Radim Vicik

Aziridine-2,3-Dicarboxylates, Peptidomimetic Cysteine Protease Inhibitors with Antileishmanial Activity

ABSTRACT 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.

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 (Ki=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.

Nonpeptidic Vinyl and Allyl Phosphonates as Falcipain-2 Inhibitors

Malaria remains one of the most deadly parasitic diseases, affecting 500 million people all over the world and causing more than one million deaths each year. The limitations of antimalarial chemotherapy underscore the urgent need to discover new drugs that are able to interact with new targets. Research efforts are currently focused on the design of inhibitors of malarial proteases, among which falcipain-2 (FP-2) plays a key role. FP-2 is a papain-family cysteine protease of the most virulent species of the malaria-causing parasite Plasmodium falciparum, and is required by mature schizonts for the cleavage of erythrocytic cytoskeletal proteins and by intraerythrocytic trophozoites for hemoglobin degradation, which provides free amino acids for parasite protein synthesis. Thus, selective and irreversible inhibition of FP-2 would be advantageous for the control and elimination of the parasite. Peptides and peptidomimetics containing an activated double bond, such as vinyl sulfones and vinyl esters, have been shown to be highly potent irreversible cysteine protease inhibitors. Whereas the former are active on papain-like enzymes, the latter are known in particular as inhibitors of viral proteases. Both inhibitor types interact with the target enzyme by forming a covalent bond with the thiol group of the active site cysteine. Peptidyl vinyl sulfones are stable and unreactive toward nucleophiles, and require the “catalytic machinery” of cysteine proteases for their activation. Peptidyl vinyl sulfones containing a homoPhe residue at the P1 site have been proven to be highly specific FP-2 inhibitors, with the aromatic side chain being a key structural requirement for greater selectivity toward the target enzyme. In this context we recently reported a new class of peptidomimetic FP-2 inhibitors based on a rigid benzodiazepine scaffold as a conformationally constrained form of the d-Ser-Gly fragment and on a terminal electrophilic vinyl sulfone moiety on the P1 site that reacts as classical Michael acceptor (such as compounds 1a–d, Figure 1). All the synthesized compounds showed a high level of inhibitory potency and are quite selective toward FP-2, as they weakly inhibit human cysteine proteases cathepsin B and L. In particular, compound 1b displayed potent enzymatic inhibition (k2=307000m min ) coupled with a good activity against cultured P. falciparum (IC50= 9.1 mm). Among other irreversible cysteine protease inhibitors containing a vinyl moiety conjugated to electron withdrawing groups (EWGs), vinyl phosphonates showed good inhibitory activity against cultured P. falciparum. On this basis, we designed and synthesized nonpeptidic unsaturated phosphonate structures 2 and 3 (Scheme 1) to evaluate their ability to inhibit FP-2 and to make a head-to-head

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 (IC50 falcipain‐2: 0.079–5.4 μM, falcipain‐3: 0.25–39.8 μM). 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.

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

Abstract The coronavirus main protease, Mpro, 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 Mpro 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.

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.

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

Abstract 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, nonradioactive reagents that selectively target enzymes involved in pathogenicity of the parasite.

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

Abstract 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.