RESUMO
Despite their widespread impact on human health, there are no approved drugs for combating alphavirus infections. The heterocyclic ß-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad-spectrum antiviral activity. Analogs of 1a that varied each of the three regions of the molecule were synthesized to establish structure-activity relationships for the inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The vinyl sulfone covalent warhead was highly sensitive to modifications. However, alterations to the core five-membered heterocycle and aryl substituent were well tolerated. The 5-(2,5-dimethoxyphenyl)pyrazole (1o) and 4-cyanopyrazole (8d) analogs exhibited kinact/Ki ratios >9000 M-1 s-1. 3-Arylisoxazole (10) was identified as an isosteric replacement for the five-membered heterocycle, which circumvented the intramolecular cyclization of pyrazole-based inhibitors like 1a. A ligand-based model of the enzyme active site was developed to aid the design of nsP2 protease inhibitors as potential therapeutics against alphaviruses.
Assuntos
Antivirais , Vírus Chikungunya , Cisteína Endopeptidases , Sulfonas , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Relação Estrutura-Atividade , Sulfonas/farmacologia , Sulfonas/química , Sulfonas/síntese química , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Humanos , Animais , Replicação Viral/efeitos dos fármacosRESUMO
Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 µM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.
Assuntos
Cisteína Endopeptidases , Simulação de Acoplamento Molecular , Proteínas de Protozoários , Triazóis , Trypanosoma cruzi , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Cisteína Endopeptidases/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/síntese química , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Desenho Assistido por Computador , Desenho de Fármacos , Humanos , Estrutura Molecular , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/síntese química , Doença de Chagas/tratamento farmacológicoRESUMO
Cathepsin L (CTSL), a cysteine cathepsin protease of the papain superfamily, plays a crucial role in cancer progression and metastasis. Dysregulation of CTSL is frequently observed in tumor malignancies, leading to the degradation of extracellular matrix and facilitating epithelial-mesenchymal transition (EMT), a key process in malignant cancer metastasis. This review mainly provides a comprehensive information about recent findings on natural inhibitors targeting CTSL and their anticancer effects, which have emerged as potent anticancer therapeutic agents or metastasis-suppressive adjuvants. Specifically, inhibitors are categorized into small-molecule and macromolecule inhibitors, with a particular emphasis on cathepsin propeptide-type macromolecules. Additionally, the article explores the molecular mechanisms of CTSL involvement in cancer metastasis, highlighting its regulation at transcriptional, translational, post-translational, and epigenetic levels. This work underscores the importance of understanding natural CTSL inhibitors and provides researchers with practical insights to advance the relevant fields and discover novel CTSL-targeting inhibitors from natural sources.
Assuntos
Produtos Biológicos , Catepsina L , Metástase Neoplásica , Humanos , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Animais , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/síntese química , Estrutura Molecular , Proliferação de Células/efeitos dos fármacosRESUMO
Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.
Assuntos
Catepsina B , Quirópteros , Cistatina A , Animais , Humanos , Catepsina B/metabolismo , Catepsina B/química , Catepsina B/genética , Catepsina B/antagonistas & inibidores , Cistatina A/metabolismo , Cistatina A/química , Cistatina A/genética , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Catepsina L/metabolismo , Catepsina L/química , Catepsina L/genética , Catepsina L/antagonistas & inibidores , Cinética , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/metabolismoRESUMO
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 MolecularAssuntos
Cisteína Proteases , Inibidores de Cisteína Proteinase , Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Humanos , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Cisteína Proteases/metabolismo , Cisteína Proteases/química , Imunoconjugados/química , Imunoconjugados/farmacologiaRESUMO
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 químicaRESUMO
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 & derivadosRESUMO
Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/farmacologia , Antivirais/química , Chlorocebus aethiops , Células Vero , Humanos , COVID-19/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Poríferos/química , Internalização do Vírus/efeitos dos fármacosRESUMO
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 farmacológicoRESUMO
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 MolecularesRESUMO
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/farmacologiaRESUMO
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 â¼104 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 , TermodinâmicaRESUMO
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/farmacologiaRESUMO
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 fármacosRESUMO
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/farmacologiaRESUMO
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/fisiologiaRESUMO
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-AtividadeRESUMO
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 terapêuticoRESUMO
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.â 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.â cruzi. In addition, cruzain inhibitors seem to occupy specific regions of the chemical space that cannot be easily correlated with T.â 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.