Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK).

Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity.

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.

Isomannide-based peptidomimetics as inhibitors for human tissue kallikreins 5 and 7.

Human kallikrein 5 (KLK5) and 7 (KLK7) are potential targets for the treatment of skin inflammation and cancer. Previously, we identified isomannide derivatives as potent and competitive KLK7 inhibitors. The introduction of N-protected amino acids into the isomannide-based scaffold was studied. Some KLK5 inhibitors with submicromolar affinity (K i values of 0.3-0.7 µM) were identified, and they were 6- to 13-fold more potent than our previous hits. Enzyme kinetics studies and the determination of the mechanism of inhibition confirmed that the new isomannide-based derivatives are competitive inhibitors of both KLK5 and KLK7. Molecular docking and MD simulations of selected inhibitors into the KLK5 binding site provide insight into the molecular mechanism by which these compounds interact with the enzyme. The promising results obtained in this study open new prospects on the design and synthesis of highly specific KLK5 and KLK7 inhibitors.

Human tissue kallikreins 3 and 5 can act as plasminogen activator releasing active plasmin.

Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated.

Isomannide derivatives as new class of inhibitors for human kallikrein 7.

Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. Of the inhibitors tested, two inhibited KLK7 with K(i) values in the low micromolar range (9g=1.8µM; 9j=3.0µM). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB)SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. The promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors.

Biological evaluation and docking studies of natural isocoumarins as inhibitors for human kallikrein 5 and 7.

Human kallikrein 5 and 7 (KLK5 and KLK7) are trypsin-like and chymotrypsin-like serine proteases, respectively, and promising targets for the treatment of skin desquamation, inflammation and cancer. In an effort to develop new inhibitors for these enzymes, we carried out enzymatic inhibition assays and docking studies with three isocoumarin compounds. Some promising inhibitors were uncovered, with vioxanthin and 8,8'-paepalantine being the most potent competitive inhibitors of KLK5 (K(i)=22.9 µM) and KLK7 (K(i)=12.2 µM), respectively. Our docking studies showed a good correlation with the experimental results, and revealed a distinct binding mode for the inhibitors at the binding sites of KLK5 and KLK7. In addition, the docking results suggested that the formation of hydrogen bonds at the oxyanion hole is essential for a good inhibitor.

Substrate specificity of kallikrein-related peptidase 13 activated by salts or glycosaminoglycans and a search for natural substrate candidates.

KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and its substrate specificity by employing a series of five substrates derived from the fluorescence resonance energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic residues with highest efficiency for R; furthermore, the S(3) to S(2)' subsites accepted most of the natural amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight peptide substrates were identified containing sequences of proteins found in testis and one with myelin basic protein sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides present in higher primates with broad antifungal and mucosal healing activities that are generated from the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclusively at R(25) (DSHAKRHHGYKRKFHEKHHSHRGYR(25)↓SNYLYDN) that is the first cleavage observed inside the salivary gland. In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs.

Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans.

We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY downward arrowIHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.

S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen.

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.

Substrate specificity of human kallikrein 6: salt and glycosaminoglycan activation effects.

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.