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1.
Molecules ; 29(11)2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38893535

RESUMO

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.


Assuntos
Cisteína Proteases , Inibidores de Cisteína Proteinase , Simulação de Acoplamento Molecular , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/metabolismo , Relação Estrutura-Atividade , Cisteína Proteases/metabolismo , Cisteína Proteases/química , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/química , Estrutura Molecular
2.
Eur J Protistol ; 94: 126085, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38703600

RESUMO

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.


Assuntos
Inibidores de Cisteína Proteinase , Proteínas Recombinantes , Tetrahymena thermophila , Inibidores de Cisteína Proteinase/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetrahymena thermophila/genética , Tetrahymena thermophila/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Leucina/análogos & derivados
3.
J Med Chem ; 67(11): 8757-8790, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38753594

RESUMO

Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M-1s-1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents.


Assuntos
Antivirais , Proteases 3C de Coronavírus , Nitrilas , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , SARS-CoV-2/efeitos dos fármacos , Nitrilas/química , Nitrilas/farmacologia , Nitrilas/síntese química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/química , Relação Estrutura-Atividade , Humanos , Compostos Macrocíclicos/farmacologia , Compostos Macrocíclicos/química , Compostos Macrocíclicos/síntese química , Tratamento Farmacológico da COVID-19 , Descoberta de Drogas , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/síntese química , Peptidomiméticos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/síntese química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/síntese química
4.
Int J Mol Sci ; 25(8)2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38673995

RESUMO

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.


Assuntos
Inibidores de Cisteína Proteinase , Nitrilas , Plasmodium falciparum , Trypanosoma brucei rhodesiense , Tripanossomíase Africana , Humanos , Antimaláricos/uso terapêutico , Antimaláricos/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Inibidores de Cisteína Proteinase/química , Malária/tratamento farmacológico , Nitrilas/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico
5.
J Med Chem ; 67(9): 7048-7067, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38630165

RESUMO

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.


Assuntos
Antivirais , Catepsina L , SARS-CoV-2 , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Animais , Chlorocebus aethiops , Células Vero , SARS-CoV-2/efeitos dos fármacos , Humanos , Relação Estrutura-Atividade , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/síntese química , Cristalografia por Raios X , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Modelos Moleculares
6.
Expert Opin Ther Pat ; 34(1-2): 17-49, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38445468

RESUMO

INTRODUCTION: Cysteine proteases are involved in a broad range of biological functions, ranging from extracellular matrix turnover to immunity. Playing an important role in the onset and progression of several diseases, including cancer, immune-related and neurodegenerative disease, viral and parasitic infections, cysteine proteases represent an attractive drug target for the development of therapeutic tools. AREAS COVERED: Recent scientific and patent literature focusing on the design and study of cysteine protease inhibitors with potential therapeutic application has been reviewed. EXPERT OPINION: The discovery of a number of effective structurally diverse cysteine protease inhibitors opened up new challenges and opportunities for the development of therapeutic tools. Mechanistic studies and the availability of X-ray crystal structures of some proteases, alone and in complex with inhibitors, provide crucial information for the rational design and development of efficient and selective cysteine protease inhibitors as preclinical candidates for the treatment of different diseases.


Assuntos
Cisteína Proteases , Doenças Neurodegenerativas , Humanos , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Patentes como Assunto , Inibidores de Proteases/farmacologia , Antivirais/farmacologia
7.
Biophys Chem ; 305: 107140, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38118338

RESUMO

Odanacatib (ODN) is a selective cathepsin K inhibitor that acts as an anti-resorptive agent to treat osteoporosis. ODN is also found effective in reducing the effect of severe periodontitis. The interaction between ODN and human serum albumin (HSA) was investigated using spectroscopic, microscopic, and in silico approaches to characterize their binding. The fluorescence intensity of HSA increased upon the addition of increasing concentrations of ODN accompanied by blueshift in the fluorescence spectrum, which suggested hydrophobic formation around the microenvironment of the fluorophores upon ODN binding. A moderate binding affinity was obtained for ODN-HSA binding, with binding constant (Ka) values of ∼104 M-1. Circular dichroism results suggested that the overall secondary and tertiary structures of HSA were both only slightly altered upon ODN binding. The surface morphology of HSA was also affected upon ODN binding, showing aggregate formation. Drug displacement and molecular docking results revealed that ODN preferably binds to site III in subdomain IB of HSA, while molecular dynamics simulations indicated formation of a stable protein complex when site III was occupied by ODN. The ODN-HSA complex was mainly stabilized by a combination of hydrogen bonding, hydrophobic interactions, and van der Waals forces. These findings provide additional information to understand the interaction mechanism of ODN in blood circulation and may help in future improvements on the adverse effects of ODN.


Assuntos
Inibidores de Cisteína Proteinase , Albumina Sérica Humana , Humanos , Albumina Sérica Humana/química , Simulação de Acoplamento Molecular , Sítios de Ligação , Ligação Proteica , Inibidores de Cisteína Proteinase/farmacologia , Espectrometria de Fluorescência , Dicroísmo Circular , Termodinâmica
8.
Viruses ; 15(7)2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37515189

RESUMO

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester (CA074me) and a reversible oxindole inhibitor. Here, we determined the X-ray crystal structure of the CA074-inhibited nsP2 protease and compared it with our E64d-inhibited structure. We found that the two inhibitors occupy different locations in the protease. We designed hybrid inhibitors with improved potency. Virus yield reduction assays confirmed that the viral titer was reduced by >5 logs with CA074me. Cell-based assays showed reductions in viral replication for CHIKV, VEEV, and WEEV, and weaker inhibition of EEEV by the hybrid inhibitors. The most potent was NCGC00488909-01 which had an EC50 of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC50 = 7.90 µM. Other compounds from the NCATS libraries such as the H1 antihistamine oxatomide (>5-log reduction), emetine, amsacrine an intercalator (NCGC0015113), MLS003116111-01, NCGC00247785-13, and MLS00699295-01 were found to effectively reduce VEEV viral replication in plaque assays. Kinetic methods demonstrated time-dependent inhibition by the hybrid inhibitors of the protease with NCGC00488909-01 (Ki = 3 µM) and NCGC00484087 (Ki = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl2, MnCl2, or MgCl2 were measured with varying concentrations of inhibitor, Mg2+ and Mn2+ slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV.


Assuntos
Cisteína Proteases , Vírus da Encefalite Equina Venezuelana , Animais , Cavalos , Replicação Viral , Inibidores de Cisteína Proteinase/farmacologia
9.
Int J Mol Sci ; 24(10)2023 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-37239824

RESUMO

Rhodesain is the main cysteine protease of Trypanosoma brucei rhodesiense, the parasite causing the acute lethal form of Human African Trypanosomiasis. Starting from the dipeptide nitrile CD24, the further introduction of a fluorine atom in the meta position of the phenyl ring spanning in the P3 site and the switch of the P2 leucine with a phenylalanine led to CD34, a synthetic inhibitor that shows a nanomolar binding affinity towards rhodesain (Ki = 27 nM) and an improved target selectivity with respect to the parent dipeptide nitrile CD24. In the present work, following the Chou and Talalay method, we carried out a combination study of CD34 with curcumin, a nutraceutical obtained from Curcuma longa L. Starting from an affected fraction (fa) of rhodesain inhibition of 0.5 (i.e., the IC50), we observed an initial moderate synergistic action, which became a synergism for fa values ranging from 0.6 to 0.7 (i.e., 60-70% inhibition of the trypanosomal protease). Interestingly, at 80-90% inhibition of rhodesain proteolytic activity, we observed a strong synergism, resulting in 100% enzyme inhibition. Overall, in addition to the improved target selectivity of CD34 with respect to CD24, the combination of CD34 + curcumin resulted in an increased synergistic action with respect to CD24 + curcumin, thus suggesting that it is desirable to use CD34 and curcumin in combination.


Assuntos
Curcumina , Trypanosoma brucei rhodesiense , Curcumina/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Nitrilas , Relação Estrutura-Atividade , Trypanosoma brucei rhodesiense/efeitos dos fármacos
10.
Mol Cell Proteomics ; 22(5): 100543, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37030595

RESUMO

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.


Assuntos
Morte Celular , Neurônios , Sinapses , Animais , Masculino , Camundongos , Ratos , Calpaína/metabolismo , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Neuroproteção , Proteoma/análise , Ratos Wistar , Acidente Vascular Cerebral/patologia , Sinapses/patologia , Sinapses/fisiologia
11.
J Am Chem Soc ; 145(18): 10015-10021, 2023 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-37104712

RESUMO

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.


Assuntos
Caspases , Cisteína , Humanos , Caspase 6 , Apoptose , Inibidores de Cisteína Proteinase/farmacologia
12.
Eur J Med Chem ; 252: 115299, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-36996716

RESUMO

Malaria is a tropical disease with significant morbidity and mortality burden caused by Plasmodium species in Africa, the Middle East, Asia, and South America. Pathogenic Plasmodium species have lately become increasingly resistant to approved chemotherapeutics and combination therapies. Therefore, there is an emergent need for identifying new druggable targets and novel chemical classes against the parasite. Falcipains, cysteine proteases required for heme metabolism in the erythrocytic stage, have emerged as promising drug targets against Plasmodium species that infect humans. This perspective discusses the biology, biochemistry, structural features, and genetics of falcipains. The efforts to identify selective or dual inhibitors and their structure-activity relationships are reviewed to give a perspective on the design of novel compounds targeting falcipains for antimalarial activity evaluating reasons for hits and misses for this important target.


Assuntos
Antimaláricos , Plasmodium , Humanos , Antimaláricos/química , Plasmodium falciparum , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Relação Estrutura-Atividade
13.
J Med Chem ; 66(4): 3088-3105, 2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36752718

RESUMO

Interest in covalent enzyme inhibitors as therapeutic agents has seen a recent resurgence. Covalent enzyme inhibitors typically possess an organic functional group that reacts with a key feature of the target enzyme, often a nucleophilic cysteine residue. Herein, the application of small, modular ReV complexes as inorganic cysteine-targeting warheads is described. These metal complexes were found to react with cysteine residues rapidly and selectively. To demonstrate the utility of these ReV complexes, their reactivity with SARS-CoV-2-associated cysteine proteases is presented, including the SARS-CoV-2 main protease and papain-like protease and human enzymes cathepsin B and L. As all of these proteins are cysteine proteases, these enzymes were found to be inhibited by the ReV complexes through the formation of adducts. These findings suggest that these ReV complexes could be used as a new class of warheads for targeting surface accessible cysteine residues in disease-relevant target proteins.


Assuntos
COVID-19 , Cisteína Proteases , Inibidores de Cisteína Proteinase , Cisteína , Rênio , SARS-CoV-2 , Humanos , Cisteína Proteases/metabolismo , Inibidores Enzimáticos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico
14.
ChemMedChem ; 18(6): e202200434, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36692246

RESUMO

Chagas disease is a neglected tropical disease caused by the protozoa Trypanosoma cruzi. Cruzain, its main cysteine protease, is commonly targeted in drug discovery efforts to find new treatments for this disease. Even though the essentiality of this enzyme for the parasite has been established, many cruzain inhibitors fail as trypanocidal agents. This lack of translation from biochemical to biological assays can involve several factors, including suboptimal physicochemical properties. In this work, we aim to rationalize this phenomenon through chemical space analyses of calculated molecular descriptors. These include statistical tests, visualization of projections, scaffold analysis, and creation of machine learning models coupled with interpretability methods. Our results demonstrate a significant difference between the chemical spaces of cruzain and T. cruzi inhibitors, with compounds with more hydrogen bond donors and rotatable bonds being more likely to be good cruzain inhibitors, but less likely to be active on T. cruzi. In addition, cruzain inhibitors seem to occupy specific regions of the chemical space that cannot be easily correlated with T. cruzi activity, which means that using predictive modeling to determine whether cruzain inhibitors will be trypanocidal is not a straightforward task. We believe that the conclusions from this work might be of interest for future projects that aim to develop novel trypanocidal compounds.


Assuntos
Doença de Chagas , Tripanossomicidas , Trypanosoma cruzi , Humanos , Cisteína Endopeptidases/química , Doença de Chagas/tratamento farmacológico , Proteínas de Protozoários , Tripanossomicidas/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química
15.
Biochimie ; 206: 24-35, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36198333

RESUMO

The tick-transmitted apicomplexan Theileria parva causes East Coast fever, a bovine disease of great economic and veterinary importance in Africa. Papain-like cysteine proteases play important roles in protozoan parasite host cell entry and egress, nutrition and host immune evasion. This study reports the identification and characterisation of a T. parva strain Muguga cathepsin L-like (C1A subfamily) cysteine protease (ThpCP). Molecular modelling confirmed the papain-like fold of ThpCP, hydrophobic character of the S2 substrate binding pocket and non-covalent interaction between the pro- and catalytic domains preceding low pH autoactivation. ThpCP was recombinantly expressed in a protease deficient E. coli (Rosetta (DE3)pLysS strain) expression host as a 46 kDa proenzyme. Following Ni-chelate affinity chromatography and acidification, the 27 kDa mature ThpCP was purified by cation-exchange chromatography. Purified ThpCP hydrolysed typical cathepsin L substrates N-α-benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methyl-coumarin (AMC) (kcat/Km = 4.49 × 105 s-1M-1) and Z-Leu-Arg-AMC (kcat/Km = 4.20 × 105 s-1M-1), but showed no activity against the cathepsin B-selective substrate Z-Arg-Arg-AMC. Recombinant ThpCP was active over a broad pH range from pH 4.5 to 7.5, thereby showing potential activity in the acidic parasite food vacuole and close to neutral pH of the host lymphocyte cytoplasm. Recombinant ThpCP was inhibited by the cysteine protease inhibitors E64, iodoacetate, leupeptin, chymostatin, Z-Phe-Ala-diazomethylketone (DMK) and Z-Phe-Phe-DMK and hydrolysed bovine proteins: haemoglobin, immunoglobulin G, serum albumin and fibrinogen as well as goat IgG at pH 6 and 7. Functional expression and characterisation of Theileria cysteine proteases should enable high throughput screening of cysteine protease inhibitor libraries against these proteases.


Assuntos
Cisteína Proteases , Theileria parva , Animais , Bovinos , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Catepsina L/metabolismo , Theileria parva/genética , Theileria parva/metabolismo , Sequência de Aminoácidos , Papaína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Éxons
16.
Eur J Med Chem ; 244: 114876, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36343429

RESUMO

Chagas disease is a major public health problem caused by Trypanosoma cruzi, with an estimated 6-7 million people infected and 70 million at risk of infection. T. brucei gambiense and T. brucei rhodesiense are two subspecies of related parasites that cause human African trypanosomiasis, a neglected tropical disease with also millions of people at risk of infection. Pharmacotherapy for both diseases suffers from low efficacy, side effects, or drug resistance. Recently, we reported a noncovalent competitive inhibitor of cruzain (IC50 26 µM, Ki 3 µM) and TbrCatL (IC50 50 µM), two cysteine proteases considered promising drug targets for trypanosomiasis. Here, we describe the design and synthesis of derivatives of our lead compound. The new thiosemicarbazone derivatives showed potency in the nanomolar concentration range against the two enzymes, but they were later characterized as aggregators. Nevertheless, the thiosemicarbazone derivatives showed promising antiparasitic activities against T. b. brucei (EC50 13-49.7 µM) and T. cruzi (EC50 0.027-0.59 µM) under in vitro conditions. The most active thiosemicarbazone was 200-fold more potent than the current anti-chagasic drug, benznidazole, and showed a selectivity index of 370 versus myoblast cells. We have identified an excellent candidate for further optimization and in vivo studies.


Assuntos
Doença de Chagas , Tiossemicarbazonas , Tripanossomicidas , Trypanosoma brucei brucei , Trypanosoma cruzi , Humanos , Tripanossomicidas/farmacologia , Tiossemicarbazonas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Relação Estrutura-Atividade , Doença de Chagas/tratamento farmacológico
17.
Bioorg Chem ; 129: 106185, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36240541

RESUMO

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. Mpro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of Mpro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against Mpro with IC50 of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 Mpro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of Mpro, and ACF has been previously reported as a PLpro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.


Assuntos
Acriflavina , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Inibidores de Cisteína Proteinase , Proflavina , SARS-CoV-2 , Inibidores de Protease Viral , Acriflavina/farmacologia , Proflavina/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Protease Viral/farmacologia , Mesocricetus , Animais , Cricetinae , Linhagem Celular , Replicação Viral/efeitos dos fármacos
18.
Int J Mol Sci ; 23(16)2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-36012257

RESUMO

Heavy metal ions can disrupt biological functions via multiple molecular mechanisms, including inhibition of enzymes. We investigate the interactions of human papain-like cysteine endopeptidases cathepsins L, K, and S with gallium and cerium ions, which are associated with medical applications. We compare these results with zinc and lead, which are known to inhibit thiol enzymes. We show that Ga3+, Ce3+, and Ce4+ ions inhibit all tested peptidases with inhibition constants in the low micromolar range (between 0.5 µM and 10 µM) which is comparable to Zn2+ ions, whereas inhibition constants of Pb2+ ions are one order of magnitude higher (30 µM to 150 µM). All tested ions are linear specific inhibitors of cathepsin L, but cathepsins K and S are inhibited by Ga3+, Ce3+, and Ce4+ ions via hyperbolic inhibition mechanisms. This indicates a mode of interaction different from that of Zn2+ and Pb2+ ions, which act as linear specific inhibitors of all peptidases. All ions also inhibit the degradation of insoluble elastin, which is a common target of these peptidases in various inflammatory diseases. Our results suggest that these ions and their compounds have the potential to be used as cysteine cathepsin inhibitors in vitro and possibly in vivo.


Assuntos
Cério , Gálio , Catepsina K/metabolismo , Catepsinas/metabolismo , Cisteína , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/metabolismo , Humanos , Íons , Cinética , Chumbo
19.
Bioorg Med Chem Lett ; 74: 128927, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35944849

RESUMO

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.


Assuntos
Osteoartrite , Animais , Osso e Ossos , Catepsina K , Catepsinas , Condrócitos , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Cães , Osteoartrite/tratamento farmacológico , Osteoclastos
20.
Microbiol Res ; 261: 127061, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35605309

RESUMO

The regulation of the activity of proteases by endogenous inhibitors is a common trend in almost all forms of life. Here, we review the endogenous inhibitors of cysteine proteases of three major pathogenic parasitic protozoa. The review focuses on members of the genus Plasmodium, Entamoeba, and Leishmania. Research in this domain has revealed the presence of only chagasin-like inhibitors of cysteine proteases that house a ß-barrel immunoglobulin-fold and inhibit the target proteases using a 3-loop inhibitory mechanism in these pathogens. Inhibitors of cysteine proteases are highly evolvable enzymes that target a broad spectrum of pathogenic cysteine proteases with a proclivity for those involved in host-parasite interactions. A common trend reflects a limited sequence homology between cysteine proteases and their inhibitors. The inhibitors are also known to participate in other housekeeping functions of the parasites. Generalizations about their roles are thus best avoided. In this review, the reader will find comprehensive information on the cellular localization of inhibitors of cysteine proteases, their structure, function, and the associated mechanisms of action. The reader will also find a thorough analysis of the role of these inhibitors in parasite pathology and the common trends interlinking them with parasite biology and evolution.


Assuntos
Cisteína Proteases , Parasitos , Sequência de Aminoácidos , Animais , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Protozoários
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