Cysteine Protease Cruzain Research Articles

Antitrypanosomal and cysteine protease inhibitory activities of alkyldiamine cryptolepine derivatives

Cryptolepine derivatives containing alkyldiamine side-chains, 2, with potent inhibitory activity against Trypanosoma brucei brucei are reported. Compounds 2 showed improved activity and selectivity to T. b. brucei when compared to the lead compound. The most selective compound, 2k, presents a selectivity index value of 6200 and an IC50 of 10nM against the parasite. These derivatives are also potent inhibitors of the trypanosome papain-like cysteine proteases cruzain, which could, at least in part, explain their antitrypanosomal activity. Overall, these compounds with good antitrypanosomal activity and selectivity provide an encouraging starting point for the rational design of new and effective antitrypanosomal agents.

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 kobs/[I] of 6080 ± 1390 M−1s−1.

The Trypanosoma cruzi protease cruzain mediates immune evasion.

Trypanosoma cruzi is the causative agent of Chagas' disease. Novel chemotherapy with the drug K11777 targets the major cysteine protease cruzain and disrupts amastigote intracellular development. Nevertheless, the biological role of the protease in infection and pathogenesis remains unclear as cruzain gene knockout failed due to genetic redundancy. A role for the T. cruzi cysteine protease cruzain in immune evasion was elucidated in a comparative study of parental wild type- and cruzain-deficient parasites. Wild type T. cruzi did not activate host macrophages during early infection (<60 min) and no increase in ∼P iκB was detected. The signaling factor NF-κB P65 colocalized with cruzain on the cell surface of intracellular wild type parasites, and was proteolytically cleaved. No significant IL-12 expression occurred in macrophages infected with wild type T. cruzi and treated with LPS and BFA, confirming impairment of macrophage activation pathways. In contrast, cruzain-deficient parasites induced macrophage activation, detectable iκB phosphorylation, and nuclear NF-κB P65 localization. These parasites were unable to develop intracellularly and survive within macrophages. IL 12 expression levels in macrophages infected with cruzain-deficient T. cruzi were comparable to LPS activated controls. Thus cruzain hinders macrophage activation during the early (<60 min) stages of infection, by interruption of the NF-κB P65 mediated signaling pathway. These early events allow T. cruzi survival and replication, and may lead to the spread of infection in acute Chagas' disease.

Quantitative Analyses of Aggregation, Autofluorescence, and Reactivity Artifacts in a Screen for Inhibitors of a Thiol Protease

The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.

Design, synthesis and cruzain docking of 3-(4-substituted-aryl)-1,2,4-oxadiazole- N-acylhydrazones as anti- Trypanosoma cruzi agents

Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a valid chemotherapeutic target, since inhibitors of this protease affect the pathology appropriately. By exploring the N-acylhydrazones (NAH) as privileged structures usually present in antiparasitic agents, we investigated a library of 16 NAH bearing the 3-(4-substituted-aryl)-1,2,4-oxadiazole scaffold (NAH 3a– h, 4a– h). The in vitro bioactivity against epimastigote and trypomastigote forms of T. cruzi was evaluated, and some NAH under study exhibited antitrypanosomal activity at concentrations that are not toxic to mammalian cells. The series of compounds based on the 3-(4-substituted-aryl)-1,2,4-oxadiazole scaffold revealed the remarkable importance of each substituent at the phenyl’s 4-position for the inhibitory activity. Non-nitrated compounds 3a and 4e were found to be as potent as the reference drug, Benznidazole. In addition, the molecular origin of the antitrypanosomal properties for these series was investigated using docking studies of the TCC structure.

Divergent Modes of Enzyme Inhibition in a Homologous Structure−Activity Series

A docking screen identified reversible, noncovalent inhibitors (e.g., 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. Detailed investigation of the SAR series subsequently revealed that many members of the oxadiazole class (and surprisingly also 1) act via divergent modes of inhibition (competitive or via colloidal aggregation) depending on the assay conditions employed.

Approaches for the Development of New Anti-Trypanosoma cruzi Agents

The recent highlights on the biochemical pathways of Trypanosoma cruzi have allowed a significant improvement in the development of new strategies for drug design and also in the understanding of the mechanisms of action of new trypanocidal agents. Several biochemical pathways of fundamental importance and validated targets (e.g. cysteine protease cruzain, trypanothione reductase, trans-sialidase) of T. cruzi have proved usefulness for drug development in many examples of new candidates to anti-T. cruzi drugs. This review will focus on some approaches used for the design of new potential trypanocidal agents, exploring modern concepts of medicinal chemistry such as bioisosterism, molecular hybridization, bioinspired design in lead compounds, as well as the complexation of transition metals with bioactive ligands. The examples discussed in this article may serve as lessons for the antitrypanosomal drug design.

Chemical Compositions and Biological Activities of Leaf Essential Oils of Twelve Species of Piper from Monteverde, Costa Rica

The leaf essential oils of twelve species of Piper (Piperaceae) from Monteverde, Costa Rica ( Piper aequale, Piper amalago, Piper biasperatum, Piper bredemeyeri, Piper dotanum, Piper fimbriulatum, Piper glabrescens, Piper imperiale, Piper lanceifolium, Piper nemorense, Piper oblanceolatum, and Piper sp. A near aereum) have been obtained by hydrodistillation and analyzed by GC-MS. The Piper leaf oils have been screened for antibacterial activity against Bacillus cereus, Staphylococcus aureus, and Escherichia coli; for Artemia salina (brine shrimp) lethality; for in-vitro cytotoxic activity against MCF-7 (human breast tumor) cells; and inhibition of the cysteine protease cruzain. A cluster analysis comparison with previously published Piper leaf oil compositions was carried out in order to discern the differences and similarities between the volatile chemical compositions of Piper species.

Cruzain Inhibition by Terpenoids: A Molecular Docking Analysis

A molecular docking analysis has been carried out using monoterpene and sesquiterpene hydrocarbons and triterpenoids that have shown enzyme inhibitory activity as ligands for the cysteine protease cruzain. The binding energies of the docked ligands roughly correlate with their inhibitory activities. The orientations of the docked ligands are consistent with a mechanism whereby these hydrophobic compounds dock into a hydrophobic pocket near the active site, thereby blocking binding of the protein target to the protease.

Cruzain Inhibitory Activity of Leaf Essential Oils of Neotropical Lauraceae and Essential Oil Components

The leaf essential oils of twenty-three species of Lauraceae from Monteverde, Costa Rica, have been screened for inhibition of the cysteine protease cruzain. Of these, nine showed promising cruzain inhibitory activity (IC50 &lt; 100 μg/mL), six showed marginal activity (IC50, 100–500 μg/mL), and eight were inactive (IC50 &gt; 500 μg/mL). The cruzain inhibitory activities of the essential oils can be attributed to active sesquiterpenoid components as well as synergistic effects between two or more components. The sesquiterpenes α-copaene, β-caryophyllene, α-humulene, and germacrene D are active (IC50 ~5–30 μg/mL) alone, but also show increased activity in combination with other essential oil components.

Dipeptidyl-α,β-epoxyesters as potent irreversible inhibitors of the cysteine proteases cruzain and rhodesain

The dipeptidyl epoxyesters 3 and 4 are potent, irreversible inhibitors of cruzain and rhodesain.

3-O-(3′-Hydroxytetradecanoyl)lupeol from Sorocea trophoides Inhibits Cruzain

The crude chloroform extract from the stem bark of Sorocea trophoides (Moraceae) showed in-vitro inhibition of the cysteine protease cruzain. Activity-directed fractionation led to isolation of the novel 3- O-(3′-hydroxytetradecanoyl)lupeol as the inhibitory agent. The structure was elucidated by analysis of NMR spectra.

Synthesis, Cruzain Docking, and in vitro Studies of Aryl‐4‐Oxothiazolylhydrazones Against Trypanosoma cruzi

Research in recent years has demonstrated that the Trypanosoma cruzi cysteine protease cruzain (TCC) is a valid chemotherapeutic target. Herein we describe a small library of aryl-4-oxothiazolylhydrazones that have been tested in assays against T. cruzi cell cultures. The docking studies carried out suggest that these compounds are potential ligands for the TCC enzyme. The most promising compound of this series, N-(4-oxo-5-ethyl-2'-thiazolin-2-yl)-N'-phenylthio-(Z)-ethylidenehydrazone (6 f), was shown to be very active at non-cytotoxic concentrations in in vitro assays with mammalian cells and has a potency comparable with reference drugs such as nifurtimox (Nfx) and benznidazole (Bdz).

Chemical Composition and Cruzain Inhibitory Activity of Croton draco Bark Essential Oil from Monteverde, Costa Rica

American trypanosomiasis, Chagas disease, is a great cause of human morbidity and mortality in the Neotropics. Although there is currently no effective treatment for this parasitic disease, a number of potential biochemical targets have been identified, including the cysteine protease cruzain. Croton draco Cham. &amp; Schldl. (Euphorbiaceae), commonly known as sangre de drago, is used in traditional medicine for a number of maladies. In this study, Croton draco bark essential oil has been shown to inhibit the activity of cruzain. The bark oil has been analyzed by GC-MS and the major components found to be β-caryophyllene (31.9%), caryophyllene oxide (22.0%), 1,8-cineole (6.2%), and α-humulene (5.6%). The major components have been tested for cruzain inhibitory activity, but show minimal activity, so it is not clear if the activity of Croton draco bark oil is due to a synergistic effect of the essential oil components or due to very active minor components. Croton draco bark oil has also been tested for cytotoxic activity against a panel of human tumor cell lines, but shows little activity.

Modeling of the Inhibition Constant (Ki) of Some Cruzain Ketone‐Based Inhibitors Using 2D Spatial Autocorrelation Vectors and Data‐Diverse Ensembles of Bayesian‐Regularized Genetic Neural Networks

AbstractThe inhibition constant (Ki) of a set of 46 ketone‐based cruzain inhibitors against cysteine protease cruzain was successfully modeled by means of data‐diverse ensembles of Bayesian‐regularized genetic neural networks. 2D spatial autocorrelation vectors were used for encoding structural information yielding a nonlinear model describing about 90 and 75% of ensemble training and test set variances, respectively. From the results of a ranking analysis of the neural network inputs, it was derived that atomic van der Waals volume distributions at topological lags 3, 5, and 6 in the 2D topological structure of the inhibitors have a high nonlinear influence on the inhibition constants. Furthermore, optimum subset of autocorrelation vectors well mapped the studied compounds according to their inhibition constant values in a Kohonen self‐organizing map.

Crystal structures of reversible ketone-Based inhibitors of the cysteine protease cruzain

The crystal structures of two hydroxymethyl ketone inhibitors complexed to the cysteine protease cruzain have been determined at 1.1 and 1.2 Å resolution, respectively. These high resolution crystal structures provide the first structures of non-covalent inhibitors bound to cruzain. A series of compounds were prepared and tested based upon the structures providing further insight into the key binding interactions.

General solid-phase method to prepare novel cyclic ketone inhibitors of the cysteine protease cruzain

A series of constrained ketone-based inhibitors has been developed that show low nanomolar Ki values. These ketone inhibitors showed promising activity towards cruzain, the cysteine protease implicated in Chagas' disease. This series of constrained inhibitors, which can be accessed quickly and efficiently using a solid-phase combinatorial strategy, should be applicable to other members of the cysteine protease class.

Synthesis and structure-activity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain.

American trypanosomiasis, or Chagas' disease, is the leading cause of heart disease in Latin America. Currently there is an urgent need to develop antitrypanosomal therapy due to the toxicity of existing agents and emerging drug resistance. A novel series of potent thio semicarbazone small-molecule inhibitors of the Trypanosoma cruzi cysteine protease cruzain have been identified. Some of these inhibitors have been shown to be trypanocidal. We initially discovered that 3'-bromopropiophenone thio semicarbazone (1i) inhibited cruzain and could cure mammalian cell cultures infected with T. cruzi. 3'-Bromopropiophenone thio semicarbazone showed no toxicity for mammalian cells at concentrations that were trypanocidal. Following this lead, more than 100 compounds were designed and synthesized. A specific structure-activity relationship (SAR) was established, and many potent analogues with IC(50) values in the low nanomolar range were identified. Eight additional analogues were trypanocidal in a cell culture assay, and this indicates that aryl thio semicarbazone is a productive scaffold for killing the parasites. Kinetic studies show that these are time-dependent inhibitors. Molecular modeling studies of the enzyme-inhibitor complex have led to a proposed mechanism of interaction as well as insight into the SAR of the thio semicarbazone series. The nonpeptide nature of this series, small size, and extremely low cost of production suggest this is a promising direction for the development of new antitrypanosome chemotherapy.

Identification of potent and selective mechanism-based inhibitors of the cysteine protease cruzain using solid-phase parallel synthesis.

Targeted libraries of ketone-based cysteine protease inhibitors were synthesized and screened against cruzain, a cysteine protease implicated in Chagas' disease. A number of single digit nanomolar, low molecular weight inhibitors were identified and optimized for solubility and potency. Specifically, the best inhibitors identified have K(i) values of 0.9-10 nM and molecular weights between 499 and 609 Da. The most effective inhibitor was also found to be greater than 1000-fold selective for cruzain relative to cathepsin B and 100-fold selective for cruzain relative to cathepsin L.

Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain

A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000 s −1 M −1 versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.