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 MolecularRESUMO
Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.
Assuntos
Glioblastoma , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/uso terapêutico , Glioblastoma/patologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Transdução de SinaisRESUMO
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 , ChumboRESUMO
Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.
Assuntos
Micorrizas , Inibidores de Cisteína Proteinase/metabolismo , Ecossistema , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Laccaria , Micorrizas/genética , Micorrizas/metabolismo , Raízes de Plantas/microbiologia , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Solo , Simbiose/genéticaRESUMO
Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacocinética , Domínio Catalítico , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinética , Células VeroRESUMO
The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe-Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/metabolismo , Chlorocebus aethiops , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Células HEK293 , Humanos , Ligação de Hidrogênio , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ligação Proteica , SARS-CoV-2/enzimologia , Estereoisomerismo , Células VeroRESUMO
Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase.
Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Endocitose/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Animais , Catepsina B/metabolismo , Catepsina L/metabolismo , Clostridium perfringens , Inibidores de Cisteína Proteinase/metabolismo , Cães , Lisossomos/metabolismo , Células Madin Darby de Rim CaninoRESUMO
Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.
Assuntos
Antivirais/metabolismo , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Isoxazóis/metabolismo , Fenilalanina/análogos & derivados , Pirrolidinonas/metabolismo , SARS-CoV-2/enzimologia , Valina/análogos & derivados , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Enterovirus Humano D/enzimologia , Ligação de Hidrogênio , Isoxazóis/química , Fenilalanina/química , Fenilalanina/metabolismo , Ligação Proteica , Pirrolidinonas/química , Eletricidade Estática , Valina/química , Valina/metabolismoRESUMO
Staphylococcus aureus is the leading cause of hospital-acquired infections. The enzyme sortase A, present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability. Inhibition of sortase A activity offers a powerful but clinically less explored therapeutic strategy, as it offers the possibility of not inducing any selective pressure on the bacteria to evolve drug-resistant strains. In this Perspective, we offer a chemical space narrative for the design of sortase A inhibitors, as delineated into three broad domains: peptidomimetics, natural products, and synthetic small molecules. This provides immense opportunities for medicinal chemists to alleviate the ever-growing crisis of antibiotic resistance.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Aminoaciltransferases/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.
Assuntos
Acrilamidas/química , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Corantes Fluorescentes/química , Acrilamidas/síntese química , Acrilamidas/metabolismo , Domínio Catalítico , Catepsina K/química , Catepsina K/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Ligação ProteicaRESUMO
Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 µM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Sulfonas/farmacologia , Ácidos Sulfônicos/farmacologia , Tripanossomicidas/farmacologia , Compostos de Vinila/farmacologia , Animais , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/toxicidade , Ensaios Enzimáticos , Feminino , Células HeLa , Humanos , Cinética , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Ligação Proteica , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/metabolismo , Sulfonas/toxicidade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/metabolismo , Ácidos Sulfônicos/toxicidade , Tripanossomicidas/síntese química , Tripanossomicidas/metabolismo , Tripanossomicidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacos , Compostos de Vinila/síntese química , Compostos de Vinila/metabolismo , Compostos de Vinila/toxicidadeRESUMO
Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.
Assuntos
Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Citosol/metabolismo , Lisossomos/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Catepsinas/metabolismo , Permeabilidade da Membrana Celular , Inibidores de Cisteína Proteinase/metabolismo , Endopeptidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Peptídeos/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Células A549 , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Benzamidas , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Coronavirus Humano OC43/fisiologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Piperidinas , Piridinas , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 has created an unprecedented global health emergency. As of July 2021, only three antiviral therapies have been approved by the FDA for treating infected patients, highlighting the urgent need for more antiviral drugs. The SARS-CoV-2 3CL protease (3CLpro) is deemed an attractive drug target due to its essential role in viral polyprotein processing and pathogenesis. Indeed, a number of peptidomimetic 3CLpro inhibitors armed with electrophilic warheads have been reported by various research groups that can potentially be developed for treating COVID-19. However, it is currently impossible to compare their relative potencies due to the different assays employed. To solve this, we conducted a head-to-head comparison of fifteen reported peptidomimetic inhibitors in a standard FRET-based SARS-CoV-2 3CLpro inhibition assay to compare and identify potent inhibitors for development. Inhibitor design and the suitability of various warheads are also discussed.
Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Peptidomiméticos/química , SARS-CoV-2/enzimologia , Antivirais/metabolismo , Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Concentração Inibidora 50 , Peptidomiméticos/metabolismo , Ligação ProteicaRESUMO
Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.
Assuntos
Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Acrilamidas/síntese química , Acrilamidas/metabolismo , Acrilatos/síntese química , Acrilatos/metabolismo , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/síntese química , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Ligação Proteica , SARS-CoV-2/química , Bibliotecas de Moléculas Pequenas/síntese químicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (Mpro ). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with Mpro has not been done. In this work, we performed molecular dynamics simulations of the MST-Mpro complex crystal structure. The effect of the protonation states of Mpro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and Mpro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.
Assuntos
Proteases 3C de Coronavírus/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , SARS-CoV-2/enzimologia , Tiazóis/metabolismo , Benzamidas , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Inibidores de Cisteína Proteinase/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Piperidinas , Ligação Proteica , Piridinas , Eletricidade Estática , Tiazóis/químicaRESUMO
Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Ácidos Sulfônicos/farmacologia , Animais , Antivirais/síntese química , Antivirais/metabolismo , Sítios de Ligação , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Micelas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , SARS-CoV-2/enzimologia , Solubilidade , Relação Estrutura-Atividade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/metabolismo , Células VeroRESUMO
The southern green stink bug, Nezara viridula is one of the primary soybean pests and causes significant economic losses around the world. In spite of the high proteases inhibitor (PI) levels, N. viridula can feed on developing seeds of field-grown soybean and reduce crop yields. Although the PI-induced responses have been extensively investigated in many pest insects, there is lack of knowledge about the mechanisms that stink bugs employ to withstand cysteine PIs of soybean seeds. This study demonstrated that feeding on developing seeds of field-grown soybean inhibited total proteases activity of N. viridula, as result of inhibition of cathepsin B-like activity in the gut. In addition, from the 30 digestive cathepsins recognized in this study, 6 were identified as cathepsin B-like. Stink bugs that fed on growing seeds of field-grown soybean had similar gut pH to those reared in the laboratory, and both cathepsin B- and L-like had an optima pH of 6.5. Therefore, using specific proteases inhibitors we found that the main proteolytic activity in the gut is from cysteine proteases when N. viridula feeds on soybean crops. Since cathepsin L-like activity was not inhibited by soybean PIs, our results suggested that N. viridula relays on cathepsin L-like to feed on soybean. To our knowledge no study before has shown the impact of seed PIs of field-grown soybean on digestive proteases (cathepsin B- and L-like) of N. viridula. This study suggests that the activity of PI-insensitive cathepsins L-like in the gut would be part of an adaptive strategy to feed on developing soybean seeds. In agreement, the expansions of cathepsin L-like complement observed in pentatomids could confer to the insects a higher versatility to counteract the effects of different PIs.
Assuntos
Catepsina B/metabolismo , Comportamento Alimentar , Heterópteros , Animais , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Heterópteros/metabolismo , Heterópteros/fisiologia , Proteínas de Insetos/metabolismo , Intestinos/fisiologia , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Glycine max/metabolismoRESUMO
In this study, chemical investigation of methanol extract of the air-dried fruits of Luffa cylindrica led to the identification of a new δ-valerolactone (1), along with sixteen known compounds (2-17). Their chemical structures including the absolute configuration were elucidated by extensive spectroscopic analysis and electronic circular dichroism analysis, as well as by comparison with those reported in the literature. For the first time in literature, we have examined the binding potential of the isolated compounds to highly conserved protein, Mpro of SARS-CoV-2 using the molecular docking technique. We found that the isolated saponins (14-17) bind to the substrate-binding pocket of SARS-CoV-2 Mpro with docking energy scores of -7.13, -7.29, -7.47, and -7.54 kcal.mol-1, respectively, along with binding abilities equivalent to an already claimed N3 protease inhibitor (-7.51 kcal.mol-1).