CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector FEBS Letters 587 (2013) 3021–3026 journal homepage: www.FEBSLetters.org Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB) ⇑ Mohammad A. Qasim ,1, Lixia Wang 2, Sabiha Qasim, Stephen Lu 3, Wuyuan Lu 4, Richard Wynn 5, Zheng-Ping Yi 6, Michael Laskowski Jr. 7 Department of Chemistry, Purdue University, 1393 Brown Building, West Lafayette, IN 47907-1393, United States article info abstract Article history: We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Received 20 May 2013 Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In Revised 15 July 2013 one, the reactive site residue (position 18) was retained and the best residues were substituted at Accepted 16 July 2013 contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact Available online 23 July 2013 positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T13E14Y15-OMTKY3 and T13E14Y15G18M21P32V36-OMTKY3, and for SGPB, S13D14Y15-OMTKY3 and 13 14 15 18 19 21 0 Edited by Robert B. Russell S D Y G I K -OMTKY3. The free energies of association (DG ) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine prote- Keywords: ases and the results are discussed. Inhibitor design Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Kazal inhibitor Serine protease Protease inhibitor Additivity 1. Introduction form a stable non-covalent complex with the serine protease [1– 4]. Based on sequence homologies and disulfide bond topologies, Standard mechanism serine protease inhibitors bind to serine eighteen families of standard mechanism serine protease inhibi- proteases like substrates but instead of getting hydrolyzed they tors have been recognized [3,5]. Despite differences in sequences, size, and disulfide bonding patterns, all of the eighteen families follow the same mechanism of inhibition commonly called the Abbreviations: SRA, sequence to reactivity algorithm; OMTKY3, turkey ovomu- coid third domain; SGPA and SGPB, Streptomyces griseus protease A and B. In standard mechanism of inhibition [1]. MEROPS database and recent literature these are listed as Streptogrisin A and B; PPE, We have been involved in the research work aimed at develop- porcine pancreatic elastase; HLE, human leukocyte elastase; CARL, subtilisin ing a sequence to reactivity algorithm (SRA) for the Kazal family of Carlsberg standard mechanism inhibitors. In the first part of our research ⇑ Corresponding author. project, ovomucoid third domains (a Kazal family inhibitor) were E-mail address: [email protected] (M.A. Qasim). 1 Present address: Department of Chemistry, Indiana U Purdue U Fort Wayne, IN prepared and purified from egg whites of a large number of species 46805, United States. of birds. The ovomucoid third domains were sequenced [6–8] and 2 Present address: Institute of Plant Protection, Chinese Academy of Agricultural free energy changes of their association (DG0) were measured with Sciences, Beijing 100193, PR China. 3 Present address: AbbVie BioResearch Center, 100 Research Drive, Worcester, MA a panel of six serine proteases [9–12]. In the second part of the pro- 01605, United States. ject all single amino acid variants at ten of the twelve consensus 4 Present address: Institute of Human Virology, University of Maryland School of contact positions of turkey ovomucoid third domain (OMTKY3) Medicine, 725 West Lombard Street, Baltimore, MD 21201, United States. (see Fig. 1) were prepared and their DG0 values were measured 5 Present address: Incyte Corporation, Experimental Station, E336/241B, Route 141 & Henry Clay Road, Wilmington, DE 19880, United States. against the same set of six serine proteases [13–15]. The culmina- 6 Present address: Department of Pharmaceutical Sciences, Eugene Applebaum tion of these two projects produced an SRA for the Kazal family of College of Pharmacy/Health Sciences, Wayne State University, Detroit, MI 48201, inhibitors, in addition to providing a large and unbiased set of United States. inhibitors for testing the algorithm. 7 Deceased. 0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.07.029 3022 M.A. Qasim et al. / FEBS Letters 587 (2013) 3021–3026 SRA. The substitution of Gly at P1 is based on overwhelming data [14,24–27] that show strong additivity of substitutions involving the P1 position of inhibitors as well as substrates. Most of the pre- dicted and the measured values were in excellent agreement. The success of these studies emphasizes the importance of including more serine proteases in further developing the sequence to reac- tivity algorithm. 2. Materials and methods 2.1. Chemicals Four of the six serine proteases used in this research namely, TLCK treated bovine a-chymotrypsin (Worthington), human leuko- cyte elastase (HLE) (Elastin Products), porcine pancreatic elastase (PPE) (Sigma), and subtilisin Carlsberg (CARL) (Sigma) were ob- tained from the commercial sources listed in parentheses. The other two serine proteases, Streptomyces griseus protease A and B, (SGPA and SGPB) were purified from a commercially obtained preparation of pronase (Sigma) as described [28]. The identity and the purity of the two proteases were established by amino acid Fig. 1. Primary structure of expressed wtOMTKY3. The first five residues are not analysis and by analytical ion exchange chromatography. shown because they are not part of the expressed OMTKY3 (and its variants) and The chromogenic and fluorogenic synthetic substrates of the have been found to have no effect on the inhibitory activity of OMTKY3. The type succinyl-ala-ala-pro-Xxx-pNA and succinyl-ala-ala-pro-Xxx- residues are numbered sequentially as well as in Schechter–Berger notation [30].In AMC were purchased from BACHEM. Other chemicals used in this Schechter–Berger notation, the reactive site residue (shown by the arrow) is labeled work were all analytical grade. as P1. Residues towards the N-terminal of the P1 residue are sequentially labeled as P2, P3, ..., Pn whereas residues C-terminal to P1 residue are labeled as P10,P20, ..., Pn0. The amino acids shown as filled circles are the consensus contact residues in inhibitor–protease complexes. The two filled circles shown in grey color represent 2.2. Construction and expression of variants the residues which in addition to being the contact residues also play a structural role. Site-directed mutagenesis was carried out to introduce amino acid substitutions in the recombinant OMTKY3. For the variant S13D14Y15, the plasmid of variant Y15 was used as template, and An important assumption in our proposal of SRA was the addi- the following primers were used to create the indicated changes: tivity of DG0 values when substitutions at the contact positions of S13D14Y15-forward primer: 50-GAC TGT AGT GAG TAC CCT AGC OMTKY3 are made. In principle, a substitution at an inhibitor con- GAT TAC TGC ACG CTG-30;S13D14Y15-reverse primer: 50-CAG CGT tact position is additive if that position is independent of other GCA GTA ATC GCT AGG GTA CTC ACT ACA GTC-30. The variant positions in the inhibitor and it also does not produce alterations plasmid could be easily distinguished from the parental plasmid through protease contact residues [16,17]. Thus, the additivity de- by the digestion with Pst I. For the mutant S13D14Y15G18I19K21, pends both on the contact position of the inhibitor as well as on the the plasmid of the variant S13D14Y15 was further used as template, serine protease being investigated. We presented extensive (400) and the following primers were used: S13D14Y15G18I19K21-forward additivity tests in our SRA paper [15]. These tests were based on primer: 50-C TGC ACG GGG ATC TAC AAA CCT CTC TGT GGA natural ovomucoid third domains that differed from OMTKY3 at TC-30;S13D14Y15G18I19K21-reverse primer: 50-GA TCC ACA GAG two or more contact positions [6,14]. Since that time we have per- AGG TTT GTA GAT CCC CGT GCA G-30. formed many more additivity tests [18]. The general consensus in For the variant T13E14Y15, the plasmid of variant Y15 was used as all additivity tests is that most contact positions, with the excep- template, and the following primers were used to create the indi- 0 13 14 15 0 tion of the contact positions P2 and P1, are additive with the six ser- cated changes: T E Y -forward primer: 5 -GAC TGT AGT GAG ine proteases that we have used [15,16,19,20]. The two important TAC CCT ACG GAG TAT TGC ACG CTG-30;T13E14Y15-reverse primer: applications of additivity-based SRA are: (i) the prediction, with 50-CAG CGT GCA ATA CTC CGT AGG GTA CTC ACT ACA GTC-30. The few restrictions, of the free energy of association of any Kazal variant plasmid could also be easily distinguished from the inhibitor of known protein or gene sequence with any of the six parental plasmid by the digestion with Pst I. For the variant serine proteases we have used, and (ii) the design of strong, spe- T13E14Y15G18M21, the plasmid of the variant T13E14Y15 was further cific, or non-specific inhibitors for the six serine proteases. used as template, and the following primers were used: T13E14 Structure based design of strong and specific drugs and ligands Y15G18M21-forward primer: 50-G TAT TGC ACG GGG GAA TAC for target proteins is an area of great academic and practical inter- ATG CCT CTC TG-30;T13E14Y15G18M21-reverse primer: 50-CA GAG est [21–23].