RESUMEN
The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.
Asunto(s)
Antivirales , Proteasas 3C de Coronavirus , Enterovirus , SARS-CoV-2 , Antivirales/química , Antivirales/farmacología , Especificidad por Sustrato , Enterovirus/enzimología , Enterovirus/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Humanos , Coronavirus/enzimología , Coronavirus/efectos de los fármacosRESUMEN
Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.
Asunto(s)
Serina Endopeptidasas , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Humanos , Diseño de Fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Animales , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/antagonistas & inhibidoresRESUMEN
Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.
RESUMEN
The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Proteínas no Estructurales Virales/genética , Péptidos/genética , Proteasas 3C de Coronavirus/genética , Inhibidores de Proteasas/química , Antivirales/farmacología , Péptido HidrolasasRESUMEN
Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.
Asunto(s)
Péptido Hidrolasas , Porphyromonas gingivalis , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Péptido Hidrolasas/metabolismo , Calpaína/genética , Calpaína/metabolismo , Especificidad por Sustrato , Endopeptidasas/metabolismoRESUMEN
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.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Catepsinas , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/química , Inhibidores de Proteasas/farmacología , Cisteína Endopeptidasas/metabolismoRESUMEN
Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage-a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues-bis(2-carbamoylaryl)phenyl diselenides-in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Metiltransferasas , Péptido Hidrolasas , Antivirales/farmacología , Antivirales/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Proteasas/farmacología , Simulación del Acoplamiento MolecularRESUMEN
The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.
RESUMEN
High levels of expression and activity of the 20S proteasome have been linked to many types of pathologies, including neoplasia, autoimmune disorders, neurodegenerative diseases and many more. Moreover, distinguishing between 20S proteasome catalytic subunits is neglected, although it may provide further insight into the development of pathologies. Several approaches have been developed to detect 20S proteasome activity, one of which is internally quenched fluorescent (IQF) substrates, which currently suffer from low efficiency and sensitivity. Previous reports focused on peptides including natural amino acids; therefore, in this report, we synthesized and analyzed IQF substrates with both natural and unnatural amino acids in the P1' and P2' positions to investigate their influences on selectivity toward 20S proteasome subunits. We found that elongation of the substrate by the P1' and P2' positions increased specificity in comparison to tetrapeptides. Moreover, we were able to obtain IQF substrates for the Ch-L subunit, which was characterized by higher selectivity than formerly used tetrapeptides. These findings may further contribute to the development of novel diagnostic tools for 20S proteasome-dependent disorders.
Asunto(s)
Péptidos , Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Péptidos/química , Aminoácidos/metabolismo , Proteolisis , Especificidad por Sustrato , Sitios de UniónRESUMEN
Several chemical approaches have been applied to develop Ub-based substrates and probes selective toward one or a narrow subset of deubiquitinases (DUBs). Since DUBs are highly specific toward ubiquitin and exhibit low activity toward shorter peptides, it is challenging to design truly selective chemical tools to investigate one DUB in biological samples. Incorporating amino acids other than canonical LRG at the P4-P2 positions in the Ub improves DUB activity and selectivity toward Ub derivatives. Here, we describe the protocol for identifying selective peptide sequences using a hybrid combinatorial substrate library (HyCoSuL) approach that can be introduced in the C-terminal motif of Ub. Furthermore, we describe the synthesis protocol of Ub-based probes and substrates containing unnatural amino acids and the application of Ub-based probes to detect DUBs in cell lysates.
Asunto(s)
Aminoácidos , Ubiquitina , Ubiquitina/metabolismo , Aminoácidos/metabolismo , Secuencia de Aminoácidos , Péptidos/química , Enzimas Desubicuitinizantes/metabolismo , UbiquitinaciónRESUMEN
Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.
Asunto(s)
COVID-19 , Aceites Volátiles , Antivirales/farmacología , Antivirales/uso terapéutico , Humanos , Aceites Volátiles/farmacología , Inhibidores de Proteasas , SARS-CoV-2RESUMEN
The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.
Asunto(s)
COVID-19 , Proteasas 3C de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Humanos , Especificidad por SustratoRESUMEN
The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.
Asunto(s)
Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteasas Similares a la Papaína de Coronavirus/ultraestructura , SARS-CoV-2/genética , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , COVID-19/genética , COVID-19/metabolismo , Proteasas Similares a la Papaína de Coronavirus/genética , Células HEK293 , Humanos , Papaína/química , Papaína/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Unión Proteica/genética , SARS-CoV-2/metabolismo , Especificidad por Sustrato/genética , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas Virales/metabolismoRESUMEN
Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.
Asunto(s)
Antifúngicos/química , Proteasas de Ácido Aspártico/antagonistas & inhibidores , Cryptococcus neoformans/enzimología , Proteínas Fúngicas/antagonistas & inhibidores , Antifúngicos/metabolismo , Antifúngicos/farmacología , Proteasas de Ácido Aspártico/genética , Proteasas de Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/efectos de los fármacos , VIH/enzimología , Proteasa del VIH/química , Proteasa del VIH/metabolismo , Simulación de Dinámica Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.
Asunto(s)
Antivirales/química , COVID-19/diagnóstico por imagen , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Células Epiteliales/virología , Inhibidores de Proteasas/química , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , COVID-19/patología , COVID-19/virología , Dominio Catalítico , Técnicas Químicas Combinatorias , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Células Epiteliales/ultraestructura , Colorantes Fluorescentes/química , Expresión Génica , Glutamina/química , Humanos , Modelos Moleculares , Nasofaringe/virología , Inhibidores de Proteasas/farmacología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , SARS-CoV-2/enzimología , Especificidad por SustratoRESUMEN
Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.
Asunto(s)
Betacoronavirus/enzimología , Diseño de Fármacos , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Secuencia de Aminoácidos , Betacoronavirus/aislamiento & purificación , Sitios de Unión , COVID-19 , Dominio Catalítico , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Cristalografía por Rayos X , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Pandemias , Neumonía Viral/patología , Neumonía Viral/virología , Inhibidores de Proteasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SARS-CoV-2 , Especificidad por Sustrato , Ubiquitinas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs-MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell lysate experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.
RESUMEN
The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.
Asunto(s)
Complejos de Coordinación/química , Elementos de la Serie de los Lantanoides/química , Sondas Moleculares/química , Péptido Hidrolasas/análisis , Línea Celular , Complejos de Coordinación/síntesis química , Humanos , Sondas Moleculares/síntesis química , Estructura Molecular , Péptido Hidrolasas/metabolismoRESUMEN
In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.
RESUMEN
West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts.