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The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies. The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies. Proteases hydrolyze amide bonds in proteins and peptides and represent one of the largest and most important protein families known. They comprise over 2% of the human genome and play diverse physiological roles (merops.sanger.ac.uk) (1Rawlings N.D. Tolle D.P. Barrett A.J. Nucleic Acids Res. 2004; 32: D160-D164Crossref PubMed Google Scholar). The substrate specificity of a protease enables the enzyme to preferentially cleave its substrates in the presence of other peptides or proteins. Therefore, specificity information can provide clues about the biological function of the protease and aid in the design of efficient substrates and potent, selective inhibitors. Various methods including both biological and chemical-based approaches to study protease specificity have been developed and were recently reviewed (2Marnett A.B. Craik C.S. Trends Biotechnol. 2005; 23: 59-64Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Positional scanning synthetic combinatorial libraries (PS-SCLs) 2The abbreviations used are: PS-SCL, positional scanning synthetic combinatorial libraries; ACC, 7-amino-4-carbamoylmethylcoumarin; DMF, N,N-dimethyl formamide; DICI, diisopropylcarbodiimide; HOBt, 1-hydroxybenzotriazole; Ac, acetyl; Z, benzyloxycarbonyl; AMC, 7-amino-4-methylcoumarin; HPLC, high pressure liquid chromatography; AOMK, acyloxylmethyl ketone; Fmoc, N-(9-fluorenyl)methoxycarbonyl. of fluorogenic substrates have emerged as useful reagents for the rapid and exhaustive determination of protease specificity (3Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1849) Google Scholar). A peptide-based PS-SCL is composed of sublibraries in which one peptide position is fixed with an amino acid, whereas the remaining positions contain an equimolar mixture of amino acids. Assaying proteases with these sublibraries rapidly establishes the amino acid preferences at the defined position. Initially, the substrate specificities of caspases and granzyme B were profiled using PS-SCLs with the P1 position fixed as an aspartic acid. The limitations of the original P1 fixed libraries were overcome through the development of a modified coumarin, 7-amino-4-carbamoylmethylcoumarin (ACC) fluorogenic leaving group. The bifunctional nature of this enables straightforward solid-phase synthesis of libraries containing any amino acid at the P1 position. Early applications involved the use of a P1-diverse PS-SCL in combination with several P1-fixed PS-SCLs to study P1 and P2-P3-P4 specificity, respectively (4Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar, 5Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 6Harris J.L. Niles A. Burdick K. Maffitt M. Backes B.J. Ellman J.A. Kuntz I. Haak-Frendscho M. Craik C.S. J. Biol. Chem. 2001; 276: 34941-34947Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7Salter J.P. Choe Y. Albrecht H. Franklin C. Lim K.C. Craik C.S. McKerrow J.H. J. Biol. Chem. 2002; 277: 24618-24624Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We report the preparation of a completely diversified PS-SCL of ACC-based substrates. This library permits the determination of P1-P2-P3-P4 specificity of proteases regardless of their P1 specificity. Using this complete diverse library, numerous proteases from various sources including humans, parasites, bacteria, and viruses have been profiled. As a representative family, we present a study of the substrate specificity of papain-like cysteine proteases. The papain-like cysteine proteases, which include plant enzymes papain and bromelain, human cysteine cathepsins (B, H, L, S, C, K, O, F, V, X, W), and parasite proteases cruzain and falcipains, have been characterized as key enzymes in many biological and pathological events (8Lecaille F. Kaleta J. Brömme D. Chem. Rev. 2002; 102: 4459-4488Crossref PubMed Scopus (448) Google Scholar, 9Brömme D. Kaleta J. Curr. Pharm. Des. 2002; 8: 1639-1658Crossref PubMed Scopus (93) Google Scholar, 10Sajid M. McKerrow J.H. Mol. Biochem. Parasitol. 2002; 120: 1-21Crossref PubMed Scopus (670) Google Scholar, 11Rosenthal P.J. Int. J. Parasitol. 2004; 34: 1489-1499Crossref PubMed Scopus (286) Google Scholar). As a result, many of them represent particularly attractive drug targets. Of particular interest to these studies is cathepsin K, a cysteine protease implicated in osteoporosis and other diseases (12Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The substrate binding pocket of these proteases can be divided into seven substrate binding subsites, S4 to S3′, that interact with P4 to P3′ residues of substrates (13Berger A. Schechter I. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1970; 257: 249-264Crossref PubMed Scopus (378) Google Scholar). Hydrolysis occurs at the scissile bond between P1 and P1′. Among these, S3 and S2′ subsites interact with substrates through only side chain contacts, and their interactions spread over a relatively wide area. In contrast, the S2, S1, and S1′ subsites involve both main chain and side chain contacts. These recognition properties in combination with the well conserved structure of the family result in broad and similar specificities for the papain-like proteases (14Turk D. Guncar G. Podobnik M. Turk B. Biol. Chem. 1998; 379: 137-147Crossref PubMed Scopus (219) Google Scholar). In this study, however, the complete diverse PS-SCL and the cluster analysis of the resulting specificity information have revealed distinctive differences between the members of this class. The utility of this library and the specificity information obtained using it is well exemplified by the development of specific ACC-based substrates and an acyloxymethyl ketone inhibitor for cathepsin K. Materials—Chemicals were obtained from commercial suppliers and used without further purification, unless otherwise stated. Rink amide AM resin and Fmoc-amino acids were purchased from Novabiochem. Anhydrous low amine content N,N-dimethyl formamide (DMF) was from EM was from acid, and were from and were purchased from and were purchased from cathepsin B was purchased from Biochem. purification, and site were as for human cathepsin D. Sci. 1996; PubMed Scopus Google K (12Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google J. Biol. Chem. Full Text PDF PubMed Google S D. Sci. 1996; PubMed Scopus Google and D. M. PubMed Scopus Google Scholar). papain-like cysteine proteases of parasite origin were from J. H. M. and C. R. from cruzain from a cathepsin protease from and cathepsin proteases and from were and as A. J. M. D. McKerrow J.H. Mol. Biochem. Parasitol. 2001; PubMed Scopus Google Scholar, G. McKerrow J.H. Craik C.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the preparation of and was as using an (4Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar, B.J. Harris J.L. Leonetti F. Craik C.S. Ellman J.A. Biotechnol. 2000; PubMed Scopus Google Scholar, Leonetti F. Backes B.J. Harris J.L. Craik C.S. Ellman J.A. J. Chem. 2002; PubMed Scopus Google Scholar). The of the resin was by a quantitative assay The Scholar). The synthesis of the library was using a synthesis the P1 of the P1 library, each of 20 Fmoc-amino acids cysteine and including to was to the of the The use of in the amino acid is to the of information provided by the substrate specificity the of as and and has a similar chain structure as it provides information in the substrate specificity of proteases. the P2, P3, and P4 an mixture of 20 amino acids to each was by the in for (4Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar, J.H. 34: PubMed Scopus Google Scholar). the resin was and then in the of the 20 each was to each well to the amino and for the the was and a of in was to the resin and for The was then by and the resin was with P2 amino acids to the P2 library, 20 individual Fmoc-amino acids were in using and in and to the for the P2 library. P2 amino acids to the P1, P3, and P4 an mixture of the 20 Fmoc-amino acids each library, was with and in The was then to each well for the P1, P3, and P4 and a for the was and the resin was with The P3 and P4 positions were in the using 20 individual Fmoc-amino acids for the P3 position of the P3 library and for the P4 position of the P4 library, whereas a mixture was used for the remaining the synthesis of the peptide was the of the P4 amino acids was and the resin in each with DMF, was with a of and in for the resin was with and then with The substrates were from the resin by for with a of and the was The were in to a of and at of Individual and an synthesis of individual peptide substrates was using the for the complete diverse PS-SCL the acid cleavage The substrates from the resin were with any was the resulting were to with a with was used to the of the substrates. The were in and at ketone was based on the library assay of cathepsin K and other cathepsins. The synthesis was using similar to P.J. A. J. Chem. PubMed Scopus Google Scholar, D. P.J. H. A. Biol. Chem. PubMed Scopus Google Scholar). The inhibitor was and its was as for the preparation of individual substrates. PS-SCL cysteine proteases were at in a containing and the of in from each of 20 sublibraries of the P1, P2, P3, and P4 libraries were to the of a plate The of each of the was in The were by the of enzyme and with a with at at and at (4Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar, J.H. 34: PubMed Scopus Google Scholar). The and of the substrates and of the substrate by a protease to the in a of the and to and of and an at is used to the of the over the of the In the fluorophore has an at the and of and The of the for the of protease of from the PS-SCL the specificity information with amino acid sequence the from the library were the from the library assay were to in a from to by a of to the amino acids that the in each library to whereas amino acids that were a of The were with the and in a by using D. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The P1, P2, P3, and P4 specificities were to with the sequence and for The amino acid sequence of protease was using the The sequences were from the or of Individual and an analysis was used to the of each substrate and protease The of substrates from and the of in the was 2% The of cathepsins K, L, and B were and were at in The of substrates was using the assay for the complete diverse library The and were used to the and and characterization of the inhibitor was as A. J. M. D. McKerrow J.H. Mol. Biochem. Parasitol. 2001; PubMed Scopus Google Scholar). of human cathepsin K cathepsin and cathepsin B were with a synthetic substrate the complete diverse library assay for the at each of inhibitor were for individual by the to the enzyme is the of over and is the of the analysis to the and the was using the and is the of to the of the the and of human cathepsins L, B, and K, a was as D. A. M. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the binding of to the proteases. This was using for quantitative of the completely diversified PS-SCL with the structure of was synthesized using ACC, a bifunctional fluorophore leaving with for peptide synthesis and to The library of P1, P2, P3, and P4 libraries in which the P1, P2, P3, or P4 position is fixed with one of 20 amino acids cysteine and including whereas the remaining positions contain an equimolar mixture of these amino acids As a result, each of the libraries has 20 sublibraries that contain a mixture of of tetrapeptide fluorogenic substrates. As a the complete diverse library 160,000 unique tetrapeptide substrates. The library was characterized using the enzymes papain, and the substrate specificities of which well The P1-diverse PS-SCL and various P1-fixed PS-SCLs were used to profile these enzymes and human cysteine cathepsins The obtained using these libraries were with obtained from the complete diverse PS-SCL The were in with each that the complete diverse library was of specificities of papain, bromelain, and human cysteine cathepsins L, V, S, K, F, and B were using the complete diverse PS-SCL The specificities of cathepsin protease from and cathepsin proteases and from S. were using the complete diverse of these proteases a preference for amino acids at the P2 position bromelain, which amino acids as human cathepsins papain in specificity by and at the P1 amino acids at the P2 and specificities at the P3 and P4 the complete diverse PS-SCL assay the differences in the chemical of the amino acids at the P2 position and P3 specificity. the P2 the substrate specificity profile of cathepsin a preference for residues over amino acids which it from cathepsins K and S and K and S have been to residues at the P2 whereas cathepsin has been to amino acids (12Brömme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, Brömme D. Sci. 1998; PubMed Scopus Google Scholar). These studies in with the complete diverse PS-SCL assay which that cathepsins K and S amino acids at the P2 position. V, which is the to cathepsin in of sequence a preference similar to cathepsin L, amino acids over amino acids and well at the P2 which is with studies D. M. PubMed Scopus Google Scholar, T. Brömme D. Biochem. 2004; PubMed Scopus Google Scholar). The P2 specificity of cathepsin was similar to that of cathepsin K for the proline preference of cathepsin K is that cathepsin aspartic acid at the P2 and P3 positions, whereas of the other cathepsins this amino acid at position. The complete diverse library assay that cathepsin B has P2 specificity. B with the P1 library with the P2 library, which with the cathepsin proteases cathepsins L, V, S, and K The library assay that cathepsin B well at the P2 whereas the other cathepsins any with this amino acid, in with studies Biochem. 2004; PubMed Scopus Google Scholar). the P3 cathepsins and S broad specificity preference for amino acids and amino acids whereas cathepsin proline and The library assay that cathepsin B has a P3 specificity it was to of Proteases of the parasite proteases tested similar P1 specificity as human cathepsins. the in the to be the and cathepsin protease from specificity that is similar to that of human cathepsins and V, whereas cathepsin proteases and from S. P2 specificity, amino acids the parasite proteases is that cathepsin protease has similar P2 and P3 specificities to human cathepsin B, it has sequence to human cathepsin B cathepsin protease This that a sequence is to the specificity of an of the complete diverse PS-SCL, cathepsin K the most substrate specificity the human cathepsins The protease amino acids at the P2 cathepsins and V, which both and amino acids. cathepsin K proline and glycine at the P2 and P3 positions, respectively, whereas of amino were by the other human cathepsins the proline preference is with a This is in with a study that that a for cathepsin K J. H. A. D.W. Brömme D. Biol. Chem. PubMed Scopus Google Scholar). of by PS-SCL and of K and B, H, and it to their substrates. the and of cathepsins K and S is that of cathepsins B, H, and As a result, information on their physiological substrates is the between the specificities by the PS-SCL and the physiological substrates for cathepsin K W. Brömme D. Biochem. J. 1998; PubMed Scopus Google and cathepsin S J.A. C. Chapman J. 1997; PubMed Scopus Google and physiological substrate cleavage sequences of human cathepsins K and by the complete diverse site by the complete diverse site in a of the specificity with the sequence papain-like cysteine proteases including papain, bromelain, human cathepsins L, V, S, K, F, B, and parasitic proteases as cathepsin and cathepsin proteases and were based on their specificity using the D. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). a amino acid sequence of these proteases is The cluster analysis was by the specificity profile or each position for each The resulting of proteases specificity and represent functional the proteases. The result of the specificity profile the sequence cathepsin and the cathepsin proteases of parasite origin were with human cathepsin S and papain the to the cathepsin by the cathepsin group. K was from the cathepsin to its substrate specificity. B and cathepsin proteases were as the from cathepsin The cluster analysis of each P1, P2, P3, and P4 specificity the P1 the specificity of the cysteine proteases to be cathepsin S was by its unique P1 specificity, and over is at this cathepsin proteases were from the cathepsin the P2 distinctive specificity were The that cathepsin proteases and V, and cathepsin amino acids amino acids to amino acids and K were relatively from cathepsin this to their unique B and S, cathepsin and papain the that broad specificity with a preference of amino acids. The unique preference for and of is well The P3 position has to the of a to amino acids. the complete diverse PS-SCL and the cluster analysis that is P3 specificities as preference for amino amino or it is as as P2 specificity The cluster analysis of the P4 specificity is at this position of Individual ACC-based provide a quantitative of the specificity information obtained by the library a of substrates of and sequence were synthesized for studies with cathepsins K, L, and B the substrate cathepsin the these cathepsins to its low of the proteases any which that interactions and cathepsin K with whereas cathepsin with These with the complete diverse PS-SCL assay a and the tetrapeptide substrates and cathepsin K a preference for whereas it with to the of the P2 amino acid contrast, cathepsin with both a result by the complete diverse PS-SCL with each that substrates interactions between the protease and its substrates. the quantitative from the study using individual ACC-based substrates were in with the substrate specificity from the library analysis of substrates with human and in a of and a for the unique specificities of cathepsin K by the library assay and glycine at the P2 and P3 positions, individual substrates and were synthesized and tested for their for cathepsin K. They were cathepsins K, S, L, V, and B, and both to be selective for cathepsin K the library assay result, cathepsin K with one of the amino acids at the P1 with is the main specificity for the papain-like cysteine proteases, it an important in and efficient of these proteases an P1 substrate specificity. inhibitor was then based on the specificity of cathepsin K to its for the protease cathepsin B was by this inhibitor to the library assay result, inhibitor analysis and that inhibitor was for cathepsin K over cathepsins and that the substrate can be used to design selective substrates to design selective particularly for cathepsin analysis of an in a The cysteine cathepsins have been implicated in a of diseases and events (8Lecaille F. Kaleta J. Brömme D. Chem. Rev. 2002; 102: 4459-4488Crossref PubMed Scopus (448) Google Scholar, B. Turk D. Turk 2000; PubMed Scopus Google Scholar, Turk B. Turk D. J. 2001; PubMed Scopus Google Scholar, T. W. A. A. J. J.A. H. C. 1998; PubMed Scopus Google Scholar). the physiological or pathological roles of each cathepsin to their conserved structure and specificity with their diverse and The complete diverse PS-SCL assay their broad and similar substrate specificity. their in specificity, the substrate library particularly at the P2 and P3 K has been to play a key in which in of through Y. Kaleta J. Brömme D. Rev. 2005; PubMed Scopus Google Scholar). this the protease has been an important drug selective substrates or for cathepsin K, it is important to the specificity of the human proteases including other cathepsins as well as cathepsin K S, L, and K have been to be to particularly cathepsins S and K, which high P2 specificity A.J. N.D. of Scholar). The complete diverse PS-SCL, however, the differences between these proteases by 20 amino acids at the P1 to P4 the library assay that cathepsin K has a unique preference for proline and glycine at the P2 and P3 positions, is that a well substrate of cathepsin K, on this selective substrates and an inhibitor of cathepsin K with at the positions were the of the ACC-based PS-SCL and the information obtained using The cluster analysis of papain-like cysteine proteases sequence distinctive specificity differences were these proteases, a functional at each This information particularly which on protein the information is to aid the of specificity in substrate binding of proteases by to proteases based on their specificity. As specificity information on protease function and structure be the for the substrate specificity of a protease from its the of proteases, is an for efficient methods to their substrate specificity. specificity studies of proteases to on analysis that is by the of substrates. substrates for protease have been developed based on the physiological substrate cleavage sequences or substrate sequences of proteases or through of substrates. The complete diverse PS-SCL can rapidly the specificity of a protease without any of its physiological or to other proteases. use is by P1 specificity, which is an over PS-SCLs (4Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7754-7759Crossref PubMed Scopus (475) Google Scholar). The complete diverse PS-SCL assay a rapid microtiter plate format to provide exhaustive and specificity information of proteases at the P1, P2, P3, and P4 In most of an papain-like protease was to its specificity The substrate library assay is and which it the to be to high or assay using a tetrapeptide substrate library completely diversified at the positions to the substrate specificity of various papain-like cysteine proteases, we unique The specificity information by the complete diverse PS-SCL development of efficient and specific for the design of selective and of the of specificity through cluster and of the protein for physiological substrates. the PS-SCL is to subsites, it can be used in with other methods to the specificity the complete diverse PS-SCL is to be useful to the development of selective reagents to the of proteases and to the physiological of We the members of the Craik for many useful with
Choe et al. (Tue,) studied this question.