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The protease prostate-specific antigen (PSA) is a marker widely used clinically for monitoring prostatic malignancies. Under normal conditions, this enzyme is mainly involved in the post ejaculation degradation of the major human seminal protein, the seminal plasma motility inhibitor precursor/semenogelin I (SPMIP/SgI), which is the predominant protein component of human semen coagulum. PSA primary structure and activity on synthetic substrates predict a chymotrypsin-like activity whose specificity remains to be established. The present study was aimed at characterizing the proteolytic processing of the SPMIP/SgI by PSA. Purified SPMIP/SgI was incubated with PSA in the presence or absence of protease inhibitors. General serine protease inhibitors, heavy metal cations (Zn2+ and Hg2+), and the heavy metal chelator 1,10-phenanthroline partially or totally inhibited the proteolytic activity of PSA toward SPMIP/SgI. Under identical conditions, other proteins, such as bovine serum albumin, ovalbumin, and casein, were very poor substrates for PSA. Hydrolysis products were separated by reverse-phase high-performance liquid chromatography, assayed for sperm motility inhibitory activity, and analyzed by immunoblotting and mass spectrometry. The region responsible for the sperm motility inhibitory activity and containing an SPMI antiserum epitope was localized to the N-terminal portion of the molecule between residues 85 and 136. On the other hand, a monoclonal antibody against a seminal vesicle-specific antigen (MHS-5) recognized fragments derived from the central part of the SPMIP/SgI (residues 198-223). PSA hydrolysis occurred almost exclusively at either leucine or tyrosine residues, demonstrating directly for the first time a restricted chymotrypsin-like activity on a physiological substrate. The results suggest that PSA is the main enzyme responsible for the processing of SPMIP/SgI in human semen and that this protease manifests unusual specificity with respect to hydrolyzable substrates and sites of hydrolysis.

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K(i) value of 2.2 x 10(-14) M. It consists of an active site blocking moiety, hirudin1-48, a fibrinogen-recognition exo-site binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by omega-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin55-65 was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human alpha-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (K(i)) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K(i) = 10(-9)-10(-10) M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990). The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds. Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 1.7-3.4 nM. The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker. The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition. Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 0.51 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length. Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM). Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

A new type of thrombin exo-site inhibitor has been designed with enhanced inhibitory potency and increased metabolic stability. With the aid of the model of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio, J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5, 77-85], cyclic analogs of the hirudin fragment (hirudin55-65) were designed and synthesized. In these analogs, the side chains of appropriately substituted residues, 58 and 61, were joined in order to restrict the conformation of the inhibitor. An analog with an 18-membered lactam ring showed higher antithrombin activity (IC50 = 0.57 microM) than the corresponding analogs with 17- or 16-membered rings and was 2-fold more potent than its linear counterpart. Even 4-fold greater enhancement was obtained when a shorter fragment, hirudin 55-62, was cyclized. This cyclization not only improved the potency but, more importantly, dramatically increased the resistance to proteolytic digestion. Remarkable enhancement of stability to proteolysis was observed for peptide bonds located in the exocyclic linear peptide segments. These results are discussed using molecular modeling.