Development of peptidomimetics with a vinyl sulfone warhead as irreversible falcipain-2 inhibitors.

This paper describes the synthesis of a new class of peptidomimetic cysteine protease inhibitors based on a 1,4-benzodiazepine scaffold and on an electrophilic vinyl sulfone moiety. The former was introduced internally to a peptide sequence that mimics the fragment D-Ser-Gly; the latter was built on the P1-P1' site and reacts as a classical "Michael acceptor", leading to an alkylated enzyme by irreversible addition of the thiol group of the active site cysteine. The introduction of the vinyl sulfone moiety has been accomplished by olefin cross-metathesis, a powerful tool for the formation of carbon-carbon double bonds. New compounds 2-3 have been proven to be potent and selective inhibitors of falcipain-2, a cysteine protease isolated from Plasmodium falciparum, displaying antiplasmodial activity.

Screening of protease inhibitors as antiplasmodial agents. Part I: Aziridines and epoxides.

A broad protease-based and cell-based screening of protease inhibitors yielded the aziridine-2-carboxylic acid derivative 2 a and the N-acylated aziridine-2,3-dicarboxylic acid derivatives 32 a and 34 b as the most potent inhibitors of falcipain-2 and falcipain-3 (IC(50) falcipain-2: 0.079-5.4 microM, falcipain-3: 0.25-39.8 microM). As the compounds also display in vitro activity against the P. falciparum parasite in the submicromolar and low micromolar range, these compound classes are leads for new antiplasmodial falcipain inhibitors.

Rational design of aziridine-containing cysteine protease inhibitors with improved potency: studies on inhibition mechanism.

To enable a rational design of improved cysteine protease inhibitors, the present work investigates trends in the inhibition potency of aziridine derivatives with a substituted nitrogen center. To predict the influence of electron-withdrawing substituents, quantum chemical computations of the ring opening of N-formylated, N-methylated, and N-unsubstituted aziridines with thiolate were performed. They revealed that the N-formyl group leads to a strong decrease of the reaction barrier and a considerable increase in exothermicity due to stabilization of the transition state. In contrast, a nucleophilic attack at the carbonyl carbon atom is characterized by very low reaction barriers, suggesting a reversible reaction, thus providing the theoretical background for the reversible inhibition of cysteine proteases by peptidyl aldehydes. Reactions of aziridine building blocks (diethyl aziridine-2,3-dicarboxylate 1, diethyl 1-formyl aziridine-2,3-dicarboxylate 2) with a model thiolate in aqueous solution which were followed by NMR spectroscopy and mass spectrometry, showed the N-formylated compound 2 to readily undergo a ring-opening reaction. In contrast, the reaction of 1 with the thiolate is much slower. Enzyme assays with the cysteine protease cathepsin L showed 2 to be a 5000-fold better enzyme inhibitor than 1. Dialysis assays clearly proved irreversible inhibition. These experiments, together with the results obtained with the model thiolate, indicate that the main inhibition mechanism of the N-formylated aziridine 2 is the ring-opening reaction rather than the reversible attack of the active site cysteine residue at the carbonyl carbon atom.

Aziridide-based inhibitors of cathepsin L: synthesis, inhibition activity, and docking studies.

A comprehensive screening of N-acylated aziridine (aziridide) based cysteine protease inhibitors containing either Boc-Leu-Caa (Caa=cyclic amino acid), Boc-Gly-Caa, or Boc-Phe-Ala attached to the aziridine nitrogen atom revealed Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)(2) (18 a) as a highly potent cathepsin L (CL) inhibitor (K(i)=13 nM) (Azy=aziridine-2-carboxylate, Azi=aziridine-2,3-dicarboxylate). Docking studies, which also accounted for the unusual bonding situations (the flexibility and hybridization of the aziridides) predict that the inhibitor adopts a Y shape and spans across the entire active site cleft, binding into both the nonprimed and primed sites of CL.

Aziridine-2,3-dicarboxylates, peptidomimetic cysteine protease inhibitors with antileishmanial activity.

Chemotherapy of leishmaniasis is mainly based on antimonials. However, they are extremely toxic and cause serious side effects, and there is a worldwide increasing frequency of chemoresistance to antimonials. These issues emphasize the urgent need for affordable alternative drugs against leishmaniasis. Leishmania cysteine proteases are essential for parasite growth, differentiation, pathogenicity, and virulence and are thus attractive targets for combating leishmaniasis. Herein we demonstrate that the cysteine protease inhibitors aziridine-2,3-dicarboxylates 13b and 13e impaired promastigote growth at mid-micromolar concentrations and decreased the infection rate of peritoneal macrophages at concentrations 8- to 13-fold lower than those needed to inhibit parasite replication. Simultaneous treatment of infected cells with compound 13b and gamma interferon resulted in an even further reduction of the concentration needed for a significant decrease in macrophage infection rate. Notably, treatment with the compounds alone modulated the cytokine secretion of infected macrophages, with increased levels of interleukin-12 and tumor necrosis factor alpha. Furthermore, the decreased infection rate in the presence of compound 13b correlated with increased nitric oxide production by macrophages. Importantly, at the concentrations used herein, compounds 13b and 13e were not toxic against fibroblasts, macrophages, or dendritic cells. Together, these results suggest that the aziridine-2,3-dicarboxylates 13b and 13e are potential antileishmanial lead compounds with low toxicity against host cells and selective antiparasitic effects.

Inhibitors of cysteine proteases.

The roles of cysteine proteases (CP) as protein degrading and protein processing enzymes both in physiological and pathological processes of mammals are well known. Furthermore, the key roles of CP;s in the life cycles of infectious agents like protozoa and viruses turn them into new important targets for anti-infective drugs. Thus, the effective inhibition of pathologically relevant cysteine proteases has raised increasing interest in drug development. One strategy to create CP inhibitors is the use of electrophilic moieties, which covalently bind to the cysteine residue of the active site of the target protease. In a previous approach we have selected the aziridine-2,3-dicarboxylic acid as weak electrophilic inhibitor fragment. In order to achieve effective enzyme inhibition this electrophile was incorporated into peptidic or peptidomimetic sequences addressing the substrate binding sites of the protease. High selectivity could be obtained with compounds, which bind into both the primed and non-primed substrate binding pockets. In a second approach the alpha,beta-unsaturated ketone of the well-known diuretic drug ethacrynic acid was found to be another appropriate electrophilic moiety. Derivatives thereof turned out to be new non-peptidic CP inhibitors. Results of inhibition assays obtained with these two inhibitor series on various proteases of human, protozoan, and viral origin, theoretical studies to investigate binding modes and inhibition mechanisms, and structure-activity relationships are presented. Furthermore, the results of in vitro assays on respective pathogens as well as the results of first toxicity studies are summarized.

Aziridine-2,3-dicarboxylate inhibitors targeting the major cysteine protease of Trypanosoma brucei as lead trypanocidal agents.

The protozoan parasite Trypanosoma brucei causes Human African trypanosomiasis, which is fatal if left untreated. Due to the toxicity of currently used drugs and emerging drug resistance, there is an urgent need for novel therapies. The major trypanosome papain-like cysteine protease expressed by the parasite (e.g., rhodesain in T. b. rhodesiense) is considered an important target for the development of new trypanocidal drugs. Series of aziridine-2,3-dicarboxylate-based cysteine protease inhibitors have been tested, most of them inhibiting rhodesain in the low micromolar range. Among these, only dibenzyl aziridine-2,3-dicarboxylates display trypanocidal activity being equipotent to the drug eflornithine. The Leu-Pro-containing aziridinyl tripeptides 13a-f are the most promising as they are not cytotoxic to macrophages up to concentrations of 125microM.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, M(pro), is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential M(pro) inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed M(pro) inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a K(i) value of 35.3 muM. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor.

The coronavirus main protease, M(pro), is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compounds were performed to identify potential M(pro) inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-containing peptide 2c was found to be the most potent inhibitor.

Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells.

The malaria parasite Plasmodium falciparum invades human red blood cells. Before infecting new erythrocytes, the merozoites have to exit their host cell to get into the blood plasma. Knowledge about the mechanism of egress is scarce, but it is thought that proteases are basically involved in this step. We have introduced a biotinylated dibenzyl aziridine-2,3-dicarboxylate (bADA) as an irreversible cysteine protease inhibitor to study the mechanism of merozoite release and to identify the proteases involved. The compound acts on parasite proteins in the digestive vacuole and in the host cell cytosol, as judged by fluorescence microscopy. The inhibitor blocks rupture of the host cell membrane, leading to clustered merozoite structures, as evidenced by immunoelectron microscopy. Interestingly, bADA did not prevent rupture of the parasitophorous vacuole membrane (PVM) that surrounds the parasite during the period of intraerythrocytic maturation. The compound appears to be a valuable template for the development of inhibitors specific for individual plasmodial proteases, which would be useful tools to dissect the molecular mechanisms underlying the process of merozoite release and consequently to develop potent antimalarial drugs.

Synthesis and antiplasmodial activity of a cysteine protease-inhibiting biotinylated aziridine-2,3-dicarboxylate.

Cysteine proteases have been implicated in a variety of processes essential for the survival and progression of the malarial parasite Plasmodium falciparum. Here, we synthesized a cysteine protease inhibitor that contains the electrophilic aziridine-2,3-dicarboxylic acid as the reactive agent and biotin as a targeting label. Diethyl ester and dibenzyl ester derivatives of the inhibitor were active against cathepsin L and the plasmodial protease falcipain 2, but only the latter displayed potent antiplasmodial activity against viable parasites. The morphological changes observed during the intraerythrocytic life stages of Plasmodium suggest that degradation of hemoglobin of the host cell is seriously affected, eventually leading to growth arrest and cell death of the parasites. After incubation of infected erythrocytes with the compound plasmodial proteins were captured, with the biotinyl group of the inhibitor serving as an affinity tag. Among these the cysteine proteases falcipain 2 and falcipain 3 were identified as potential target proteins of the compound as evidenced by tandem mass spectrometry. Apparently, the compound gets access to intracellular compartments and therein targets plasmodial cysteine proteases. Accordingly, the reagent described here appears to be a valuable template to develop cell-permeable, non-radioactive reagents that selectively target enzymes involved in pathogenicity of the parasite.