Patent Application: US-43737603-A

Abstract:
a modified peptide comprises the formula : wherein r 1 is a threonine side chain , r 2 is a side chain of any amino acid , r 3 is a side chain of proline , r 4 is an acetyl group or an n - protecting group , y is a halogen , r 5 is a natural amino acid side chain , and wherein x is an electrophilic group having an ability to undergo nucleophilic attack with active site cysteine residues in cysteine proteinase enzymes .

Description:
all protected amino acids , wang resin and coupling reagents used for peptide synthesis were purchased from calbiochemnovabiochem ( nottingham , u . k .). all solvents for peptide synthesis were obtained from applied biosystems ( warrington , cheshire , u . k .). s . aureus ( oxford strain ) was from the a . t . c . c . 9144 ( manassas , va ., u . s . a .). luria - bertani ( lb ) broth was purchased from oxoid ( basingstoke , u . k .). the pet - 3d expression system , and the antibiotics ampicillin and chloramphenicol were purchased from novagen ( madison , wis ., u . s . a .). nit + nitrilotriacetate ( ni - nta )- agarose was purchased from qiagen ( crawley , west sussex , u . k .). biotin , bsa , the substrates 5 - bromo - 4 - chloroindol - 3 - yl phosphate and nitro blue tetrazolium , isopropyl p - d - thiogalactoside and goat anti - rabbit igg alkaline phosphatase were from sigma - aldrich ( poole , dorset , u . k .). novex ® sds / page gels were from invitrogen ( groningen , the netherlands ), whereas streptavidin alkaline phosphatase was from vector laboratories ( peterborough , u . k .). rabbit antiserum raised against recombinant sortase srta δn was a gift from fusion antibodies ltd . ( belfast , u . k .). fig1 to 3 outline the detailed synthetic approaches for the preparation of inhibitors which utilise a series of solid - and solution - phase synthetic protocols . essentially , n - α - protected threonine derivatives incorporating c - terminal electrophilic functions are incorporated onto solid supports , either via the c - terminal functionality ( vinyl and trans enedione acids ) or via the β - hydroxyl functionality ( vinyl sulphone , diazomethyl ketone , chioromethyl ketone , acyloxymethyl ketone ). peptide targeting sequences are synthesised using standard orthogonal fmoc -/ tbu strategies . we have utilized these sequences ( incorporating either an n - terminal acetyl - or biotinyl -( aminohexanoyl ) 2 - group ) as starting templates and subsequently synthesised peptide fragments of the form leu - pro - ala - thr - with positional scanning at each of the p2 - p4 positions . this affords a considerable degree of molecular diversity within the final library of biotinylated affinity labels and allow the exploration of the importance of each of these 3 regions in individually disclosed sortase - like species . the synthetic schemes reflect the divergent nature of the synthetic protocols . essentially , the classes of inhibitors are prepared from orthogonally protected n - α - fmoc - threonine diazomethyl ketone or aldehyde starting synthons . n - α - protecting group strategies are dependent on application of the synthons in divergent synthetic protocols . thus , those inhibitors incorporating the vinyl sulphone and amides employ n - α - allyoxycarbonyl ( alloc )- protected threonine as the starting synthons . this approach facilitates application to the wittig olefination reaction , which would affect 9 - fluorenylmethoxycarbonyl ( fmoc ) protecting groups which will be used for all other syntheses . n - α alloc - thr ( obut )- oh is converted , via an unsymmetrical anhydride intermediate , into the corresponding diazomethyl ketone ( alloc - thr ( obut )- chn 2 ). treatment with dimethyl dioxirane in moist acetone affords the α - keto - β - aldehyde intermediate in quantitative yield , which readily undergoes a wittig olefination type reaction with a series of carboxyalkyl triphenylphosphoranes to yield trans enedione esters ( 14 ). the trans enedione methyl ester will be hydrolysed ( 1m lioh / thf ) to afford the free trans enedione acid , which will be employed for the synthesis of trans enedione amides ( fig3 ). in the presence of soluble rhodium ( ii ) catalysts , diazomethyl ketones are also known to undergo insertion reactions with carboxylates to yield acyloxymethyl ketones ( 15 ). this approach is exploited for the synthesis of the orthogonally protected bis 2 , 6 ( trifluoromethyl ) benzyloxymethyl ketone analogue of threonine . synthesis of the n - protected - threonine - based starting synthons is accomplished in solution , after coupling to 2 - chlorotrityl chloride , peptide sequences are elaborated on an advanced chemtech vantage ™ automated synthesiser employing standard solid - phase synthetic protocols . n - α - alloc - thr ( obut )- oh is converted to its corresponding aldehyde derivative via lialh 4 - mediated reduction of the corresponding weinreb amide ( alloc - thr -( obut )- n ( meo ) me ). this is readily converted via wittig olefination type reactions into either vinyl sulphone ( 16 ) or vinyl esters ( 17 ). the c - terminal trans enedione ester , acyloxymethyl ketone , vinyl sulphone and vinyl ester analogues of threonine , described above , are generated using standard solution - phase methodologies . following removal of their 0 - butyl ether protecting group ( 50 % tfa / dcm ), each derivative is attached to a hmp - or sasrin resin - based solid support . this is achieved by a mitsunobu reaction in each instance . following attachment to the solid - phase , peptidyl derivatives of each warhead are synthesised using standard orthogonal fmoc / tbu protocols ( 18 ). in each instance , after completion of synthesis , inhibitors are cleaved from the solid support via acidolytic cleavage ( 95 % tfa / appropriate scavengers ). newly generated compounds are purified by reversed phase hplc and characterized by 1 h nmr and electrospray mass spectral analysis the substrate - derived inhibitor sequences illustrated in fig4 were synthesized using a combination of solid - phase and solution ethodologies previously reported ( fig5 ; [ 19 - 21 ]). in essence , the leu - pro - ala - thr - portion of each inhibitor was synthesized ( 0 . 5 mmol scale ) using standard solid - phase synthesis protocols on acid - sensitive wang resin , previously derivatized with fluoren - 9 - ylmethoxycarbonyl ( fmoc )- thr ( obut )- oh as the first amino acid [ 18 ]. each subsequent amino acid was incorporated via single 2 -( 1h - benzotriazol - 1 - yl )- 1 , 1 , 3 , 3tetramethyluronium hexafluorophosphate ( hbtu )- mediated couplings . both alanine and proline residues were incorporated as their n - a - fmoc derivatives . for inhibitor sequences i and ii , the common leucine residue was incorporated as an n - protected benzyloxycarbonyl ( cbz ) derivative . for the biotinylated sequence iii , the leucine residue was incorporated as an n - a - fmoc derivative , as was the aminohexanoic linker residue . biotin was incorporated into the sequence using a double - coupling step . on completion of each synthesis , the peptides were cleaved from the solid support by treatment with a 10 ml solution of 95 % ( v / v ) trifluoroacetic acid containing 2 . 5 % ( v / v ) double - distilled water and 2 . 5 % ( v / v ) tri - isopropyl silane . each cleavage reaction was concentrated to approx . 0 . 5 ml by evaporation under reduced pressure . the peptide product was then precipitated by dilution ( 1 : 20 ) with chilled diethyl ether and isolated by centrifugation ( 2000 g for 10 min ). the products ( yields of 80 %) were dried in vacuo , for 12 h , before the next stage of the synthesis . the peptides ( obtained as their c - terminal free acids ) were then activated , via a mixed carbonic anhydride , and converted into their respective diazomethanes by reaction with ethereal diazomethane [ 19 ]. the peptidyl - chloromethane inhibitor ( ii ) was prepared ( in quantitative yield ) by treating a sample ( 0 . 1 mmol ) of inhibitor i with a 10 ml solution of anhydrous ethereal hci , for 15 min at 0 ° c . [ 23 ]. the purity and identity of each product was confirmed by reverse - phase hplc and matrix - assisted laser - desorption ionization - time - of - flight ( maldi - tof )- ms . srta is believed to be a membrane - associated protease , with an n - terminal membrane anchor . to facilitate purification and solubility , a recombinant form of sortase ( srta δn ) has previously been described and used in which the n - terminal membrane anchor segment of the enzyme ( residues 2 - 25 ) was replaced with a hexahistidine ( his 6 ) sequence [ 8 , 9 , 12 ]. in the present work , a similar strategy was adopted to create a construct for srta δn by replacing the n - terminal t7 tag in a pet - 3d expression vector with a his 6 tag . the region of the srta gene lacking the n - terminal membrane domain was then pcr - amplified from the genomic dna of s . aureus ( oxford strain ) using the primers orf5n - ds - b ( 5 ′- aaaggatccaaaccacatatcgataattatc - 3 ′) ( seq id no : 1 ) and orf5c - b ( 5 ′- aaggatccttatttgacttctgtagctacaa - 3 ′) ( seq id no : 2 )[ 8 ], and the resulting amplicon was cloned into the expression vector at a unique bamhi restriction site . competent escherichia coli cells ( jm109 ) were then transformed and selected on lb agar plates containing ampicillin ( 100 , ug / ml ). after selection and verification of positive clones by dideoxynucleotide dna sequencing , the vector was transformed into the e . coli expression strain hms174 ( de3 ) plyss . transformed bacteria were propagated at 37 ° c . in lb broth containing ampicillin ( 100 , ug / ml ) and chloroamphenicol ( 35 , ug / ml ), and the expression was induced with 1 mm isopropyl / 7 - d - thiogalactoside . cultures were harvested after a 4 h post - induction incubation at 37 ° c ., and the recombinant enzyme was purified on a ni - ntaagarose column as described previously [ 8 ]. confirmation of srta δn was achieved using a monoclonal antibody to the his 6 tag ( sigma - aldrich ) and ion - trap ms on peptides recovered from “ in - gel ” tryptic digests of the recombinant protein kinetic analysis of inhibitors cbz - leu - pro - ala - thr - chn z ( i ) and cbz - leu - pro - ala - thr - ch z ci ( ii ) using methods described previously [ 8 , 9 ], srta δn activity was measured with the internally quenched substrate 4 ([ 4 -( dimethylamino ) phenyl ] azo )- benzoyl ( dabcyl )- gln - ala - leu - pro - glu - thr - gly - glu - glu -[( 2 - aminoethyl )- amino ] napthalenel - sulphonyl ( edans ) ( prepared by standard fmoc solid - phase synthesis ) on a cytofluor 4000 ® multi - well fluorimeter ( perseptive biosystems , foster city , calif ., u . s . a .). all srta an assays were performed at 37 ° c . ( in triplicate ) in 50 mm tris / hci ( ph 7 . 5 ) containing 150 mm nacl , 5 mm cacl2 , 5 mm nh ,- gly 3 and 5 mm dithiothreitol ( srta buffer ). wells contained approx . 10 pm of purified srta on and 50 pm of substrate , in a final volume of 200 , ul . the k m and k cat values for the srta δn - catalysed cleavage of the internally quenched sub - strate were calculated by fitting the data points directly into the michaelis - menten equation for substrate hydrolysis , using grafit ® software ( erithacus software , horley , surrey , u . k .). affinity labelling of recombinant and wild - type srta with biotin - ahx ( aminohexanoyl )- leu - pro - ala - thr - chn z ( iii ) for the detection and disclosure of srta δn with biotin - ahx - leupro - ala - thr - chn 2 ( iii ), crude lysates of transfected e . coli cells ( freshly induced ) were prepared , followed by purification of the recombinant enzyme on a ni - nta - agarose column as previously described [ 8 ]. for the disclosure of wild - type srta in s . aureus , a 50 ml overnight culture of the organism in lb broth was prepared . the bacterial cells were centrifuged at 3000 g for 15 min and the resulting pellet was washed in 5 ml of srta buffer . the washed bacteria were then resuspended in 1 ml of srta buffer containing 0 . 1 g of glass beads . the suspension was vortex - mixed continuously for 5 min followed by centrifugation at 1500 g for 15 min to remove the beads and unbroken cells . to 100 pl of purified srta δn and the crude bacterial preparations , the inhibitor biotin - ahx - leu - pro - ala - thr - chn 2 ( iii ) was added ( final concentration 50 pm ) before incubation at 37 ° c . for 30 min . to detect the affinity - labelled recombinant and wild - type srta , samples were analysed by western - blot analysis [ 19 , 24 - 26 ]. briefly , samples were treated with denaturing treatment buffer and the proteins separated by sds / page on 4 - 20 % ( w / v ) linear gradient gels , followed by semi - dry transfer to a nitrocellulose membrane ( schleicher and schiill , dassel , germany ). after transfer , non - specific binding sites were quenched with a 3 % ( w / v ) solution of bsa in 20 mm tris / hci ( ph 7 . 4 ), containing 150 mm nacl . to detect the biotin group , the membrane was incubated with a streptavidin - alkaline phosphatase conjugate ( 1 : 500 ). to confirm the identity of affinity - labeled recombinant and wild - type srta , the expressed protein and crude s . aureus extracts were respectively incubated with a rabbit antiserum ( 1 : 5000 ) raised against srta δn . bound antibody was detected with a goat anti - rabbit igg alkaline phosphatase conjugate ( 1 : 20000 ). all protein bands were revealed after incubation of the membranes with the substrates 5 - bromo - 4 - chloroindol - 3 - yl phosphate and nitro blue tetrazolium . progress curves for srta δn - catalysed hydrolysis of the internally quenched substrate dabcyl - gln - ala - leu - pro - glu - thrgly - glu - glu - edans in the presence of different concentrations of cbz - leu - pro - ala - thr - chn 2 ( i ) and cbz - leu - pro - ala - thrch 2 cl ( ii ) typified the action of active site - directed irreversible inhibitors operating via the mechanism illustrated in fig7 [ 28 , 29 ]. data from the curves were fitted , using non - linear regression analysis [ 29 ], to the integrated rate equation [ p ]= pj1 - e “- pp ”). this equation represents a first - order rate process for the formation of product p as a function of time , where k app is the apparent rate constant and p . represents the concentration of product at a time approaching infinity . using five different concentrations for each inhibitor ( 25 , 50 , 100 , 150 and 200 / μm ), the values k app and p ∞ were determined and utilized to evaluate the apparent second - order rate constant a for the inactivation of srta δn in the presence of the substrate [ 28 ]. the individual kinetic constants k i and k i were then evaluated for both inhibitors from a plot of 1 / a versus inhibitor concentration [ i ] [ 28 ], and the specificity constant k ;/ k ; was calculated . fig6 shows the plot for inactivation of srta δn by the peptidyl - chloromethane inhibitor cbz - leu - pro - ala - thr - ch , cl ( ii ). all the kinetic constants determined for inhibitors i and ii are given in table 1 below . table 1 kinetic constants for the inactivation of srta on by the substratederived peptidyl - diazomethane ( i ) and peptidyl - chloromethane ( ii ) inhibitors data from the progress curves for srt δn - catalysed hydrolysis of the internally quenched substrate dabcyl - gln - ala - leu - pro - glu - thr - gly - glu - glu - edans in the presence of five different concentrations of inhibitors i and ii ( 25 , 50 , 100 , 150 and 200 , w ) were fitted by non - linear regression analysis to the integrated rate equation [ p ]= p ∞ ( 1 - e − kappt ). the p ∞ . and kapp values were determined and used to evaluate the apparent second - order rate constant a for inactivation of srt δn in the presence of substrate . the individual kinetic constants k and k were then evaluated from a plot of 1 / a versus inhibitor concentration [ i ], and the specificity constant for each inhibitor ( k i k i ) calculated . values represent the means ± s . e . m . for four determinations . inhibitor k 1 ( min − 1 ) k i ( m ) k i / k i ( m − 1 − min − 1 ) i 5 . 8 ± 0 . 6 × 10 − 3 2 . 2 ± 0 . 2 × 10 − 7 2 . 2 ± 0 . 2 × 10 4 ii 1 . 1 ± 0 . 1 × 10 − 2 2 . 1 ± 0 . 2 × 10 − 7 5 . 3 ± 0 . 6 × 10 4 the first point of interest was the observation that the peptidylchloromethane inhibitor ( ii ) inactivates srta δn by approx . 2 - fold more rapidly than the analogous diazomethane sequence ( 1 ), as revealed by the specificity constants k i / k i = 5 . 3 × 10 4 and 2 . 2 × 10 4 m − 1 - min − 1 respectively . both inhibitors i and 11 exhibited almost identical , sub - micromolar inhibitor constants k i = 2 . 2 × 10 − 1 and 2 . 1 × 10 − 1 m respectively . this implies high affinity of interaction between the common tetrapeptide motif of each inhibitor and the srta δn enzyme . the greater effectiveness of the chloromethane inhibitor ( ii ) can be attributed almost exclusively to the increased rate at which it covalently modifies srta δn . therefore the peptidyl - diazomethane ( i ) covalently modifies srta δn with a first - order rate constant k i = 5 . 8 × 10 − 3 min − 1 , whereas the peptidyl - chloromethane ( ii ) covalently modifies the enzyme by approx . 2 - fold more rapidly ( k i = 1 . 1 × 10 − 2 min − 1 ). this observation fits the known relative chemical reactivity of the electrophilic groupings of both inhibitors towards thiol nucleophiles [ 27 , 30 ]. the first - order rate constants observed are considerably smaller than those previously determined for the inactivation of cysteine proteases by active - site directed peptidyl - diazomethanes and peptidyl - chloromethanes . for example , the peptidyl - chloromethane inhibitor cbz - phe - phe - ch , cl alkylates the active - site cysteine residue of cathepsin b with k i = 12 . 5 min − 1 [ 30 ]. this is approx . 1000 - fold greater than the irreversible modification of srta δn by the peptidyl - chloromethane sequence ( ii ). the potential of a biotinylated inhibitor of srta for the detection of functionally active forms of srta - like species in gram - positive organisms was evaluated . towards this end , the biotinylated inhibitor sequence biotin - ahx - leu - pro - ala - thr - chn 2 ( iii ) was utilised . the strategy underlying the design of iii was based on two tenets . first , the peptidyl - diazomethane ( i ) was chosen over the slightly more active chloromethane sequence ( 11 ), as the latter has potential to cause non - specific alkylation of biomolecules [ 27 ]. secondly , an aminohexanoic spacer group was incorporated between the recognition motif of the inhibitor and the biotin moiety to maximize the interaction with streptavidin alkaline phosphatase , which was used for disclosure of affinitylabelled proteolytic species after sds / page and western - blot analysis . during initial western - blotting experiments , biotin - ahx - leupro - ala - thr chn 2 ( iii ) ( 50 , μm ) was examined for its ability to disclose srta δn in e . coli cells transfected with the srta gene from s . aureus ( within the pet - 3d expression vector ; see fig5 a ). as shown in fig8 lane 1 contained a crude lysate prepared from a freshly induced culture of the transfected e . coli cells , whereas lane 2 contained the ‘ flow - through ’ from the ninta - agarose column used to purify the expressed his s - tagged srta δn . in lane 3 , the bound fraction eluted from the column with 10 mm imidazole ( purified srta δn ) was also analysed . in both lanes 1 and 2 , the intensely stained protein species with an apparent molecular mass of 22 kda was the endogenous biotin carbonyl carrier protein ( bccp ). this species was totally absent in the fraction selectively eluted from the ni - nta - agarose column ( fig8 lane 3 ). the band with an apparent molecular mass of approx . 30 kda , which was present in both the crude cell lysate ( fig8 lane 1 ) and eluted fraction ( fig8 lane 3 ), was his 6 - tagged srta δn . the identity of the affinity - labelled srta δn band was confirmed with a monoclonal antibody to the his , sequence and a rabbit antiserum raised against the recombinant enzyme ( results not shown ). the molecular mass determined for srta δn by sds / page was in agreement with the previous reports of ton - that et al . [ 8 ]. in lane 4 , pre - treatment of the purified srt δn sample with the thiol - directed inhibitor phmb , before ‘ probing ’ with biotin - ahx - leu - pro - ala - thrchn 2 ( iii ), resulted in complete diminution of srta δn labelling . as the enzyme contains only a single cysteine residue ( cys 184 ) essential for catalytic activity [ 8 , 9 ], we can confidently conclude that the binding of this biotinylated inhibitor to srta δn is indeed active - site - directed . western - blotting experiments were performed with biotinahx - leu - pro - ala - thr - chn 2 ( iii ) to detect the presence of wildtype srta in crude cell lysates from s . aureus ( oxford strain ; see fig8 b ). in the duplicate control lanes 1 and 2 , which contained unprobed cell lysate , the bccp protein ( 22 kda ) was again observed . in the duplicate lanes 3 and 4 , which contained cell lysate “ probed ” with biotin - ahx - leu - pro - ala - thr - chn 2 ( iii ), the bccp protein was detected along with an additional protein of apparent molecular mass 24 kda . this molecular mass was consistent with the predicted value for wild - type srta . that this protein band was indeed affinity - labelled srta was confirmed with an antiserum raised against the recombinant form of the protein ( see fig8 b ). the discrete labelling of only recombinant and wild - type forms of srta in crude cell lysates prepared from transfected e . coli cells and s . aureus respectively provides convincing evidence for the selectivity of action of the biotin - ahx - leu - pro - ala - thr - chn 2 inhibitor . as expected , biotin - ahx - leu - pro - ala - thr - chn 2 ( iii ) selectively disclosed wild - type srta , but not srtb in crude s . aureus extracts . two aspects explain the specificity of our affinity label for srta only . first , srta is a constitutively expressed protease in s . aureus , whereas srtb expression is tightly regulated by the concentration of iron , which was not added to our culture media [ 11 ]. secondly , the previous studies of mazmanian et al . [ 11 ] demonstrated the exquisite specificity of srta and srtb towards cognate synthetic peptides modelled on their respective sorting signals . when tested against internally quenched fluorescent peptides , srtb was shown only to cleave peptides containing the - asn - pro - gln - thr ↓ asn - sorting sequence , and not the - leu - pro - xaa - thr ↓ gly - sequence recognized by srta . in contrast , srta displayed exactly the contrary specificity . consequently , we would not have expected srtb to be inhibited by our biotinylated affinity label , even under conditions where the enzyme was expressed . biotinylated versions of the inhibitors of the invention may be used to detect additional sortase species . identification of such species in gram positive organisms could lead to their validation as therapeutic targets . the inhibitors of the invention may also be used to block bacterial adherence in animals and humans and thus limit infection . when used in combination with other therapeutic regimes , they could lead to more rapid clearing of infection . the inhibitors could also be used in combination with vaccine approaches to prevent the display of decoy or cloaking proteins , or be used as adjuvant therapies in limiting bacterial infection in burns patients , by blocking adherence to exposed sub - epithelial tissue . the inhibitors could also be used to reduce bacterial adherence to medical devices such as stents , catheters and prosthesis . for pharmaceutical use , the inhibitor may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition . pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents , excipients , solvents , adjuvants , or vehicles , for parenteral injection , for intranasal or sublingual delivery , for oral administration , for rectal or topical administration or the like . for oral administration , the medicament according to the invention may be in the form of , for example , a tablet , capsule suspension or liquid . the medicament is preferably made in the form of a dosage unit containing a particular amount of the active ingredient . examples of such dosage units are capsules , tablets , powders , granules or a suspension , with conventional additives such as lactose , mannitol , corn starch or potatoes starch ; with binders such as crystalline cellulose , cellulose derivatives , acacia , corn starch or gelatins ; with disintegrators such as corn starch , potaote starch or sodium carboxymethyl - cellulose ; and with lubricants such as talc or magnesium stearate . the active ingredient may also be administered by injection as a composition wherein , for example , saline , dextrose or water may be used as a suitable carrier . for intravenous , intramuscular , subcutaneous , or intraperitioneal administration , the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient . such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride , glycine , and the like , and having a buffered ph compatible with physiological conditions to produce an aqueous solution , and rendering said solution sterile . the formulations may be present in unit or multi - dose containers such as seated ampoules or vials . if the disease or condition to be treated is localized in the g . i . tract , the compound may be formulated with acid - stable , base - liable coatings known in the art which began to dissolve in the high ph intestine . formulations to enhance local pharmacologic effects and reduce systemic uptake are preferred . formulations suitable for administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic . preparations for injections may also be formulated by suspending or emulsifying the compounds in non - aqueous solvent , such as vegetable oil , synthetic aliphatic acid glycerides , esters of higher aliphatic acids or propylene glycol . formulations for topical use include known gels , creams , oils , and the like . for aerosol delivery , the compounds may be formulated with known aerosol exipients , such as saline and administered using commercially available nebulizers . formulation in a fatty acid source may be used to enhance biocompatibility . for rectal administration , the active ingredient may be formulated into suppositories using bases which are solid at room temperature and melt and dissolve at body temperature . commonly used bases include cocoa butter , glycerinated gelatin , hydrogenated vegetable oil , polyethylene glycols of various molecular weights , and fatty esters of polyethylene stearate . the dosage form and amount can be readily established by reference to known anti - bacterial or vaccination treatment or prophylactic regiments . the amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this invention depends on a variety of factors , including the age , weight , sex and medical condition of the subject , the severity of the disease , the route and frequency of administration , and the particular compound employed , the location of the disease or condition , as well as the pharmacokinetic properties of the individual treated , and thus may vary widely . the dosage will generally be lower if the compounds are administered locally rather than systemically , and for prevention rather than for treatment . such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician . one of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual . the pharmaceutical compositions may contain active ingredient in the range of about 0 . 1 to 2000 mg , preferably in the range of about 0 . 5 to 500 mg and most preferably between about 1 and 200 mg . a daily dose of about 0 . 01 to 100 mg / kg body weight , preferably between about 0 . 1 and about 50 mg / kg body weight , may be appropriate . the daily dose can be administered in one to four doses per day . the invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention . 1 . navarre , w . w . and schneewind , 0 . 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