RESUMO
The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.
Assuntos
Domínio Catalítico , Proteases 3C de Coronavírus , Cisteína , Dissulfetos , Oxirredução , SARS-CoV-2 , Dissulfetos/química , Dissulfetos/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteases 3C de Coronavírus/metabolismo , Proteases 3C de Coronavírus/química , Cisteína/química , Cisteína/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Multimerização Proteica , COVID-19/virologiaRESUMO
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
Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Catepsinas , Antivirais/farmacologia , Antivirais/uso terapêutico , Antivirais/química , Inibidores de Proteases/farmacologia , Cisteína Endopeptidases/metabolismoRESUMO
The author wishes to make the following correction to this paper [...].
RESUMO
SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , Sítio Alostérico , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus , Humanos , Papaína/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2RESUMO
The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.
RESUMO
The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced ß-catenin binding resulting in increased cytosolic and nuclear ß-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/ß-catenin signaling.
Assuntos
Antígenos CD/genética , Neoplasias Encefálicas/genética , Caderinas/genética , Carcinoma/genética , Neoplasias Neuroepiteliomatosas/genética , Adenoma/genética , Adenoma/patologia , Compostos de Anilina/uso terapêutico , Animais , Diversidade de Anticorpos , Neoplasias Encefálicas/tratamento farmacológico , Carcinoma/tratamento farmacológico , DNA de Neoplasias/genética , Técnicas de Introdução de Genes , Variação Genética , Células HEK293 , Humanos , Neoplasias Neuroepiteliomatosas/tratamento farmacológico , Oligodendroglioma/genética , Oligodendroglioma/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Sequenciamento Completo do GenomaRESUMO
The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.
Assuntos
Sítio Alostérico , Antivirais/química , Domínio Catalítico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Desenvolvimento de Medicamentos , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.
Assuntos
Actomiosina/química , Difosfato de Adenosina/química , Dictyostelium/química , Fosfatos/química , Proteínas de Protozoários/química , Actomiosina/genética , Trifosfato de Adenosina/química , Regulação Alostérica , Dictyostelium/genética , Proteínas de Protozoários/genéticaRESUMO
The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.
Assuntos
Difosfato de Adenosina/química , Domínio Catalítico , Compostos Heterocíclicos de 4 ou mais Anéis/química , Simulação de Dinâmica Molecular , Miosinas/química , Actinas/química , Trifosfato de Adenosina/química , Cristalização , Hidrólise , Conformação Proteica em Folha betaRESUMO
Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7â Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P.â aeruginosa infections.
Assuntos
Ligases/química , Ligases/metabolismo , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/metabolismo , Quinolonas/metabolismo , Percepção de Quorum , ortoaminobenzoatos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Pseudomonas aeruginosa/enzimologia , ortoaminobenzoatos/químicaRESUMO
Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated ß-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.Gram-negative bacteria assemble biofilms from amyloid fibres, which translocate across the outer membrane as unfolded amyloid precursors through a secretion system. Here, the authors characterise the structural details of the amyloid transporter FapF in Pseudomonas.