Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors

This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA cysteine protease inhibitors. The structure of the inhibitors consists of a R₁-Phe-R₂-AS-Ph scaffold (AS?=?allyl sulfone). R₁ was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R₂ was varied with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine proteases cathepsin B and calpain I with a k_obs/[I] of 6080?±?1390?M^?1s^?1.     

Aryl sulfones: a new class of gamma-secretase inhibitors

The development of a novel series of 4-aryl, 4-phenylsulfonyl cyclohexananone-derived gamma-secretase inhibitors for the potential treatment of Alzheimer's disease is described.     

3-Acylamino-azetidin-2-one as a novel class of cysteine proteases inhibitors

A new class of inhibitors for cysteine proteases cathepsin B, L, K and S is described. These inhibitors are based on the beta-lactam ring designed to interact with the nucleophilic thiol of the cysteine in the active site of cysteine proteases. Some 3-acylamino-azetidin-2-one derivatives showed very potent inhibition activities for cathepsins L, K and S at the nanomolar or subnanomolar IC(50) values.     

Peptidyl sulfonium salts. A new class of protease inhibitors

The possibility has been examined that peptidylmethyl sulfonium salts might affinity label proteases by an alkyl transfer from sulfur to an active center residue. The synthesis of a number of agents of this type is described as well as initial results of their effect on cysteinyl proteases, papain and cathepsin B. These are readily inactivated by reagents in which the peptidyl portion contains features that promote binding to the proteases such as a penultimate phenylalanine residue. Irreversible inactivation ensues by transfer of the peptidyl portion, not methyl groups. Peptidylmethyl sulfonium salts lose a proton to form an ylide structure which may be the prevalent form at physiological pH values. The ylide may also be the active affinity labeling form of the reagent since the rate of inactivation of cathepsin B increases with pH. In contrast, the action of another affinity labeling reagent for cathepsin B, benzyloxycarbonyl-Phe-AlaCHN2, a diazomethyl ketone, is relatively independent of pH.     

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL^pro and PL^pro, viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1–7) act as time dependent inhibitors of PL^pro, but no improved inhibition was observed following preincubation with the 3CL^pro. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC₅₀ = 0.7 μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.     

Fighting an enemy within: cytoplasmic inhibitors of bacterial cysteine proteases

The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases.     

Structural studies of cysteine proteases and their inhibitors.

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.     

Otubains: a new family of cysteine proteases in the ubiquitin pathway

The modification of cellular proteins by ubiquitin (Ub) is an important event that underlies protein stability and function in eukaryotes. Protein ubiquitylation is a dynamic and reversible process; attached Ub can be removed by deubiquitylating enzymes (DUBs), a heterogeneous group of cysteine proteases that cleave proteins precisely at the Ub–protein bond. Two families of DUBs have been identified previously. Here, we describe new, highly specific Ub iso-peptidases, that have no sequence homology to known DUBs, but which belong to the OTU (ovarian tumour) superfamily of proteins. Two novel proteins were isolated from HeLa cells by affinity purification using the DUB-specific inhibitor, Ub aldehyde (Ubal). We have named these proteins otubain 1 and otubain 2, for OTU-domain Ubal-binding protein. Functional analysis of otubains shows that the OTU domain contains an active cysteine protease site.     

Defense against own arms: staphylococcal cysteine proteases and their inhibitors

Staphylococcus aureus is a human pathogen causing a wide range of diseases. Most staphylococcal infections, unlike those caused by other bacteria are not toxigenic and very little is known about their pathogenesis. It has been proposed that a core of secreted proteins common to many infectious strains is responsible for colonization and infection. Among those proteins several proteases are present and over the years many different functions in the infection process have been attributed to them. However, little direct, in vivo data has been presented. Two cysteine proteases, staphopain A (ScpA) and staphopain B (SspB) are important members of this group of enzymes. Recently, two cysteine protease inhibitors, staphostatin A and staphostatin B (ScpB and SspC, respectively) were described in S. aureus shedding new light on the complexity of the processes involving the two proteases. The scope of this review is to summarize current knowledge on the network of staphylococcal cysteine proteases and their inhibitors in view of their possible role as virulence factors.     

(S)-Thiirancarboxylic acid as a reactive building block for a new class of cysteine protease inhibitors

For (S)-thiirancarboxylic acid a second-order rate constant of k2nd = 222 M(-1) min(-1) for the irreversible inhibition of papain was determined. The ethyl and methyl ester do not inhibit the enzyme time-dependently. An improved synthesis of enantiomerically pure thiirancarboxylic acid is described. It is shown that thiirancarboxylates can be substrates for serine proteases (alpha-chymotrypsin) and esterases (pig liver esterase) and even for metallo proteases (thermolysin).     

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.     

Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.     

The Bsmoc group as a novel scaffold for the design of irreversible inhibitors of cysteine proteases

Carbamate and ester derivatives of the 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) scaffold react readily with thiols via a Michael addition at rates not significantly affected by the nature of the carboxylic or carbamic acid leaving group. These Michael acceptors are irreversible inhibitors of the cysteine proteases papain and human liver cathepsin B, displaying first-order kinetics with respect to inhibitor concentration. In contrast, none of the Bsmoc derivatives inhibited porcine pancreatic elastase, a serine protease.     

Tetraketones: a new class of tyrosinase inhibitors

Twenty-eight tetraketones (1-28) with variable substituents at C-7 were synthesized and evaluated as tyrosinase inhibitors. Remarkably compounds 25 (IC(50)=2.06 microM), 11 (IC(50)=2.09 microM), 15 (IC(50)=2.61 microM), and 27 (IC(50)=3.19 microM) were found to be the most active compounds of the series, even better than both standards kojic acid (IC(50)=16.67 microM) and L-mimosine (IC(50)=3.68 microM). This study may lead to the discovery of therapeutically potent agents against clinically very important dermatological disorders including hyperpigmentation as well as skin melanoma.     

Synthesis and evaluation of chloromethyl sulfoxides as a new class of selective irreversible cysteine protease inhibitors

The synthesis and biological evaluation of a new class of selective irreversible cysteine protease inhibitors is described. A set of amino acid based chloromethyl sulfoxides was prepared and they were found to inhibit irreversibly the cysteine protease papain. They were selective for cysteine proteases since no inhibition was found for the serine protease chymotrypsin.     

Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases

Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.     

Aromatic Tris-amidines. A new class of highly active inhibitors of trypsin-like proteases.

A number of novel aromatic Tris-amidines have been synthesized and investigated for their antiproteolytic property. The basic structure of the compounds is that of mesitylene where each of the methyl groups has been substituted with a 3- or 4-amidinophenoxy moiety. The compounds displayed considerable activity against trypsin (EC 3.4.21.4) and thrombin (EC 3.4.21.5), but proved most effective against porcine pancreatic kallikrein (EC 3.4.21.8). With this enzyme a Ki value of 2.43-10(-8) M was recorded for alpha,alpha',alpha'-tris(4-amidino-2-bromophenoxy)mesitylene at pH 8.1 and 37 degrees C. The most potent thrombin inhibitor, alpha,alpha',alpha'-tris(3-amidinophenoxy)mesitylene, had a Ki value of 6.51-10(-7) M and was also a strong overall anticoagulant. The inhibitors were able to interfere with the kinin release by human plasma kallikrein at concentrations as low as 1-10(-10) M. However, despite this remarkable antikallikrein effect and the known importance of plasma kallikrein in the activation of Hageman factor (factor XII), the compounds had only little influence on the early stages of blood coagulation.