Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2
Pengxuan RenChangyue YuRuxue ZhangTianqing NieQiaoyu HuHui LiXianglei ZhangXueyuan ZhangShiwei LiLu LiuWenhao DaiJian LiYechun XuHaixia SuLeike ZhangHong LiuFang Bai
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Peptidomimetic
Cysteine protease
Cysteine Proteinase Inhibitors
Abstract Insects are capable of readjusting their digestive regimes in response to dietary challenge. Cowpea bruchids ( Callosobruchus maculatus ) strongly induce C. maculatus cathepsin B‐like cysteine protease 1 ( CmCatB1 ) transcripts when fed diet containing a soybean cysteine protease inhibitor soyacystatin N (scN). CmCatB1 shares significant sequence similarity with cathepsin B‐like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1 , but lacking a 70‐amino‐acid internal section. CmCatB1 and CmCatB2 probably resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. Further, this expression was only detectable in larvae, which is the developmental stage associated with food ingestion. The scN‐activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter‐defence against antinutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH‐dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E‐64 and the cathepsin B‐specific inhibitor CA‐074, verifying its cathepsin B‐like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. Together, we have provided functional evidence suggesting that CmCatB1 confers inhibitor‐insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors.
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Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.
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Entamoeba
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ABSTRACT The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba . In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba .
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In comparison to the huge number of peptidic and peptidomimetic inhibitors of cysteine proteases which have been developed during the last twenty years the number of non-peptidic compounds with cysteine protease inhibiting properties is restricted to a few substance classes. In contrast to peptidic and peptidomimetic inhibitors the non-peptidic lead structures have mainly been discovered by computational or enzymatic industrial screenings and not by a rational approach. But, the growing number of resolved X-ray structures of the target enzymes as well as molecular modeling methods have supported the further development of potent inhibitors beginning from these lead structures. In this review we will focus on new non-peptidic cysteine protease inhibitors which have been developed during the last years. Discovery, structure-activity-relationship and inhibition mechanisms will be discussed.
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The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per monomer which are highly conserved among viral isolates. However, these cysteine residues are not essential for catalytic activity which raises the question of why they are conserved. We have found previously that these cysteine residues are unusually susceptible to oxidation by metal ions, and this results in inhibition of protease activity. Recombinant protease mutants (C67A, C95A, and the double mutant C67A,C95A) were prepared to assess the possible role of these cysteines in redox regulation of the enzyme. Mixed disulfides were formed between the cysteine residues of the enzymes and low molecular weight thiols. Enzyme activity was lost when a mixed disulfide was formed between 5,5'-dithiobis(2-nitrobenzoic acid) and cysteine 95, while the same mixed disulfide at cysteine 67 reduced activity by 50%. This effect was reversible as normal activity could be restored when the enzyme was treated with dithiothreitol. The cysteines could also be modified with the common cellular thiol glutathione. Modification with glutathione was verified by mass spectrometry of the protein peaks obtained from HPLC separation. Glutathiolation of cysteine 95 abolished activity whereas modification at cysteine 67 increased the k(cat) by more than 2-fold with no effect on K(m). In addition, glutathiolation at cysteine 67 markedly stabilized the enzyme activity presumably by reducing autoproteolysis. These results demonstrate one possible mechanism for regulation of the HIV-1 protease through cysteine modification and identify additional targets for affecting protease activity other than the active site.
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Cysteine Metabolism
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Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
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The synthesis of a new class of peptidomimetics 1a−j, based on a 1,4-benzodiazepine scaffold and on a C-terminal aspartyl aldehyde building block, is described. Compounds 1a−j provided significant inhibitory activity against falcipains 2A and 2B (FP-2A and FP-2B), two cysteine proteases from Plasmodium falciparum.
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Clonorchis sinensis
Cysteine protease
Cysteine Proteinase Inhibitors
Cathepsin L
Cystatin
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