obtained by reaction of aminobenzolamide (5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) with tri-/tetra-substituted pyrilium salts possessing alkyl-, aryl- or combinations of alkyl and aryl groups at the pyridinium ring. These new compounds are membrane-impermeant due to their salt-like character and were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, the membrane-anchored bCA IV and the membrane-bound, tumor associated isozyme hCA IX. The high affinity of these new derivatives for the tumor-associated isozyme CA IX and their membrane impermeability, make this type of CA inhibitors interesting candidates for the selective inhibition of only the tumor associated isozyme and not the cytosolic ones, for which they also show high potency.  
         [0464]    Results  
         [0465]    CA inhibition. Inhibition data against isozymes I, II, IV and IX with compounds 71-91 reported here are shown in Table 3. 
                                                                                                                                                                                                   TABLE 3                           Inhibition of isozymes hCA I, hCA II, bCA IV and hCA IX with the       pyridinium salts 71-91.                B                                                                              K I *(nM)                                    hCA                       R 1     R 2     R 3     R 4     R 5     I a     hCA II a     bCA IV b     hCA IX c                      71   Me   H   Me   H   Me   4   0.26   2.1   3       72   i-Pr   H   Me   H   Me   4   0.39   3.0   5       73   i-Pr   H   Me   H   i-Pr   7   1.54   4.7   16       74   t-Bu   H   Me   H   t-Bu   11   3.13   9.4   34       75   Me   H   Ph   H   Me   3   0.20   2.0   6       76   Et   H   Ph   H   Et   4   0.21   2.3   9       77   n-Pr   H   Ph   H   n-Pr   9   3.45   8.1   35       78   n-Bu   H   Ph   H   n-Bu   10   4.62   10.3   40       79   i-Pr   H   Ph   H   i-Pr   5   1.61   4.1   30       80   Me   H   Ph   H   Ph   4   1.21   3.0   24       81   Et   H   Ph   H   Ph   5   1.14   3.8   29       82   n-Pr   H   Ph   H   Ph   8   3.90   6.0   40       83   i-Pr   H   Ph   H   Ph   6   3.74   4.5   32       84   n-Bu   H   Ph   H   Ph   8   4.95   8.4   45       85   t-Bu   H   Ph   H   Ph   12   4.11   7.0   43       86   Ph   H   Me   H   Ph   6   4.78   5.8   12       87   Ph   H   Ph   H   Ph   5   5.96   5.6   12       88   Ph   H   H   H   Ph   5   4.93   5.4   16       89   Me   Me   Me   H   Me   3   0.30   2.4   5       90   Me   Me   Ph   H   Me   3   1.24   5.2   15            91   Me   R 3 ,R 5  =   Me   3   1.37   4.6   12               (CH 2 ) 9 ;               R 4  = Me            aminobenzolamide   6   2.04   5.1   38       acetazolamide   250   12   70   25       methazolamide   50   14   36   27       dichlorophenamide   1200   38   380   50       indisulam   30   15   65   24                                                  
 
         [0466]    Ex vivo penetration through red blood cells. Levels of sulfonamides in red blood cells after incubation of human erythrocytes with millimolar solutions of inhibitor for 30-60 min (both classical as well as positively-charged sulfonamides were used in such experiments) are shown in Table 4 [4, 12, 36, 45, 53, 54, 58, 59, 84, 116, 118].  
                                                                                                   TABLE 4                           Levels of sulfonamide CA inhibitors (μM) in red blood       cells at 30 and 60 min, after exposure of 10 mL of blood       to solutions of sulfonamide (2 mM sulfonamide in 5 mM       Tris buffer, pH 7.4). The concentrations of sulfonamide       has been determined by three methods: HPLC; electronic       spectroscopy (ES) and the enzymatic method       (EI) - see Experimental for details.                [sulfonamide], μM*                t = 30 min   t = 60 min            Inhibitor   HPLC a     ES b     EI c     HPLC a     ES b     EI c                      AAZ   136   139   140   160   167   163       MZA   170   169   165   168   168   167       Benzolamide   110   108   112   148   146   149       Aminobenzolamide   125   127   122   154   156   158       71   0.3   0.5   0.5   0.4   0.5   0.3       76   1.0   1.1   1.0   1.1   1.2   1.1       89   0.3   0.2   0.5   0.3   0.6   0.4       91   0.4   0.3   0.5   0.3   0.6   0.5                          
 
         [0467]    The new compounds reported in the present work were characterized by standard chemical and physical methods (elemental analysis, within ±0.4% of the theoretical values; IR and NMR spectroscopy) that confirmed their structure (see Materials and Methods and Table 5 below for details) and were assayed for the inhibition of isozymes hCA I, hCA II, bCA IV and hCA IX.  
                                               TABLE 5                           Elemental analysis data for the       compounds described in Example 3                Elemental analysis data (calc./found)            No   Formula   % C   % H   % N               71   C 16 H 18 N 5 O 4 S 3   +  ClO 4   −     35.59/35.32   3.36/3.62   12.97/12.93       72   C 18 H 22 N 5 O 4 S 3   +  ClO 4   −     38.06/37.95   3.90/4.16   12.33/12.18       73   C 20 H 26 N 5 O 4 S 3   +  ClO 4   −     40.30/39.99   4.40/4.54   11.75/11.63       74   C 22 H 30 N 5 O 4 S 3   +  ClO 4   −     42.34/42.56   4.84/4.76   11.22/11.03       75   C 21 H 20 N 5 O 4 S 3   +  ClO 4   −     41.89/42.02   3.35/3.03   11.63/11.48       76   C 23 H 24 N 5 O 4 S 3   +  ClO 4   −     43.84/43.88   3.84/3.62   11.11/10.95       77   C 25 H 28 N 5 O 4 S 3   +  ClO 4   −     45.62/45.60   4.29/4.36   10.64/10.50       78   C 27 H 32 N 5 O 4 S 3   +  ClO 4   −     47.26/47.45   4.70/4.89   10.21/10.14       79   C 25 H 28 N 5 O 4 S 3   +  ClO 4   −     45.62/45.49   4.29/4.18   10.64/10.61       80   C 26 H 22 N 5 O 4 S 3   +  ClO 4   −     47.02/46.79   3.34/3.33   10.55/10.23       81   C 27 H 24 N 5 O 4 S 3   +  ClO 4   −     47.82/47.73   3.57/3.73   10.33/10.40       82   C 28 H 26 N 5 O 4 S 3   +  ClO 4   −     48.59/48.83   3.79/3.91   10.12/10.24       83   C 28 H 26 N 5 O 4 S 3   +  ClO 4   −     48.59/48.27   3.79/3.82   10.12/10.05       84   C 29 H 28 N 5 O 4 S 3   +  ClO 4   −     49.32/49.59   4.00/4.23   9.92/9.67       85   C 29 H 28 N 5 O 4 S 3   +  ClO 4   −     49.32/49.16   4.00/3.94   9.92/9.71       86   C 26 H 22 N 5 O 4 S 3   +  ClO 4   −     47.02/47.25   3.34/3.18   10.55/10.46       87   C 31 H 24 N 5 O 4 S 3   +  ClO 4   −     51.27/51.50   3.33/3.60   9.64/9.67       88   C 25 H 20 N 5 O 4 S 3   +  ClO 4   −     46.19/46.28   3.10/2.95   10.77/10.67       89   C 17 H 20 N 5 O 4 S 3   +  ClO 4   −     36.86/36.72   3.64/3.53   12.64/12.45       90   C 22 H 22 N 5 O 4 S 3   +  ClO 4   −     42.89/42.70   3.60/3.84   11.37/11.15       91   C 24 H 32 N 5 O 4 S 3   +  ClO 4   −     44.34/44.57   4.96/4.99   10.77/10.51                  
 
         [0468]    Conclusions  
         [0469]    We report here a general approach for the preparation of positively-charged, membrane-impermeant sulfonamide CA inhibitors with high affinity for the cytosolic isozymes CA I and CA II, as well as for the membrane-bound ones CA IV and CA IX. They were obtained by attaching substituted-pyridinium moieties to aminobenzolamide, a very potent CA inhibitor itself. Ex vivo studies showed the new class of inhibitors reported here to discriminate for the membrane-bound versus the cytosolic isozymes. Correlated with the low nanomolar affinity of some of these compounds for the tumor-associated isozyme CA IX, this report constitutes the basis of selectively inhibiting only the target, tumor-associated CA IX in vivo, whereas the cytosolic isozymes would remain unaffected.  
         [0470]    Characterization of Compounds 71-91 (For preparation, see Materials and Methods Section)  
         [0471]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,4,6-trimethyl-pyridinium perchlorate 71: white crystals, mp&gt;300° C.; IR (KBr), cm −1  (bands in italics are due to the anion): 595, 625, 664, 787, 803, 884, 915, 1100, 1150, 1190, 1200, 1285, 1360, 1495, 1604, 3065;  1 H-NMR (D 2 O), δ, ppm: 3.08 (s, 6H, 2,6-Me 2 ); 3.11 (s, 3H, 4-Me), 7.30-8.06 (m, AA′BB′,4H, ArH from phenylene); 9.05 (s,2H, ArH, 3,5-H from pyridinium); in this solvent the sulfonamido protons are not seen, being in fast exchange with the solvent. Anal C 16 H 18 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0472]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-iso-propyl-4,6-dimethylpyridinium perchlorate 72, colorless crystals, mp 29o-1° C.; IR (KBr), cm −1 : 625, 680, 720, 1100, 1165, 1330, 1640, 3020, 3235;  1 H-NMR (TFA), δ, ppm: 1.50 (d, 6H, 2Me from i-Pr); 2.80 (s, 3H, 6-Me); 2.90 (s, 3H, 4-Me); 3.49 (heptet, 1H, CH from i-Pr); 7.25-8.43 (m, AA′BB′, 4H, ArH from 1,4-phenylene); 7.98 (s, 2H, ArH, 3,5-H from pyridinium). Anal C 18 H 22 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0473]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-iso-propyl-4-methylpyridinium perchlorate 73, tan crystals, mp 278-9° C.; IR (KBr), cm −1 : 625, 685, 820, 1100, 1165, 1340, 1635, 3030, 3250;  1 H-NMR (TFA), δ, ppm: 1.51 (d, 12H, 4Me from 2 i-Pr); 2.83 (s, 3H, 4-Me); 3.42 (heptet, 2H, 2CH from 2 i-Pr); 7.31-8.51 (m, AA′BB′, 4H, ArH from 1,4-phenylene); 8.05 (s, 2H, ArH, 3,5-H from pyridinium). Anal C 20 H 26 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0474]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenylpyridinium perchlorate 75, white crystals, mp&gt;300° C.; IR (KBr), cm −1 : 625, 690, 770, 1100, 1170, 1330, 1635, 3030, 3260, 3330;  1 H-NMR (TFA), δ, ppm: 2.62 (s, 6H, 2,6-(Me) 2 ); 8.10-9.12 (m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). Anal C 21 H 20 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0475]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-diethyl-4-phenylpyridinium perchlorate 76, tan crystals, mp 267-8° C.; IR (KBr), cm −1 : 625, 695, 765, 1100, 1180, 1340, 1630, 3040, 3270, 3360;  1 H-NMR (TFA), δ, ppm: 1.43 (t, 6H, 2 Me from ethyl); 2.82 (q, 4H, 2 CH 2  from Et); 7.68-8.87 (m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). Anal C 23 H 24 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0476]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-n-propyl-4-phenylpyridinium perchlorate 77, colorless crystals, mp 235-7° C.; IR (KBr), cm −1 : 625, 695, 770, 1100, 1180, 1340, 1630, 3050, 3220, 3315;  1 H-NMR (TFA), δ, ppm: 1.06 (t, 6H, 2 Me from propyl); 1.73 (sextet, 4H, 2CH 2  (β) from n-Pr); 2.84 (t, 4H, 2 CH 2  (α) from n-Pr); 7.55-8.71 (m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). Anal C 25 H 28 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0477]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-isopropyl-4-phenylpyridinium perchlorate 79, white crystals, mp 278-9° C.; IR (KBr), cm −1 : 625, 690, 765, 1100, 1180, 1340, 1625, 3040, 3270, 3315;  1 H-NMR (TFA), δ, ppm: 1.45 (d, 12H, 4 Me from i-Pr); 2.95 (heptet, 2H, 2 CH from i-Pr); 7.92-8.97 (m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). Anal C 25 H 28 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0478]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-methyl-4,6-diphenylpyridinium perchlorate 80, white crystals, mp 298-99° C.; IR (KBr), cm −1 : 625, 710, 770, 1100, 1170, 1345, 1625, 3040, 3245, 3350;  1 H-NMR (TFA), δ, ppm: 2.75 (s, 3H, 2-Me); 7.53-8.70 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph 2 ). Anal C 26 H 22 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0479]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-ethyl-4,6-diphenylpyridinium perchlorate 81, white crystals, mp 254-5° C.; IR (KBr), cm −1 : 625, 700, 770, 1100, 1180, 1340, 1620, 3040, 3250, 3350;  1 H-NMR (TFA), δ, ppm: 1.52 (t, 3H, Me from ethyl); 2.97 (q, 2H, CH 2 ); 7.40-8.57 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph 2 ). Anal C 27 H 24 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0480]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-n-propyl-4,6-diphenylpyridinium perchlorate 82, white crystals, mp 214-5° C.; IR (KBr), cm −1 : 625, 700, 770, 1100, 1180, 1340, 1620, 3030, 3270, 3350;  1 H-NMR (TFA), δ, ppm: 1.03 (t, 3H, Me from propyl); 1.95 (sextet, 2H, β-CH 2  from n-Pr); 2.88 (t, 2H, α-CH 2  from n-Pr); 7.39-8.55 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph 2 ). Anal C 28 H 26 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0481]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-iso-propyl-4,6-diphenylpyridinium perchlorate 83, white crystals, mp 186-8° C.; IR (KBr), cm −1 : 625, 700, 770, 1100, 1170, 1340, 1620, 3040, 3250, 3360;  1 H-NMR (TFA), δ, ppm: 1.51 (d, 6H, 2 Me from i-propyl); 2.50-3.27 (m, 1H, CH from i-Pr); 7.32-8.54 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph 2 ). Anal C 28 H 26 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0482]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-n-butyl-4,6-diphenylpyridinium perchlorate 84, white crystals, mp 241-3° C.; IR (KBr), cm −1 : 625, 710, 770, 1100, 1180, 1335, 1625, 3040, 3260, 3345;  1 H-NMR (TFA), δ, ppm: 0.93 (t, 3H, Me from butyl); 1.12-2.14 (m, 4H, CH 3 —CH 2 —CH 2 —CH 2  from n-Bu); 2.96 (t, 2H, α-CH 2  from n-Bu); 7.21-8.50 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph 2 ). Anal C 29 H 28 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0483]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-tert-butyl-4,6-diphenylpyridinium perchlorate 85, white crystals, mp 203-5° C.; IR (KBr), cm −1 : 625, 705, 765, 1100, 1160, 1310, 1620, 3060, 3270;  1 H-NMR (TFA), δ, ppm: 1.91 (s, 9H, t-Bu); 6.80-8.74 (m, 16H, ArH from 1,4-phenylene, 4,6-Ph 2  and 3,5-H from pyridinium). Anal C 29 H 28 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0484]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,4,6-triphenhyl-pyridinium perchlorate 87: pale yellow crystals, mp&gt;300° C.; IR (KBr),  −1  (bands in italics are due to the anion): 625, 635, 703, 785, 896, 1100, 1150, 1204, 1355, 1410, 1520, 1600, 3065;  1 H-NMR (D 2 O), δ, ppm: 7.50-8.60 (m, 19H, ArH, 3Ph +C 6 H 4 ); 9.27 (s,2H, ArH, 3,5-H from pyridinium); in this solvent the sulfonamido protons are not seen, being in fast exchange with the solvent. Anal C 31 H 24 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0485]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-diphenylpyridinium perchlorate 88, yellow crystals, mp 218-20° C.; IR (KBr), cm −1 : 625, 705, 765, 1100, 1160, 1335, 1615, 3050, 3260;  1 H-NMR (TFA), δ, ppm: 6.75-8.43 (m, 17H, ArH from 1,4-phenylene, 2,6-Ph 2  and 3,4,5-H from pyridinium). Anal C 25 H 20 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0486]    1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,3,4,6-tetramethylpyridinium perchlorate 89, tan crystals, mp&gt;300° C.; IR (KBr), cm −1 : 625, 800, 1100, 1165, 1330, 1630, 3030, 3305;  1 H-NMR (TFA), δ, ppm: 2.62 (s, 3H, 4-Me); 2.74 (s, 3H, 3-Me); 2.88 (s, 6H, 2,6-(Me) 2 ); 7.21-8.50 (m, AA′BB′, 4H, ArH from 1,4-phenylene); 7.93 (s, 1H, ArH, 5-H from pyridinium). Anal C 17 H 20 N 5 O 4 S 3   + ClO 4   −  (C, H, N).  
         [0487]    The description of the foregoing embodiments of the invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described in order to explain the principles of the invention and its practical application to enable thereby others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.  
         [0488]    All references cited herein are hereby incorporated by reference.  
     
       
       
         1 
         
           
             9  
           
           
             1  
             1522  
             DNA  
             Homo sapiens  
             
               CDS  
               (13)..(1389)  
             
             
               mat_peptide  
               (124)..(1389)  
             
           
            1 

acagtcagcc gc atg gct ccc ctg tgc ccc agc ccc tgg ctc cct ctg ttg     51 
              Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu 
                      -35                 -30                 -25 

atc ccg gcc cct gct cca ggc ctc act gtg caa ctg ctg ctg tca ctg       99 
Ile Pro Ala Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu 
                -20                 -15                 -10 

ctg ctt ctg atg cct gtc cat ccc cag agg ttg ccc cgg atg cag gag      147 
Leu Leu Leu Met Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu 
            -5              -1  1               5 

gat tcc ccc ttg gga gga ggc tct tct ggg gaa gat gac cca ctg ggc      195 
Asp Ser Pro Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly 
    10                  15                  20 

gag gag gat ctg ccc agt gaa gag gat tca ccc aga gag gag gat cca      243 
Glu Glu Asp Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro 
25                  30                  35                  40 

ccc gga gag gag gat cta cct gga gag gag gat cta cct gga gag gag      291 
Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu 
                45                  50                  55 

gat cta cct gaa gtt aag cct aaa tca gaa gaa gag ggc tcc ctg aag      339 
Asp Leu Pro Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys 
            60                  65                  70 

tta gag gat cta cct act gtt gag gct cct gga gat cct caa gaa ccc      387 
Leu Glu Asp Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro 
        75                  80                  85 

cag aat aat gcc cac agg gac aaa gaa ggg gat gac cag agt cat tgg      435 
Gln Asn Asn Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp 
    90                  95                  100 

cgc tat gga ggc gac ccg ccc tgg ccc cgg gtg tcc cca gcc tgc gcg      483 
Arg Tyr Gly Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala 
105                 110                 115                 120 

ggc cgc ttc cag tcc ccg gtg gat atc cgc ccc cag ctc gcc gcc ttc      531 
Gly Arg Phe Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe 
                125                 130                 135 

tgc ccg gcc ctg cgc ccc ctg gaa ctc ctg ggc ttc cag ctc ccg ccg      579 
Cys Pro Ala Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro 
            140                 145                 150 

ctc cca gaa ctg cgc ctg cgc aac aat ggc cac agt gtg caa ctg acc      627 
Leu Pro Glu Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr 
        155                 160                 165 

ctg cct cct ggg cta gag atg gct ctg ggt ccc ggg cgg gag tac cgg      675 
Leu Pro Pro Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg 
    170                 175                 180 

gct ctg cag ctg cat ctg cac tgg ggg gct gca ggt cgt ccg ggc tcg      723 
Ala Leu Gln Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser 
185                 190                 195                 200 

gag cac act gtg gaa ggc cac cgt ttc cct gcc gag atc cac gtg gtt      771 
Glu His Thr Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val 
                205                 210                 215 

cac ctc agc acc gcc ttt gcc aga gtt gac gag gcc ttg ggg cgc ccg      819 
His Leu Ser Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro 
            220                 225                 230 

gga ggc ctg gcc gtg ttg gcc gcc ttt ctg gag gag ggc ccg gaa gaa      867 
Gly Gly Leu Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu 
        235                 240                 245 

aac agt gcc tat gag cag ttg ctg tct cgc ttg gaa gaa atc gct gag      915 
Asn Ser Ala Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu 
    250                 255                 260 

gaa ggc tca gag act cag gtc cca gga ctg gac ata tct gca ctc ctg      963 
Glu Gly Ser Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu 
265                 270                 275                 280 

ccc tct gac ttc agc cgc tac ttc caa tat gag ggg tct ctg act aca     1011 
Pro Ser Asp Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr 
                285                 290                 295 

ccg ccc tgt gcc cag ggt gtc atc tgg act gtg ttt aac cag aca gtg     1059 
Pro Pro Cys Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val 
            300                 305                 310 

atg ctg agt gct aag cag ctc cac acc ctc tct gac acc ctg tgg gga     1107 
Met Leu Ser Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly 
        315                 320                 325 

cct ggt gac tct cgg cta cag ctg aac ttc cga gcg acg cag cct ttg     1155 
Pro Gly Asp Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu 
    330                 335                 340 

aat ggg cga gtg att gag gcc tcc ttc cct gct gga gtg gac agc agt     1203 
Asn Gly Arg Val Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser 
345                 350                 355                 360 

cct cgg gct gct gag cca gtc cag ctg aat tcc tgc ctg gct gct ggt     1251 
Pro Arg Ala Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly 
                365                 370                 375 

gac atc cta gcc ctg gtt ttt ggc ctc ctt ttt gct gtc acc agc gtc     1299 
Asp Ile Leu Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val 
            380                 385                 390 

gcg ttc ctt gtg cag atg aga agg cag cac aga agg gga acc aaa ggg     1347 
Ala Phe Leu Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly 
        395                 400                 405 

ggt gtg agc tac cgc cca gca gag gta gcc gag act gga gcc             1389 
Gly Val Ser Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala 
    410                 415                 420 

tagaggctgg atcttggaga atgtgagaag ccagccagag gcatctgagg gggagccggt   1449 

aactgtcctg tcctgctcat tatgccactt ccttttaact gccaagaaat tttttaaaat   1509 

aaatatttat aat                                                      1522 

 
           
             2  
             459  
             PRT  
             Homo sapiens  
           
            2 

Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala 
        -35                 -30                 -25 

Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu 
    -20                 -15                 -10 

Met Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro 
-5              -1  1               5                   10 

Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp 
            15                  20                  25 

Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu 
        30                  35                  40 

Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro 
    45                  50                  55 

Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp 
60                  65                  70                  75 

Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn 
                80                  85                  90 

Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly 
            95                  100                 105 

Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe 
        110                 115                 120 

Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala 
    125                 130                 135 

Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu 
140                 145                 150                 155 

Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro 
                160                 165                 170 

Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln 
            175                 180                 185 

Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr 
        190                 195                 200 

Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser 
    205                 210                 215 

Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu 
220                 225                 230                 235 

Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala 
                240                 245                 250 

Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser 
            255                 260                 265 

Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp 
        270                 275                 280 

Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys 
    285                 290                 295 

Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser 
300                 305                 310                 315 

Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp 
                320                 325                 330 

Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg 
            335                 340                 345 

Val Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala 
        350                 355                 360 

Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu 
    365                 370                 375 

Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu 
380                 385                 390                 395 

Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser 
                400                 405                 410 

Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala 
            415                 420 

 
           
             3  
             10898  
             DNA  
             Homo sapiens  
             
               gene  
               (1)..(10898)  
             
             
               misc_feature  
               (1974)..(1974)  
               n is a, c, g, or t  
             
           
            3 

ggatcctgtt gactcgtgac cttaccccca accctgtgct ctctgaaaca tgagctgtgt     60 

ccactcaggg ttaaatggat taagggcggt gcaagatgtg ctttgttaaa cagatgcttg    120 

aaggcagcat gctcgttaag agtcatcacc aatccctaat ctcaagtaat cagggacaca    180 

aacactgcgg aaggccgcag ggtcctctgc ctaggaaaac cagagacctt tgttcacttg    240 

tttatctgac cttccctcca ctattgtcca tgaccctgcc aaatccccct ctgtgagaaa    300 

cacccaagaa ttatcaataa aaaaataaat ttaaaaaaaa aatacaaaaa aaaaaaaaaa    360 

aaaaaaaaaa gacttacgaa tagttattga taaatgaata gctattggta aagccaagta    420 

aatgatcata ttcaaaacca gacggccatc atcacagctc aagtctacct gatttgatct    480 

ctttatcatt gtcattcttt ggattcacta gattagtcat catcctcaaa attctccccc    540 

aagttctaat tacgttccaa acatttaggg gttacatgaa gcttgaacct actaccttct    600 

ttgcttttga gccatgagtt gtaggaatga tgagtttaca ccttacatgc tggggattaa    660 

tttaaacttt acctctaagt cagttgggta gcctttggct tatttttgta gctaattttg    720 

tagttaatgg atgcactgtg aatcttgcta tgatagtttt cctccacact ttgccactag    780 

gggtaggtag gtactcagtt ttcagtaatt gcttacctaa gaccctaagc cctatttctc    840 

ttgtactggc ctttatctgt aatatgggca tatttaatac aatataattt ttggagtttt    900 

tttgtttgtt tgtttgtttg tttttttgag acggagtctt gcatctgtca tgcccaggct    960 

ggagtagcag tggtgccatc tcggctcact gcaagctcca cctcccgagt tcacgccatt   1020 

ttcctgcctc agcctcccga gtagctggga ctacaggcgc ccgccaccat gcccggctaa   1080 

ttttttgtat ttttggtaga gacggggttt caccgtgtta gccagaatgg tctcgatctc   1140 

ctgacttcgt gatccacccg cctcggcctc ccaaagttct gggattacag gtgtgagcca   1200 

ccgcacctgg ccaatttttt gagtctttta aagtaaaaat atgtcttgta agctggtaac   1260 

tatggtacat ttccttttat taatgtggtg ctgacggtca tataggttct tttgagtttg   1320 

gcatgcatat gctacttttt gcagtccttt cattacattt ttctctcttc atttgaagag   1380 

catgttatat cttttagctt cacttggctt aaaaggttct ctcattagcc taacacagtg   1440 

tcattgttgg taccacttgg atcataagtg gaaaaacagt caagaaattg cacagtaata   1500 

cttgtttgta agagggatga ttcaggtgaa tctgacacta agaaactccc ctacctgagg   1560 

tctgagattc ctctgacatt gctgtatata ggcttttcct ttgacagcct gtgactgcgg   1620 

actatttttc ttaagcaaga tatgctaaag ttttgtgagc ctttttccag agagaggtct   1680 

catatctgca tcaagtgaga acatataatg tctgcatgtt tccatatttc aggaatgttt   1740 

gcttgtgttt tatgctttta tatagacagg gaaacttgtt cctcagtgac ccaaaagagg   1800 

tgggaattgt tattggatat catcattggc ccacgctttc tgaccttgga aacaattaag   1860 

ggttcataat ctcaattctg tcagaattgg tacaagaaat agctgctatg tttcttgaca   1920 

ttccacttgg taggaaataa gaatgtgaaa ctcttcagtt ggtgtgtgtc cctngttttt   1980 

ttgcaatttc cttcttactg tgttaaaaaa aagtatgatc ttgctctgag aggtgaggca   2040 

ttcttaatca tgatctttaa agatcaataa tataatcctt tcaaggatta tgtctttatt   2100 

ataataaaga taatttgtct ttaacagaat caataatata atcccttaaa ggattatatc   2160 

tttgctgggc gcagtggctc acacctgtaa tcccagcact ttgggtggcc aaggtggaag   2220 

gatcaaattt gcctacttct atattatctt ctaaagcaga attcatctct cttccctcaa   2280 

tatgatgata ttgacagggt ttgccctcac tcactagatt gtgagctcct gctcagggca   2340 

ggtagcgttt tttgtttttg tttttgtttt tcttttttga gacagggtct tgctctgtca   2400 

cccaggccag agtgcaatgg tacagtctca gctcactgca gcctcaaccg cctcggctca   2460 

aaccatcatc ccatttcagc ctcctgagta gctgggacta caggcacatg ccattacacc   2520 

tggctaattt ttttgtattt ctagtagaga cagggtttgg ccatgttgcc cgggctggtc   2580 

tcgaactcct ggactcaagc aatccaccca cctcagcctc ccaaaatgag ggaccgtgtc   2640 

ttattcattt ccatgtccct agtccatagc ccagtgctgg acctatggta gtactaaata   2700 

aatatttgtt gaatgcaata gtaaatagca tttcagggag caagaactag attaacaaag   2760 

gtggtaaaag gtttggagaa aaaaataata gtttaatttg gctagagtat gagggagagt   2820 

agtaggagac aagatggaaa ggtctcttgg gcaaggtttt gaaggaagtt ggaagtcaga   2880 

agtacacaat gtgcatatcg tggcaggcag tggggagcca atgaaggctt ttgagcagga   2940 

gagtaatgtg ttgaaaaata aatataggtt aaacctatca gagcccctct gacacataca   3000 

cttgcttttc attcaagctc aagtttgtct cccacatacc cattacttaa ctcaccctcg   3060 

ggctccccta gcagcctgcc ctacctcttt acctgcttcc tggtggagtc agggatgtat   3120 

acatgagctg ctttccctct cagccagagg acatgggggg ccccagctcc cctgcctttc   3180 

cccttctgtg cctggagctg ggaagcaggc cagggttagc tgaggctggc tggcaagcag   3240 

ctgggtggtg ccagggagag cctgcatagt gccaggtggt gccttgggtt ccaagctagt   3300 

ccatggcccc gataaccttc tgcctgtgca cacacctgcc cctcactcca cccccatcct   3360 

agctttggta tgggggagag ggcacagggc cagacaaacc tgtgagactt tggctccatc   3420 

tctgcaaaag ggcgctctgt gagtcagcct gctcccctcc aggcttgctc ctcccccacc   3480 

cagctctcgt ttccaatgca cgtacagccc gtacacaccg tgtgctggga caccccacag   3540 

tcagccgcat ggctcccctg tgccccagcc cctggctccc tctgttgatc ccggcccctg   3600 

ctccaggcct cactgtgcaa ctgctgctgt cactgctgct tctggtgcct gtccatcccc   3660 

agaggttgcc ccggatgcag gaggattccc ccttgggagg aggctcttct ggggaagatg   3720 

acccactggg cgaggaggat ctgcccagtg aagaggattc acccagagag gaggatccac   3780 

ccggagagga ggatctacct ggagaggagg atctacctgg agaggaggat ctacctgaag   3840 

ttaagcctaa atcagaagaa gagggctccc tgaagttaga ggatctacct actgttgagg   3900 

ctcctggaga tcctcaagaa ccccagaata atgcccacag ggacaaagaa ggtaagtggt   3960 

catcaatctc caaatccagg ttccaggagg ttcatgactc ccctcccata ccccagccta   4020 

ggctctgttc actcagggaa ggaggggaga ctgtactccc cacagaagcc cttccagagg   4080 

tcccatacca atatccccat ccccactctc ggaggtagaa agggacagat gtggagagaa   4140 

aataaaaagg gtgcaaaagg agagaggtga gctggatgag atgggagaga agggggaggc   4200 

tggagaagag aaagggatga gaactgcaga tgagagaaaa aatgtgcaga cagaggaaaa   4260 

aaataggtgg agaaggagag tcagagagtt tgaggggaag agaaaaggaa agcttgggag   4320 

gtgaagtggg taccagagac aagcaagaag agctggtaga agtcatctca tcttaggcta   4380 

caatgaggaa ttgagaccta ggaagaaggg acacagcagg tagagaaacg tggcttcttg   4440 

actcccaagc caggaatttg gggaaagggg ttggagacca tacaaggcag agggatgagt   4500 

ggggagaaga aagaagggag aaaggaaaga tggtgtactc actcatttgg gactcaggac   4560 

tgaagtgccc actcactttt tttttttttt tttttgagac aaactttcac ttttgttgcc   4620 

caggctggag tgcaatggcg cgatctcggc tcactgcaac ctccacctcc cgggttcaag   4680 

tgattctcct gcctcagcct ctagccaagt agctgcgatt acaggcatgc gccaccacgc   4740 

ccggctaatt tttgtatttt tagtagagac ggggtttcgc catgttggtc aggctggtct   4800 

cgaactcctg atctcaggtg atccaaccac cctggcctcc caaagtgctg ggattatagg   4860 

cgtgagccac agcgcctggc ctgaagcagc cactcacttt tacagaccct aagacaatga   4920 

ttgcaagctg gtaggattgc tgtttggccc acccagctgc ggtgttgagt ttgggtgcgg   4980 

tctcctgtgc tttgcacctg gcccgcttaa ggcatttgtt acccgtaatg ctcctgtaag   5040 

gcatctgcgt ttgtgacatc gttttggtcg ccaggaaggg attggggctc taagcttgag   5100 

cggttcatcc ttttcattta tacaggggat gaccagagtc attggcgcta tggaggtgag   5160 

acacccaccc gctgcacaga cccaatctgg gaacccagct ctgtggatct cccctacagc   5220 

cgtccctgaa cactggtccc gggcgtccca cccgccgccc accgtcccac cccctcacct   5280 

tttctacccg ggttccctaa gttcctgacc taggcgtcag acttcctcac tatactctcc   5340 

caccccaggc gacccgccct ggccccgggt gtccccagcc tgcgcgggcc gcttccagtc   5400 

cccggtggat atccgccccc agctcgccgc cttctgcccg gccctgcgcc ccctggaact   5460 

cctgggcttc cagctcccgc cgctcccaga actgcgcctg cgcaacaatg gccacagtgg   5520 

tgagggggtc tccccgccga gacttgggga tggggcgggg cgcagggaag ggaaccgtcg   5580 

cgcagtgcct gcccgggggt tgggctggcc ctaccgggcg gggccggctc acttgcctct   5640 

ccctacgcag tgcaactgac cctgcctcct gggctagaga tggctctggg tcccgggcgg   5700 

gagtaccggg ctctgcagct gcatctgcac tggggggctg caggtcgtcc gggctcggag   5760 

cacactgtgg aaggccaccg tttccctgcc gaggtgagcg cggactggcc gagaaggggc   5820 

aaaggagcgg ggcggacggg ggccagagac gtggccctct cctaccctcg tgtccttttc   5880 

agatccacgt ggttcacctc agcaccgcct ttgccagagt tgacgaggcc ttggggcgcc   5940 

cgggaggcct ggccgtgttg gccgcctttc tggaggtacc agatcctgga caccccctac   6000 

tccccgcttt cccatcccat gctcctcccg gactctatcg tggagccaga gaccccatcc   6060 

cagcaagctc actcaggccc ctggctgaca aactcattca cgcactgttt gttcatttaa   6120 

cacccactgt gaaccaggca ccagccccca acaaggattc tgaagctgta ggtccttgcc   6180 

tctaaggagc ccacagccag tgggggaggc tgacatgaca gacacatagg aaggacatag   6240 

taaagatggt ggtcacagag gaggtgacac ttaaagcctt cactggtaga aaagaaaagg   6300 

aggtgttcat tgcagaggaa acagaatgtg caaagactca gaatatggcc tatttaggga   6360 

atggctacat acaccatgat tagaggaggc ccagtaaagg gaagggatgg tgagatgcct   6420 

gctaggttca ctcactcact tttatttatt tatttatttt tttgacagtc tctctgtcgc   6480 

ccaggctgga gtgcagtggt gtgatcttgg gtcactgcaa cttccgcctc ccgggttcaa   6540 

gggattctcc tgcctcagct tcctgagtag ctggggttac aggtgtgtgc caccatgccc   6600 

agctaatttt tttttgtatt tttagtagac agggtttcac catgttggtc aggctggtct   6660 

caaactcctg gcctcaagtg atccgcctga ctcagcctac caaagtgctg attacaagtg   6720 

tgagccaccg tgcccagcca cactcactga ttctttaatg ccagccacac agcacaaagt   6780 

tcagagaaat gcctccatca tagcatgtca atatgttcat actcttaggt tcatgatgtt   6840 

cttaacatta ggttcataag caaaataaga aaaaagaata ataaataaaa gaagtggcat   6900 

gtcaggacct cacctgaaaa gccaaacaca gaatcatgaa ggtgaatgca gaggtgacac   6960 

caacacaaag gtgtatatat ggtttcctgt ggggagtatg tacggaggca gcagtgagtg   7020 

agactgcaaa cgtcagaagg gcacgggtca ctgagagcct agtatcctag taaagtgggc   7080 

tctctccctc tctctccagc ttgtcattga aaaccagtcc accaagcttg ttggttcgca   7140 

cagcaagagt acatagagtt tgaaataata cataggattt taagagggag acactgtctc   7200 

taaaaaaaaa aacaacagca acaacaaaaa gcaacaacca ttacaatttt atgttccctc   7260 

agcattctca gagctgagga atgggagagg actatgggaa cccccttcat gttccggcct   7320 

tcagccatgg ccctggatac atgcactcat ctgtcttaca atgtcattcc cccaggaggg   7380 

cccggaagaa aacagtgcct atgagcagtt gctgtctcgc ttggaagaaa tcgctgagga   7440 

aggtcagttt gttggtctgg ccactaatct ctgtggccta gttcataaag aatcaccctt   7500 

tggagcttca ggtctgaggc tggagatggg ctccctccag tgcaggaggg attgaagcat   7560 

gagccagcgc tcatcttgat aataaccatg aagctgacag acacagttac ccgcaaacgg   7620 

ctgcctacag attgaaaacc aagcaaaaac cgccgggcac ggtggctcac gcctgtaatc   7680 

ccagcacttt gggaggccaa ggcaggtgga tcacgaggtc aagagatcaa gaccatcctg   7740 

gccaacatgg tgaaacccca tctctactaa aaatacgaaa aaatagccag gcgtggtggc   7800 

gggtgcctgt aatcccagct actcgggagg ctgaggcagg agaatggcat gaacccggga   7860 

ggcagaagtt gcagtgagcc gagatcgtgc cactgcactc cagcctgggc aacagagcga   7920 

gactcttgtc tcaaaaaaaa aaaaaaaaaa gaaaaccaag caaaaaccaa aatgagacaa   7980 

aaaaaacaag accaaaaaat ggtgtttgga aattgtcaag gtcaagtctg gagagctaaa   8040 

ctttttctga gaactgttta tctttaataa gcatcaaata ttttaacttt gtaaatactt   8100 

ttgttggaaa tcgttctctt cttagtcact cttgggtcat tttaaatctc acttactcta   8160 

ctagaccttt taggtttctg ctagactagg tagaactctg cctttgcatt tcttgtgtct   8220 

gttttgtata gttatcaata ttcatattta tttacaagtt attcagatca ttttttcttt   8280 

tctttttttt tttttttttt ttttttacat ctttagtaga gacagggttt caccatattg   8340 

gccaggctgc tctcaaactc ctgaccttgt gatccaccag cctcggcctc ccaaagtgct   8400 

gggattcatt ttttcttttt aatttgctct gggcttaaac ttgtggccca gcactttatg   8460 

atggtacaca gagttaagag tgtagactca gacggtcttt cttctttcct tctcttcctt   8520 

cctcccttcc ctcccacctt cccttctctc cttcctttct ttcttcctct cttgcttcct   8580 

caggcctctt ccagttgctc caaagccctg tacttttttt tgagttaacg tcttatggga   8640 

agggcctgca cttagtgaag aagtggtctc agagttgagt taccttggct tctgggaggt   8700 

gaaactgtat ccctataccc tgaagcttta agggggtgca atgtagatga gaccccaaca   8760 

tagatcctct tcacaggctc agagactcag gtcccaggac tggacatatc tgcactcctg   8820 

ccctctgact tcagccgcta cttccaatat gaggggtctc tgactacacc gccctgtgcc   8880 

cagggtgtca tctggactgt gtttaaccag acagtgatgc tgagtgctaa gcaggtgggc   8940 

ctggggtgtg tgtggacaca gtgggtgcgg gggaaagagg atgtaagatg agatgagaaa   9000 

caggagaaga aagaaatcaa ggctgggctc tgtggcttac gcctataatc ccaccacgtt   9060 

gggaggctga ggtgggagaa tggtttgagc ccaggagttc aagacaaggc ggggcaacat   9120 

agtgtgaccc catctctacc aaaaaaaccc caacaaaacc aaaaatagcc gggcatggtg   9180 

gtatgcggcc tagtcccagc tactcaagga ggctgaggtg ggaagatcgc ttgattccag   9240 

gagtttgaga ctgcagtgag ctatgatccc accactgcct accatcttta ggatacattt   9300 

atttatttat aaaagaaatc aagaggctgg atggggaata caggagctgg agggtggagc   9360 

cctgaggtgc tggttgtgag ctggcctggg acccttgttt cctgtcatgc catgaaccca   9420 

cccacactgt ccactgacct ccctagctcc acaccctctc tgacaccctg tggggacctg   9480 

gtgactctcg gctacagctg aacttccgag cgacgcagcc tttgaatggg cgagtgattg   9540 

aggcctcctt ccctgctgga gtggacagca gtcctcgggc tgctgagcca ggtacagctt   9600 

tgtctggttt ccccccagcc agtagtccct tatcctccca tgtgtgtgcc agtgtctgtc   9660 

attggtggtc acagcccgcc tctcacatct cctttttctc tccagtccag ctgaattcct   9720 

gcctggctgc tggtgagtct gcccctcctc ttggtcctga tgccaggaga ctcctcagca   9780 

ccattcagcc ccagggctgc tcaggaccgc ctctgctccc tctccttttc tgcagaacag   9840 

accccaaccc caatattaga gaggcagatc atggtgggga ttcccccatt gtccccagag   9900 

gctaattgat tagaatgaag cttgagaaat ctcccagcat ccctctcgca aaagaatccc   9960 

cccccctttt tttaaagata gggtctcact ctgtttgccc caggctgggg tgttgtggca  10020 

cgatcatagc tcactgcagc ctcgaactcc taggctcagg caatcctttc accttagctt  10080 

ctcaaagcac tgggactgta ggcatgagcc actgtgcctg gccccaaacg gcccttttac  10140 

ttggctttta ggaagcaaaa acggtgctta tcttacccct tctcgtgtat ccaccctcat  10200 

cccttggctg gcctcttctg gagactgagg cactatgggg ctgcctgaga actcggggca  10260 

ggggtggtgg agtgcactga ggcaggtgtt gaggaactct gcagacccct cttccttccc  10320 

aaagcagccc tctctgctct ccatcgcagg tgacatccta gccctggttt ttggcctcct  10380 

ttttgctgtc accagcgtcg cgttccttgt gcagatgaga aggcagcaca ggtattacac  10440 

tgaccctttc ttcaggcaca agcttccccc acccttgtgg agtcacttca tgcaaagcgc  10500 

atgcaaatga gctgctcctg ggccagtttt ctgattagcc tttcctgttg tgtacacaca  10560 

gaaggggaac caaagggggt gtgagctacc gcccagcaga ggtagccgag actggagcct  10620 

agaggctgga tcttggagaa tgtgagaagc cagccagagg catctgaggg ggagccggta  10680 

actgtcctgt cctgctcatt atgccacttc cttttaactg ccaagaaatt ttttaaaata  10740 

aatatttata ataaaatatg tgttagtcac ctttgttccc caaatcagaa ggaggtattt  10800 

gaatttccta ttactgttat tagcaccaat ttagtggtaa tgcatttatt ctattacagt  10860 

tcggcctcct tccacacatc actccaatgt gttgctcc                          10898 

 
           
             4  
             59  
             PRT  
             Homo sapiens  
           
            4 

Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp Leu Pro Ser Glu 
1               5                   10                  15 

Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu Pro 
            20                  25                  30 

Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Glu Val Lys Pro 
        35                  40                  45 

Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu 
    50                  55 

 
           
             5  
             257  
             PRT  
             Homo sapiens  
           
            5 

Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp Pro 
1               5                   10                  15 

Arg Val Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro Val Asp Ile 
            20                  25                  30 

Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu Arg Pro Leu Glu Leu 
        35                  40                  45 

Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu Arg Leu Arg Asn Asn 
    50                  55                  60 

Gly His Ser Val Gln Leu Thr Leu Pro Pro Gly Leu Glu Met Ala Leu 
65                  70                  75                  80 

Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln Leu His Leu His Trp Gly 
                85                  90                  95 

Ala Ala Gly Arg Pro Gly Ser Glu His Thr Val Glu Gly His Arg Phe 
            100                 105                 110 

Pro Ala Glu Ile His Val Val His Leu Ser Thr Ala Phe Ala Arg Val 
        115                 120                 125 

Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu Ala Val Leu Ala Ala Phe 
    130                 135                 140 

Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala Tyr Glu Gln Leu Leu Ser 
145                 150                 155                 160 

Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser Glu Thr Gln Val Pro Gly 
                165                 170                 175 

Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg Tyr Phe Gln 
            180                 185                 190 

Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys Ala Gln Gly Val Ile Trp 
        195                 200                 205 

Thr Val Phe Asn Gln Thr Val Met Leu Ser Ala Lys Gln Leu His Thr 
    210                 215                 220 

Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp Ser Arg Leu Gln Leu Asn 
225                 230                 235                 240 

Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg Val Ile Glu Ala Ser Phe 
                245                 250                 255 

Pro 

 
           
             6  
             37  
             PRT  
             Homo sapiens  
           
            6 

Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala 
1               5                   10                  15 

Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu 
            20                  25                  30 

Met Pro Val His Pro 
        35 

 
           
             7  
             377  
             PRT  
             Homo sapiens  
           
            7 

Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro Leu Gly Gly Gly Ser 
1               5                   10                  15 

Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp Leu Pro Ser Glu Glu 
            20                  25                  30 

Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu Pro Gly 
        35                  40                  45 

Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Glu Val Lys Pro Lys 
    50                  55                  60 

Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp Leu Pro Thr Val Glu 
65                  70                  75                  80 

Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn Ala His Arg Asp Lys 
                85                  90                  95 

Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp 
            100                 105                 110 

Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro Val Asp 
        115                 120                 125 

Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu Arg Pro Leu Glu 
    130                 135                 140 

Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu Arg Leu Arg Asn 
145                 150                 155                 160 

Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro Gly Leu Glu Met Ala 
                165                 170                 175 

Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln Leu His Leu His Trp 
            180                 185                 190 

Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr Val Glu Gly His Arg 
        195                 200                 205 

Phe Pro Ala Glu Ile His Val Val His Leu Ser Thr Ala Phe Ala Arg 
    210                 215                 220 

Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu Ala Val Leu Ala Ala 
225                 230                 235                 240 

Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala Tyr Glu Gln Leu Leu 
                245                 250                 255 

Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser Glu Thr Gln Val Pro 
            260                 265                 270 

Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg Tyr Phe 
        275                 280                 285 

Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys Ala Gln Gly Val Ile 
    290                 295                 300 

Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser Ala Lys Gln Leu His 
305                 310                 315                 320 

Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp Ser Arg Leu Gln Leu 
                325                 330                 335 

Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg Val Ile Glu Ala Ser 
            340                 345                 350 

Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala Ala Glu Pro Val Gln 
        355                 360                 365 

Leu Asn Ser Cys Leu Ala Ala Gly Asp 
    370                 375 

 
           
             8  
             20  
             PRT  
             Homo sapiens  
           
            8 

Ile Leu Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala 
1               5                   10                  15 

Phe Leu Val Gln 
            20 

 
           
             9  
             25  
             PRT  
             Homo sapiens  
           
            9 

Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser Tyr Arg 
1               5                   10                  15 

Pro Ala Glu Val Ala Glu Thr Gly Ala 
            20                  25