Application of Molecular Modeling to Urokinase Inhibitors Development

Urokinase-type plasminogen activator (uPA) plays an important role in the regulation of diverse physiologic and pathologic processes. Experimental research has shown that elevated uPA expression is associated with cancer progression, metastasis, and shortened survival in patients, whereas suppression of proteolytic activity of uPA leads to evident decrease of metastasis. Therefore, uPA has been considered as a promising molecular target for development of anticancer drugs. The present study sets out to develop the new selective uPA inhibitors using computer-aided structural based drug design methods. Investigation involves the following stages: computer modeling of the protein active site, development and validation of computer molecular modeling methods: docking (SOL program), postprocessing (DISCORE program), direct generalized docking (FLM program), and the application of the quantum chemical calculations (MOPAC package), search of uPA inhibitors among molecules from databases of ready-made compounds to find new uPA inhibitors, and design of new chemical structures and their optimization and experimental examination. On the basis of known uPA inhibitors and modeling results, 18 new compounds have been designed, calculated using programs mentioned above, synthesized, and tested in vitro. Eight of them display inhibitory activity and two of them display activity about 10 μM.

Related papers:

title: The urokinase plasminogen activator system: role in malignancy.
abstract: The urokinase plasminogen activator (uPA) system consists of the serine protease uPA, its glycolipid-anchored receptor, uPAR and its 2 serpin inhibitors, plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). Recent findings suggest that the uPA system is causally involved at multiple steps in cancer progression. In particular, uPA has been implicated in remodelling of the extracellular matrix, enhancing both cell proliferation and migration and modulating cell adhesion. Consistent with its role in cancer progression, multiple groups have shown that high levels of uPA in primary breast cancers are independently associated with adverse outcome. Paradoxically, high levels of PAI-1 also correlate with poor prognosis in patients with breast cancer. The prognostic value of uPA/PAI-1 in axillary node-negative breast cancer patients was recently validated using both a prospective randomised trial and a pooled analysis, i.e., in 2 different Level 1 Evidence studies. Assay of uPA and PAI-1 may thus help identify low risk node-negative patients for whom adjuvant chemotherapy is unnecessary. Finally, preclinical studies show that either inhibition of uPA catalytic activity or prevention of uPA binding to its receptor reduces tumor growth, angiogenesis and metastasis.

title: Urokinase plasminogen activator system: a multifunctional role in tumor progression and metastasis.
abstract: The urokinase plasminogen activator (uPA) system is central to a spectrum of biologic processes including fibrinoloysis, inflammation, atherosclerotic plaque formation, matrix remodeling during wound healing, tumor invasion, angiogenesis, and metastasis. Binding of uPA with its receptor (uPAR) initiates a proteolytic cascade that results in the conversion of plasminogen to plasmin. Plasmin through its own proteolytic function degrades a range of extracellular basement membrane components and activates others such as the metalloproteinases. Independent of catalytic activity, uPAR also is involved in cell signaling, interactions with integrins, cell motility, adhesion and invasion, and angiogenesis. Over expression of uPA or uPAR is a feature of malignancy and is correlated with tumor progression and metastasis. In contrast, inhibition of expression of these components leads to a reduction in the invasive and metastatic capacity of many tumors. Strategies that target uPA or its receptor with the aim of disrupting the interaction between the two or the ligand independent actions of uPAR include antisense technology, monoclonal antibodies, cytotoxic antibiotics, and synthetic inhibitors of uPA. Targeted therapy is a goal of future cancer treatment and the uPA system is a likely candidate for manipulation.

title: uPAR: a versatile signalling orchestrator
abstract: The plasminogen system has been implicated in clot lysis, wound healing, tissue regeneration, cancer and many other processes that affect health and disease. The urokinase receptor uPAR was originally thought to assist the directional invasion of migrating cells, but it is now becoming increasingly evident that this proteinase receptor elicits a plethora of cellular responses that include cellular adhesion, differentiation, proliferation and migration in a non-proteolytic fashion.

title: Urokinase plasminogen activator enhances neointima growth and reduces lumen size in injured carotid arteries
abstract: Increases in urokinase plasminogen activator (uPA) have been reported in tissues undergoing remodelling, but its effects on the vessel intima formation are not known. We investigated its effects on carotid artery intima, media and lumen size, as well as smooth muscle cell (SMC) proliferation and migration in vivo.  Carotid arteries of rats were distended with an inflated balloon catheter and uPA, or uPA-neutralizing antibodies were applied perivascularly in pluronic gel; control rats received vehicle. Carotid artery structure, cell migration and proliferation were assessed after 4 days by quantitative morphometry and immunohistochemistry.  Four days after increasing vessel uPA, the intima/media ratio was double compared to that in control rats (both P < 0.05). The size of the lumen reduced by 75%, compared to the vehicle-treated vessels (P < 0.05). The elevation in uPA also increased SMC numbers in the intima and media, compared to the vehicle-treated vessels (both P < 0.05). Antibody neutralizing endogenous uPA attenuated the growth responses in the distended arteries, reduced neointimal SMC numbers by approximately 50% and prevented much of the reduction in lumen size.  Thus, local increases in uPA in distended, injured arteries augment SMC migratory and proliferative responses, leading to increases in the thickness of the carotid artery intima and media and a reduction in lumen size; effects at least partially attributable to its proteolytic properties.

title: The urokinase plasminogen activator system: a target for anti-cancer therapy.
abstract: The urokinase plasminogen activator (uPA) system (uPAS) consists of the uPA, its cognate receptor (uPAR) and two specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and 2 (PAI-2). The uPA converts the proenzyme plasminogen in the serine protease plasmin, involved in a number of physiopathological processes requiring basement membrane (BM) and/or extracellular matrix (ECM) remodelling, including tumor progression and metastasis. Data accumulated over the past years have made increasingly clear that the uPAS has a multifunctional task in the neoplastic evolution, affecting tumor angiogenesis, malignant cell proliferation, adhesion and migration, intravasation and growth at the metastatic site. In agreement with their role in cancer progression and metastasis, an increased expression of uPA, uPAR, and PAI-1 has been documented in several malignant tumors, and a positive correlation between the levels of one or more uPAS members and a poor prognosis has been frequently reported. This is particularly evident in breast cancer, for which uPA has been demonstrated to be the most potent independent prognostic factor described to date. The involvement of the uPAS in cancer progression identifies its components as suitable targets for anti-cancer therapy. Several therapeutical approaches aimed at inhibiting the uPA/uPAR functions have been shown to possess anti-tumor effects in xenograft models, including selective inhibitors of uPA activity, antagonist peptides, monoclonal antibodies able to prevent uPA binding to uPAR and gene therapy techniques silencing uPA/uPAR expression. All these strategies, however, although promising, need definitive confirmation in humans as, up to now, only few uPA inhibitors entered clinical trial.

title: Intraperitoneal Injection of a Hairpin RNA–Expressing Plasmid Targeting Urokinase-Type Plasminogen Activator (uPA) Receptor and uPA Retards Angiogenesis and Inhibits Intracranial Tumor Growth in Nude Mice
abstract: The purpose of this study was to evaluate the therapeutic potential of using plasmid-expressed RNA interference (RNAi) targeting urokinase-type plasminogen activator (uPA) receptor (uPAR) and uPA to treat human glioma.  In the present study, we have used plasmid-based RNAi to simultaneously down-regulate the expression of uPAR and uPA in SNB19 glioma cell lines and epidermal growth factor receptor (EGFR)–overexpressing 4910 human glioma xenografts in vitro and in vivo, and evaluate the i.p. route for RNAi-expressing plasmid administered to target intracranial glioma.  Plasmid-mediated RNAi targeting uPAR and uPA did not induce OAS1 expression as seen from reverse transcription-PCR analysis. In 4910 EGFR-overexpressing cells, down-regulation of uPAR and uPA induced the down-regulation of EGFR and vascular endothelial growth factor and inhibited angiogenesis in both in vitro and in vivo angiogenic assays. In addition, invasion and migration were inhibited as indicated by in vitro spheroid cell migration, Matrigel invasion, and spheroid invasion assays. We did not observe OAS1 expression in mice with preestablished intracranial tumors, which were given i.p. injections of plasmid-expressing small interfering RNA–targeting uPAR and uPA. Furthermore, the small interfering RNA plasmid targeting uPAR and uPA caused regression of preestablished intracranial tumors when compared with the control mice.  In conclusion, the plasmid-expressed RNAi targeting uPAR and uPA via the i.p. route has potential clinical applications for the treatment of glioma.

title: Anti-cancer therapies targeting the tumor stroma
abstract: For anti-tumor therapy different strategies have been employed, e.g., radiotherapy, chemotherapy, or immunotherapy. Notably, these approaches do not only address the tumor cells themselves, but also the tumor stroma cells, e.g., endothelial cells, fibroblasts, and macrophages. This is of advantage, since these cells actively contribute to the proliferative and invasive behavior of the tumor cells via secretion of growth factors, angiogenic factors, cytokines, and proteolytic enzymes. In addition, tumor stroma cells take part in immune evasion mechanisms of cancer. Thus, approaches targeting the tumor stroma attract increasing attention as anti-cancer therapy. Several molecules including growth factors (e.g., VEGF, CTGF), growth factor receptors (CD105, VEGFRs), adhesion molecules (αvβ3 integrin), and enzymes (CAIX, FAPα, MMPs, PSMA, uPA) are induced or upregulated in the tumor microenvironment which are otherwise characterized by a restricted expression pattern in differentiated tissues. Consequently, these molecules can be targeted by inhibitors as well as by active and passive immunotherapy to treat cancer. Here we discuss the results of these approaches tested in preclinical models and clinical trials.

title: Targeting uPA/uPAR in prostate cancer.
abstract: Prostate cancer (CaP) is one of the most common malignancies in men, with an increasing incidence. Despite significant advances in surgery, chemotherapy and radiotherapy to treat CaP, many patients unfortunately succumb to secondary disease (metastases). The invasive ability of tumour cells plays a key role in CaP metastasis and is a major cause of treatment failure. Urokinase plasminogen activator (uPA) and its receptor (uPAR)-mediated signalling have been implicated in tumour cell invasion, survival, and metastasis in a variety of cancers including CaP. Both uPA and uPAR are expressed at much higher levels in CaP tissues than in benign and normal prostate tissues. They are used as diagnostic markers as well as therapeutic targets due to their aberrant and unique expression pattern during cancer progression. Current therapeutic options for patients with metastatic hormone-refractory CaP (HRPC) are very limited. Therefore, much effort is currently being directed toward targeting aberrant uPA or uPAR activity in CaP. This review summarizes some important new findings supporting the role of uPA/uPAR in CaP progression and establishing the potential therapeutic efficacy of uPA/uPAR-targeted therapies in CaP.

title: The crystal structure of the catalytic domain of human urokinase-type plasminogen activator.
abstract: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data.



The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease.



The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

title: CD87 (urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review
abstract: The analysis of CD87 (urokinase-type plasminogen activator receptor – uPAR) expression has a potential role in the diagnostic or prognostic work-up of several hematological malignancies, particularly acute leukemia and multiple myeloma. The distribution of CD87 in acute myeloid leukemia (AML) varies according to the FAB subtype (highest expression in M5 and lowest in M0). Functionally, it is conceivable that the expression of CD87 could contribute to the invasive properties of the leukemic cells towards the skin and mucosal tissues as reflected by the clinical behavior of CD87 high cases. The lack of or weaker expression of CD87 on blast cells from ALL patients supports the concept that CD87 investigation might help in the distinction of AMLs from lymphoid malignancies. Among lymphoproliferative disorders, the expression of CD87 is exclusively found in pathological plasma cells. Since plasma cells also coexpress some adhesion molecules such as CD138 and CD56, this observation is consistent with the capacity of these cells to home in the bone compartment. High levels of soluble uPAR appear to represent an independent factor predicting worse prognosis and extramedullary involvement in multiple myeloma.

title: The plasminogen activator system: biology and regulation
abstract: The regulation of plasminogen activation involves genes for two plasminogen activators (tissue type and urokinase type), two specific inhibitors (type 1 and type 2), and a membrane-anchored urokinase-type plasminogen-activator-specific receptor. This system plays an important role in various biological processes involving extracellular proteolysis. Recent studies have revealed that the system, through interplay with integrins and the extracellular matrix protein vitronectin, is also involved in the regulation of cell migration and proliferation in a manner independent of proteolytic activity. The genes are expressed in many different cell types and their expression is under the control of diverse extracellular signals. Gene expression reflects the levels of the corresponding mRNA, which should be the net result of synthesis and degradation. Thus, modulation of mRNA stability is an important factor in overall regulation. This review summarizes current understanding of the biology and regulation of genes involved in plasminogen activation at different levels.

title: Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis
abstract: Extracellular proteolysis is an absolute requirement for new blood vessel formation (angiogenesis). This review examines the role of the matrix metalloproteinase (MMP) and plasminogen activator (PA)-plasmin systems during angiogenesis. Specifically, a role for gelatinases (MMP-2, MMP-9), membrane-type 1 MMP (MMP-14), the urokinase-type PA receptor, and PA inhibitor 1 has been clearly defined in a number of model systems. The MMP and PA-plasmin systems have also been implicated in experimental vascular tumor formation, and their role during this process will be examined. Antiproteolysis, particularly in the context of angiogenesis, has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization.

title: Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.
abstract: Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 x 10(-10) M and 2.8 x 10(4) binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal anti-u-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H]thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphate-inactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.

title: Antibodies to plasminogen activator inhibit human tumor metastasis
abstract: The human carcinoma HEp3 grows on the chorioallantoic membrane and metastasizes to the chicken embryo with kinetics that are quantitatively predictable. We have used this experimental system to test whether plasminogen activator produced by the tumor is required for metastasis. Rabbit antibodies were raised against human urinary urokinase; these cross-reacted with and blocked the catalytic activity of HEp3-PA but did not inhibit chicken PA. When administered intravenously to embryos that had received an inoculum of HEp3 cells, the anti-urokinase antibodies did not inhibit tumor growth at the site of primary inoculation on the chorioallantoic membrane, but they either prevented or strongly inhibited metastasis to the embryo lung. Antibody treatment delayed the onset of pulmonary metastasis, indicating that plasminogen activator is required during early stages of the process.

title: Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.
abstract: To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

title: Amiloride selectively inhibits the urokinase‐type plasminogen activator
abstract: The diuretic drug amiloride, an inhibitor of Na+ uptake, competitively inhibits the catalytic activity of the urokinase‐type plasminogen activator (u‐PA), with a K i of 7 × 10−6 M. Generation of plasmin, cleavage of peptide substrates, and interaction of u‐PA with a specific macromolecular proteinase inhibitor are all prevented in the presence of the drug. In contrast, amiloride does not affect the activity of either tissue‐type plasminogen activator, plasmin, plasma kallikrein or thrombin. The inhibition of u‐PA by amiloride may be related to the previously reported inhibition of u‐PA‐type enzymes by Na+. Amiloride or related compounds could prove useful in selectively controlling u‐PA‐catalyzed extracellular proteolysis.

title: Inhibition of urokinase by 4-substituted benzo[b]thiophene-2-carboxamidines: an important new class of selective synthetic urokinase inhibitor.
abstract: Urokinase-type plasminogen activator (uPA) is an important mediator of cellular invasiveness. Specifically, cell surface receptor-bound uPA activates plasminogen to the potent general protease plasmin, which then degrades extracellular matrix or basement membrane either directly or via proteolytic activation of latent collagenases. Thus, cell surface uPA initiates an extracellular proteolytic cascade with which invasive cells eliminate barriers to movement. Since cellular invasiveness plays important roles in several disease states, including cancer metastasis and invasion, arthritis and inflammation, and diabetic retinal neovascularization, the development of synthetic uPA inhibitors is an attractive therapeutic goal. Here we show that 4-substituted benzo[b]thiophene-2-carboxamidines represent an important new class of potent and selective synthetic uPA inhibitor. Two compounds in this class, B428 and B623, inhibit human uPA in plasminogen-linked assays with median inhibition concentration (IC50) values of 0.32 and 0.07 microM, respectively. This level of inhibition represents 20- and 100-fold increases in potency, respectively, relative to the 6-7 microM potencies reported for amiloride and 4-chlorophenylguanidine, the two most potent selective synthetic uPA inhibitors previously described. Importantly, both compounds show > 300-fold selectivity for uPA relative to tissue-type plasminogen activator and > 1000-fold selectivity relative to plasmin. Lineweaver-Burk analyses show uPA inhibition by B428 and B623 to be competitive in nature with inhibition constants (Ki) of 0.53 and 0.16 microM, respectively. Since it is cell surface uPA and not free or secreted uPA that is primarily responsible for cellular invasiveness, biologically effective uPA inhibitors must be capable of inhibiting cell surface uPA. B428 and B623 meet this criterion by inhibiting cell surface uPA on HT1080 human fibrosarcoma cells with IC50 values of 0.54 and 0.20 microM, respectively. Moreover, degradation of [3H]fibronectin by HT1080 cells via cell surface uPA-mediated, plasminogen-dependent mechanisms is inhibited by B428 and B623, with IC50 values of 1.5 and 0.39 microM, respectively. In summary, 4-substituted benzo[b]thiophene-2-carboxamidines such as B428 and B623 represent the most potent class of competitive synthetic uPA inhibitors currently known. Their ability to selectively inhibit both free and cell surface uPA as well as cell surface uPA-mediated cellular degradative functions suggests that this class of compounds may hold significant promise for further development as antiinvasiveness drugs.

title: Naphthamidine urokinase plasminogen activator inhibitors with improved pharmacokinetic properties.
abstract: A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus previously described amide-linked inhibitors.

title: 4-Aminoarylguanidine and 4-aminobenzamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.
abstract: The structure-based design of potent and selective urokinase-type plasminogen activator (uPA) inhibitors with 4-aminoarylamidine or 4-aminoarylguanidine S1 binding groups, is described.

title: Selective urokinase-type plasminogen activator (uPA) inhibitors. Part 1: 2-Pyridinylguanidines.
abstract: The identification of 2-pyridinylguanidines (e.g., 27 and 28) as selective inhibitors of urokinase-type plasminogen activator (uPA) is described. The X-ray crystal structure of 27 has been determined, and modelling has been used to predict binding in the enzyme active site.

title: Selective urokinase-type plasminogen activator (uPA) inhibitors. Part 3: 1-isoquinolinylguanidines.
abstract: A series of 1-isoquinolinylguanidines are shown to be potent inhibitors of uPA with selectivity over tPA and plasmin. Potency is enhanced by the presence of a 4-halo and a 7-aryl substituent, particularly when substituted by a 3-carboxylic acid group. Compound 13j (UK-356,202) combines excellent potency and selectivity, and has been selected as a candidate for clinical evaluation.

title: Selective urokinase-type plasminogen activator inhibitors. 4. 1-(7-sulfonamidoisoquinolinyl)guanidines.
abstract: 1-isoquinolinylguanidines were previously disclosed as potent and selective inhibitors of urokinase-type plasminogen activator (uPA). Further investigation of this template has revealed that incorporation of a 7-sulfonamide group furnishes a new series of potent and highly selective uPA inhibitors. Potency and selectivity can be achieved with sulfonamides derived from a variety of amines and is further enhanced by the incorporation of sulfonamides derived from amino acids. The binding mode of these 1-isoquinolinylguanidines has been investigated by X-ray cocrystallization studies. uPA inhibitor 26 was selected for further evaluation based on its excellent enzyme potency (Ki 10 nM) and selectivity profile (4000-fold versus tPA and 2700-fold versus plasmin). In vitro, compound 26 is able to inhibit exogenous uPA in human chronic wound fluid (IC50=0.89 microM). In vivo, in a porcine acute excisional wound model, following topical delivery, compound 26 is able to penetrate into pig wounds and inhibit exogenous uPA activity with no adverse effect on wound healing parameters. On the basis of this profile, compound 26 (UK-371,804) was selected as a candidate for further preclinical evaluation for the treatment of chronic dermal ulcers.

title: Fragment-based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase-type plasminogen activator.
abstract: Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA.

title: Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator
abstract: Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents. alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups.  This study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.

title: Identification of a novel inhibitor of urokinase-type plasminogen activator
abstract: Urokinase-type plasminogen activator (uPA), a highly restricted serine protease, plays an important role in the regulation of diverse physiologic and pathologic processes. Strong clinical and experimental evidence has shown that elevated uPA expression is associated with cancer progression, metastasis, and shortened survival in patients. uPA has been considered as a promising molecular target for development of anticancer drugs. Here, we report the identification of several new uPA inhibitors using a high-throughput screen from a chemical library. From these uPA inhibitors, molecular modeling and docking studies identified 4-oxazolidinone as a novel lead pharmacophore. Optimization of the 4-oxazolidinone pharmacophore resulted in a series of structurally modified compounds with improved potency and selectivity. One of the 4-oxazolidinone analogues, UK122, showed the highest inhibition of uPA activity. The IC50 of UK122 in a cell-free indirect uPA assay is 0.2 μmol/L. This compound also showed no or little inhibition of other serine proteases such as thrombin, trypsin, plasmin, and the tissue-type plasminogen activator, indicating its high specificity against uPA. Moreover, UK122 showed little cytotoxicity against CFPAC-1 cells (IC50 >100 μmol/L) but significantly inhibited the migration and invasion of this pancreatic cancer cell line. Our data show that UK122 could potentially be developed as a new anticancer agent that prevents the invasion and metastasis of pancreatic cancer.

title: Structure-based design, synthesis and SAR of a novel series of thiopheneamidine urokinase plasminogen activator inhibitors.
abstract: The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.

title: (4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase.
abstract: Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure-activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1'/S2' subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

title: Inhibition of the invasion capacity of carcinoma cells by WX‐UK1, a novel synthetic inhibitor of the urokinase‐type plasminogen activator system
abstract: The overall survival rate of patients suffering from carcinomas has remained poor and nearly unchanged over the last decades. This is mainly due to the so‐called minimal residual disease, i.e., remaining tumor cells that overcome surgery and/or radiotherapy and are the cause of locoregional and distant metastases. To metastasize, tumor cells take advantage of proteases to invade and remodel surrounding tissues. Here, we analyzed the efficiency of WX‐UK1, a novel 3‐amidinophenylalanine‐based inhibitor of the uPA system, at inhibiting the invasive capacity of carcinoma cells. First, uPAR expression was characterized in different carcinoma cell lines, including SCCHN, breast and cervical carcinoma. Thereafter, the invasive potential of these cell lines was determined using Matrigel invasion chambers and a spheroid cocultivation model with human fibroblasts. uPAR expression levels correlated positively with invasion capacity, which could be significantly inhibited by WX‐UK1. A decrease of tumor cell invasion by up to 50% was achieved in both models with the SCCHN line FaDu and the cervical carcinoma line HeLa after treatment with WX‐UK1. Thus, our results demonstrate the potential of WX‐UK1 in vitro as a promising adjuvant antimetastatic therapy of carcinomas.

title: Application of the Docking Program SOL for CSAR Benchmark
abstract: This paper is devoted to results obtained by the docking program SOL and the post-processing program DISCORE at the CSAR benchmark. SOL and DISCORE programs are described. SOL is the original docking program developed on the basis of the genetic algorithm, MMFF94 force field, rigid protein, precalculated energy grid including desolvation in the frame of simplified GB model, vdW, and electrostatic interactions and taking into account the ligand internal strain energy. An important SOL feature is the single- or multi-processor performance for up to hundreds of CPUs. DISCORE improves the binding energy scoring by the local energy optimization of the ligand docked pose and a simple linear regression on the base of available experimental data. The docking program SOL has demonstrated a good ability for correct ligand positioning in the active sites of the tested proteins in most cases of CSAR exercises. SOL and DISCORE have not demonstrated very exciting results on the protein-ligand binding free energy estimation. Nevertheless, for some target proteins, SOL and DISCORE were among the first in prediction of inhibition activity. Ways to improve SOL and DISCORE are discussed.

title: Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94
abstract: This article introduces MMFF94, the initial published version of the Merck molecular force field (MMFF). It describes the objectives set for MMFF, the form it takes, and the range of systems to which it applies. This study also outlines the methodology employed in parameterizing MMFF94 and summarizes its performance in reproducing computational and experimental data. Though similar to MM3 in some respects, MMFF94 differs in ways intended to facilitate application to condensed‐phase processes in molecular‐dynamics simulations. Indeed, MMFF94 seeks to achieve MM3‐like accuracy for small molecules in a combined “organic/protein” force field that is equally applicable to proteins and other systems of biological significance. A second distinguishing feature is that the core portion of MMFF94 has primarily been derived from high‐quality computational data—ca. 500 molecular structures optimized at the HF/6‐31G* level, 475 structures optimized at the MP2/6‐31G* level, 380 MP2/6‐31G* structures evaluated at a defined approximation to the MP4SDQ/TZP level, and 1450 structures partly derived from MP2/6‐31G* geometries and evaluated at the MP2/TZP level. A third distinguishing feature is that MMFF94 has been parameterized for a wide variety of chemical systems of interest to organic and medicial chemists, including many that feature frequently occurring combinations of functional groups for which little, if any, useful experimental data are available. The methodology used in parameterizing MMFF94 represents a fourth distinguishing feature. Rather than using the common “functional group” approach, nearly all MMFF parameters have been determined in a mutually consistent fashion from the full set of available computational data. MMFF94 reproduces the computational data used in its parameterization very well. In addition, MMFF94 reproduces experimental bond lengths (0.014 Å root mean square [rms]), bond angles (1.2° rms), vibrational frequencies (61 cm−1 rms), conformational energies (0.38 kcal/mol/rms), and rotational barriers (0.39 kcal/mol rms) very nearly as well as does MM3 for comparable systems. MMFF94 also describes intermolecular interactions in hydrogen‐bonded systems in a way that closely parallels that given by the highly regarded OPLS force field.

title: Merck molecular force field. III. Molecular geometries and vibrational frequencies for MMFF94
abstract: This article describes the parameterization and performance of MMFF94 for molecular geometries and deformations. It defines the form used for the valence‐coordinate terms that represent variations in bond lengths and angles, and it describes the derivation of quadratic force constants from HF/6‐31G* data and the derivation of reference bond lengths and angles from fits to MP2/6‐31G*‐optimized geometries. Comparisons offered show that MMFF94 accurately reproduces the computational data used in its parameterization and demonstrate that its derivation from such data simultaneously confers the ability to reproduce experiment. In particular, MMFF94 reproduces experimentally determined bond lengths and angles for 30 organic molecules with root mean square (rms) deviations of 0.014 Å and 1.2°, respectively. MM3 reproduces bond angles to the same accuracy, but reproduces experimental bond lengths more accurately, in part because it was fit directly to thermally averaged experimental bond lengths; MMFF94, in contrast, was fit to (usually shorter) energy‐minimum values, as is proper for an anharmonic force field intended for use in molecular‐dynamics simulations. The comparisons also show that UFF and a recent version of CHARMm (QUANTA 3.3 parameterization) are less accurate for molecular geometries than either MMFF94 or MM3. For vibrational frequencies, MMFF94 and MM3 give comparable overall rms deviations versus experiment of 61 cm−1 and 57 cm−1, respectively, for 15 small, mostly organic molecules. In a number of instances, MM3's derivation employed observed frequencies that differ substantially—by nearly 400 cm−1 in one case—from other published frequencies which had themselves been confirmed theoretically by good‐quality ab initio calculations. Overall, the comparisons to experimental geometries and vibrational frequencies demonstrate that MMFF94 achieves MM3‐like accuracy for organic systems for which MM3 has been parameterized. Because MMFF94 is derived mainly from computational data, however, it has been possible to parameterize MMFF94 with equal rigor for a wide variety of additional systems for which little or no useful experimental data exist. Equally good performance can be expected for such systems.

title: Merck molecular force field. IV. conformational energies and geometries for MMFF94
abstract: This article describes the parameterization and performance of MMFF94 for conformational energies, rotational barriers, and equilibrium torsion angles. It describes the derivation of the torsion parameters from high‐quality computational data and characterizes MMFF94's ability to reproduce both computational and experimental data, the latter particularly in relation to MM3. The computational data included: (i) ∼ 250 comparisons of conformational energy based on “MP4SDQ/TZP” calculations (triple‐zeta plus polarization calculations at a defined approximation to the highly correlated MP4SDQ level) at MP2/6‐31G* geometries; and (ii) ∼ 1200 MP2/TZP comparisons of “torsion profile” structures at geometries derived from MP2/6‐31G* geometries. The torsion parameters were derived in restrained least‐squares fits that used the complete set of available computational data, thereby ensuring that a fully optimal set of parameters would be obtained. The final parameters reproduce the “MP4SDQ/TZP” and MP2/TZP computational data with root mean square (rms) deviations of 0.31 and 0.50 kcal/mol, respectively. In addition, MMFF94 reproduces a set of 37 experimental gas‐phase and solution conformational energies, enthalpies, and free energies with a rms deviation of 0.38 kcal/mol; for comparison, the “MP4SDQ/TZP” calculations and MM3 each gives a rms deviation of 0.37 kcal/mol. Furthermore, MMFF94 reproduces 28 experimentally determined rotational barriers with a rms deviation of 0.39 kcal/mol. Given the diverse nature of the experimental conformational energies and rotational barriers and the clear indications of experimental error in some cases, the MMFF94 results appear excellent. Nevertheless, MMFF94 encounters somewhat greater difficulty in handling multifunctional compounds that place highly polar functional groups in close proximity, probably because it, like other commonly used force fields, too greatly simplifies the description of electrostatic interactions. Some suggestions for enhancements to MMFF94's functional form are discussed.

title: Merck molecular force field. V. Extension of MMFF94 using experimental data, additional computational data, and empirical rules
abstract: This article describes the extension of the Merck Molecular Force Field (MMFF94) to a much broader range of organic systems. It also describes a preliminary parameterization of MMFF94 for the hydronium and hydroxide ions and for various halide, alkalai, and alkalai earth ions as well as for such “protein” metals as Zn2+, Ca2+, Cu2+, Cu+, Fe2+, and Fe3+. The extension employed computational data on charge distributions, molecular geometries, and conformational energies for a series of oxysulfur (particularly sulfonamide) and oxyphosphorous compounds and for a diverse set of small molecules and ions not covered in the core parameterization. It also employed experimental data for approximately 2800 good‐quality structures extracted from the Cambridge Structural Database (CSD). Some of the additional computational data were used to extend the explicit parameterization of electrostatic interactions and to more widely define a useful additive approximation for the “bond polarity” parameters (bond charge increments) used in MMFF94. Both the experimental and computational data served to define reference bond lengths and angles that the extended force field uses in conjunction with force constants obtained from carefully calibrated empirical rules. The extended torsion parameters consist partly of explicit parameters derived to reproduce MP2/6‐31G* conformational energies and partly of “default parameters” provided by empirical rules patterned after those used in DREIDING and UFF but calibrated, where possible, against computationally derived MMFF94 torsion parameters. Comparisons to experimental data show that MMFF94 reproduces crystallographic bond lengths and bond angles with relatively modest root mean square (rms) deviations of approx. 0.02 Å and 2°, respectively.

title: Surface Generalized Born Method: A Simple, Fast, and Precise Implicit Solvent Model beyond the Coulomb Approximation
abstract: We present the surface generalized born (S-GB) method, based on expansion of energy in a surface integral series. The method can be parametrized for simultaneous reproduction of the reaction field energy for small molecules as well as macromolecules. For a set of 195 small molecules, our S-GB model gives a root mean square (rms) of 0.13 kcal/mol relative to the rigorous polarizable continuum model (PCM).

title: Numerical Optimization
abstract: Numerical Optimization presents a comprehensive and up-to-date description of the most effective methods in continuous optimization. It responds to the growing interest in optimization in engineering, science, and business by focusing on the methods that are best suited to practical problems. For this new edition the book has been thoroughly updated throughout. There are new chapters on nonlinear interior methods and derivative-free methods for optimization, both of which are used widely in practice and the focus of much current research. Because of the emphasis on practical methods, as well as the extensive illustrations and exercises, the book is accessible to a wide audience. It can be used as a graduate text in engineering, operations research, mathematics, computer science, and business. It also serves as a handbook for researchers and practitioners in the field. The authors have strived to produce a text that is pleasant to read, informative, and rigorous - one that reveals both the beautiful nature of the discipline and its practical side.

title: Can the calculation of ligand binding free energies be improved with continuum solvent electrostatics and an ideal‐gas entropy correction?
abstract: The prediction of a ligand binding constant requires generating three‐dimensional structures of the complex concerned and reliably scoring these structures. Here, the scoring problem is investigated by examining benzamidine‐like inhibitors of trypsin, a system for which errors in the structures are small. Precise and consistent binding free energies for the inhibitors are determined experimentally for this test system. To examine possible improvement of scoring methods, we test the suitability of continuum electrostatics to account for solvation effects and use an ideal‐gas entropy correction to account for the changes in the degrees of freedom of the ligand. The small observed root‐mean‐square deviation of 0.55 kcal/mol of the calculated relative to the experimental values indicates that the essentials of the binding process have been captured. Even though all six ligands make the same salt bridge and H‐bonds to the protein, the electrostatic contribution varies among the ligands by as much as 2 kcal/mol. Moreover, although the ligands are rigid and similar in size, the entropic terms also significantly affect the relative binding affinities (by up to 2.7 kcal/mol). The present approach to solvation and entropy may allow the ranking of the ligands to be considerably improved at a cost that makes the method applicable to the optimization of lead compounds or to the screening of small collections of ligands.

title: Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters
abstract: Modern semiempirical methods are of sufficient accuracy when used in the modeling of molecules of the same type as used as reference data in the parameterization. Outside that subset, however, there is an abundance of evidence that these methods are of very limited utility. In an attempt to expand the range of applicability, a new method called PM7 has been developed. PM7 was parameterized using experimental and high-level ab initio reference data, augmented by a new type of reference data intended to better define the structure of parameter space. The resulting method was tested by modeling crystal structures and heats of formation of solids. Two changes were made to the set of approximations: a modification was made to improve the description of noncovalent interactions, and two minor errors in the NDDO formalism were rectified. Average unsigned errors (AUEs) in geometry and ΔHf for PM7 were reduced relative to PM6; for simple gas-phase organic systems, the AUE in bond lengths decreased by about 5 % and the AUE in ΔHf decreased by about 10 %; for organic solids, the AUE in ΔHf dropped by 60 % and the reduction was 33.3 % for geometries. A two-step process (PM7-TS) for calculating the heights of activation barriers has been developed. Using PM7-TS, the AUE in the barrier heights for simple organic reactions was decreased from values of 12.6 kcal/mol-1 in PM6 and 10.8 kcal/mol-1 in PM7 to 3.8 kcal/mol-1. The origins of the errors in NDDO methods have been examined, and were found to be attributable to inadequate and inaccurate reference data. This conclusion provides insight into how these methods can be improved.

title: Semiempirical Quantum Chemical PM6 Method Augmented by Dispersion and H-Bonding Correction Terms Reliably Describes Various Types of Noncovalent Complexes.
abstract: Because of its construction and parametrization for more than 80 elements, the semiempirical quantum chemical PM6 method is superior to other similar methods. Despite its advantages, however, the PM6 method fails for the description of noncovalent interactions, specifically the dispersion energy and H-bonding. Upon inclusion of correction terms for dispersion and H-bonding, the performance of the method was found to be dramatically improved. The former correction included two parameters in the damping function that were parametrized to reproduce the benchmark interaction energies [CCSD(T)/complete basis set (CBS) limit] of the dispersion-bonded complexes from the S22 data set. The latter correction was parametrized on an extended set of H-bonded stabilization energies determined at the MP2/cc-pVTZ level. The resulting PM6-DH method was tested on the S22 data set, for which chemical accuracy (error < 1 kcal/mol) was achieved, and also on the JSCH2005 set, for which significant improvement over the original PM6 method was also obtained. Implementation of analytical gradients allows very efficient geometry optimization, which, for all complexes, provides better agreement with the benchmark data. Excellent results were also achieved for small peptides, and here again, chemical accuracy was obtained (i.e., the error with respect to CCSD(T)/CBS results was smaller than 1 kcal/mol). The performance of the technique was finally demonstrated on extended complexes, namely, the porphine dimer and various graphene models with DNA bases and base pairs, where the PM6-DH stabilization energies agree very well with available benchmark data obtained with DFT-D, SCS-MP2, and MP2.5 methods. The PM6-DH calculations are very efficient and can be routinely applied for systems of up to 1000 atoms. For nonaromatic systems, the use of a linear scaling version of the SCF procedure based on localized orbitals speeds up the method significantly and allows one to investigate systems with several thousand atoms. The method can thus replace force fields, which face basic problems for the description of quantum effects, in many applications.

title: A Transferable H-Bonding Correction for Semiempirical Quantum-Chemical Methods.
abstract: Semiempirical methods could offer a feasible compromise between ab initio and empirical approaches for the calculation of large molecules with biological relevance. A key problem for attempts in this direction is the rather bad performance of current semiempirical methods for noncovalent interactions, especially hydrogen-bonding. On the basis of the recently introduced PM6-DH method, which includes empirical corrections for dispersion (D) and hydrogen-bond (H) interactions, we have developed an improved and transferable H-bonding correction for semiempirical quantum chemical methods. The performance of the improved correction is evaluated for PM6, AM1, OM3, and SCC-DFTB (enhanced by standard empirical dispersion corrections) with several test sets for noncovalent interactions and is shown to reach the quality of current DFT-D approaches for these types of problems.

title: Third-Generation Hydrogen-Bonding Corrections for Semiempirical QM Methods and Force Fields
abstract: Computational modeling of biological systems is a rapidly evolving field that calls for methods that are able to allow for extensive sampling with systems consisting of thousands of atoms. Semiempirical quantum chemical (SE) methods are a promising tool to aid with this, but the rather bad performance of standard SE methods for noncovalent interactions is clearly a limiting factor. Enhancing SE methods with empirical corrections for dispersion and hydrogen-bonding interactions was found to be a big improvement, but for the hydrogen-bonding corrections the drawback of breaking down in the case of substantial changes to the hydrogen bond, e.g., proton transfer, posed a serious limitation for its general applicability. This work presents a further improved hydrogen-bonding correction that can be generally included in parameter fitting procedures, as it does not suffer from the conceptual flaws of previous approaches: hydrogen bonds are now treated as an interaction term between electronegative acceptor and d...

title: Antigen recognition by conformational selection
abstract: Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an ‘induced fit’, which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an ‘induced fit’ requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross‐reacts with the wild‐type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

title: Conformational isomerism and the diversity of antibodies.
abstract: The fact that one cell encodes a single antibody sequence does not necessarily mean that the resulting antibody folds into a single structure, although this is a common assumption. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site. The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex hapten-binding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that ligand binding involved isomerization, as well as associative steps. The existence of an equilibrium between at least two antibody conformations, with ligands binding preferentially to one form, was deduced from the variation with hapten concentration of the apparent rate of each phase.

title: Folding and binding cascades: Dynamic landscapes and population shifts
abstract: Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713–720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181–1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970–9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three‐dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.

title: Folding and binding cascades: shifts in energy landscapes.
abstract: Recently, the funnel shape energy landscape theory has been successfully utilized to describe the folding (1–4) and binding (5) behavior in proteins. While in general the free energy landscape is depicted by a funnel-like shape, the details of the landscape surface of a folding funnel will be affected by changes in the surrounding environment. On the basis of Freire’s work in this issue of the Proceedings (6), we describe a shift in the energy landscape of a folding funnel, caused by a binding event. Shifts in energy landscapes portray shifts in the populations of the substates.

title: Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase.
abstract: New chromogenic tripeptide substrates have been used for the determination of kallikreins and urokinase. The conditions have been optimized. It is possible to determine prekallikrein in plasma after activation with Cephotest. No significant loss in activity caused by plasma kallikrein inhibitors is observed at the dilutions used.

title: Synthetic peptide substrate assays for hemostasis testing.
abstract: Publisher Summary This chapter discusses several assays for hemostasis testing. The chapter emphasizes those areas where hemostasis test procedure has reached through its potential and those where more work is needed so that it can be accepted in routine laboratory hemostasis testing. Hemostasis is a complex process by which blood remains fluid in the vascular system of mammals. Over the last decade, a new era in hemostasis testing has occurred with the introduction of synthetic substrate procedures. The progress during this period for the better understanding of the fibrinolysis, kallikrein-kinin, and protease inhibitor systems are greatly assisted by the availability of specific amidolytic assays. Most of the factors involved in blood coagulation and fibrinolysis systems are proteolytic enzymes. Proteolytic enzymes are members of a class of catalysis known as serine proteases and share a common basic mechanism of action. These particular enzymes exist in mammalian blood as zymogens along with their activators, inhibitors, and natural substrates. This system is accurately balanced by interdependent positive and negative feedback reactions. The chapter analyzes natural substrates versus synthetic substrates by comparing bioassays with chemical assays.

title: Finding more needles in the haystack: A simple and efficient method for improving high-throughput docking results.
abstract: The technology underpinning high-throughput docking (HTD) has developed over the past few years to where it has become a vital tool in modern drug discovery. Although the performance of various docking algorithms is adequate, the ability to accurately and consistently rank compounds using a scoring function remains problematic. We show that by employing a simple machine learning method (naïve Bayes) it is possible to significantly overcome this deficiency. Compounds from the Available Chemical Directory (ACD), along with known active compounds, were docked into two protein targets using three software packages. In cases where HTD alone was able to show some enrichment, the application of naïve Bayes was able to improve upon the enrichment. The application of this methodology to enrich HTD results can be carried out without a priori knowledge of the activity of compounds and results in superior enrichment of known actives compared to the use of scoring methods alone.

title: Identification of novel inhibitors of urokinase via NMR-based screening.
abstract: Using an NMR-based screen, a novel class of urokinase inhibitors were identified that contain a 2-aminobenzimidazole moiety. The inhibitory potency of this family of inhibitors is similar to that of inhibitors containing a guanidine or amidine group. However, unlike previously described guanidino- or amidino-based inhibitors which have pK(a) values greater than 9.0, urokinase inhibitors containing a 2-aminobenzimidazole have pK(a) values of 7.5. Thus, 2-aminobenzimidazoles may have improved pharmacokinetic properties which could increase the bioavailability of inhibitors which contain this moiety. A crystal structure of one of the lead inhibitors, 2-amino-5-hydroxybenzimidazole, complexed with urokinase reveals the electrostatic and hydrophobic interactions that stabilize complex formation and suggests nearby subsites that may be accessed to increase the potency of this new series of urokinase inhibitors.

title: Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets.
abstract: Involved or implicated in a wide spectrum of diseases, trypsin-like serine proteases comprise well studied drug targets and anti-targets that can be subdivided into two major classes. In one class there is a serine at position 190 at the S1 site, as in urokinase type plasminogen activator (urokinase or uPA) and factor VIIa, and in the other there is an alanine at 190, as in tissue type plasminogen activator (tPA) and factor Xa. A hydrogen bond unique to Ser190 protease-arylamidine complexes between O gamma(Ser190) and the inhibitor amidine confers an intrinsic preference for such inhibitors toward Ser190 proteases over Ala190 counterparts.



Based on the structural differences between the S1 sites of Ser190 and Ala190 protease-arylamidine complexes, we amplified the selectivity of amidine inhibitors toward uPA and against tPA, by factors as high as 220-fold, by incorporating a halo group ortho to the amidine of a lead inhibitor scaffold. Comparison of K(i) values of such halo-substituted and parent inhibitors toward a panel of Ser190 and Ala190 proteases demonstrates pronounced selectivity of the halo analogs for Ser190 proteases over Ala190 counterparts. Crystal structures of Ser190 proteases, uPA and trypsin, and of an Ala190 counterpart, thrombin, bound by a set of ortho (halo, amidino) aryl inhibitors and of non-halo parents reveal the structural basis of the exquisite selectivity and validate the design principle.



Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

title: New Synthetic Thrombin Inhibitors: Molecular Design and Experimental Verification
abstract: The development of new anticoagulants is an important goal for the improvement of thromboses treatments.  The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration.  Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured.  New compounds that are both effective direct thrombin inhibitors (the best KI was <1 nM) and strong anticoagulants in plasma (an IC50 in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD50 values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C.  The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

title: Discovering novel ligands for macromolecules using X-ray crystallographic screening
abstract: The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography–driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.