TL;DR
Findings establish that both catalytic and allosteric sites play essential roles in regulating 3CL pro function, and targeting allosteric regions such as the Asn28-associated pocket offers a promising approach for antiviral development.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a crucial therapeutic target for anti-coronavirus disease 2019 (COVID-19) drug development, as it is essential for viral replication. However, mutations within the active site have compromised the efficacy of current competitive inhibitors, prompting the exploration of alternative inhibition strategies. In this study, we systematically investigated the allosteric inhibition mechanism of SARS-CoV-2 Mpro by pelitinib and leveraged this insight for new inhibitor discovery. Through extensive molecular dynamics simulations, we showed that pelitinib exerts allosteric inhibition via the L141-S144-C145-H41 interaction network: it restricts the flexibility of L141 through CH-π interactions, transmits this effect to C145 via S144, stabilizes the hydrogen bond between C145 and H41, and thereby reduces the flexibility of the S3 helix (residues 40-60). This series of conformational changes induces the contraction of the Mpro catalytic pocket from ~1200 ų to ~800 ų, impairs substrate binding, and ultimately appears to impair Mpro activity. Based on this mechanism, we performed structure-based virtual screening and identified a novel compound (Cpd-1). Biological evaluations showed that Cpd-1 exhibits superior Mpro inhibitory activity compared to pelitinib, with negligible off-target binding to human EGFR and Myt1 kinase, low cytotoxicity (cell viability > 60% at 200 μM), and predicted inhibitory activity against clinically relevant Mpro-resistant mutants based on computational analysis. Our findings provide mechanistic insights into a key allosteric mechanism for Mpro inhibition but also provide a promising chemical scaffold for further development as an Mpro-targeting inhibitor.
ABSTRACT Diseases caused by picornaviruses pose a serious threat to society due to their high contagiousness and widespread prevalence. Beyond the poliovirus vaccine, antiviral therapeutics remain unavailable for most picornaviral infections. Moreover, existing inhibitors under development generally exhibit narrow‐spectrum activity and low barriers to resistance owing to the high mutability of RNA viral proteins. To address these issues, this study constructed a proteolysis‐targeting chimera (PROTAC) targeting the 3C protease (3CPro) — a key viral enzyme essential for picornavirus protein processing, host shutoff, and immune suppression. In enterovirus 71 (EV71), a representative member of the picornavirus family, experimental results showed that PROTAC molecule effectively inhibits viral replication through a dual mechanism of action: directly inhibiting the catalytic function of the 3CPro and inducing its degradation via the ubiquitin‐proteasome pathway. Moreover, this PROTAC molecule not only demonstrated effective degradation of previously reported and AI‐predicted potential drug‐resistant EV71 mutants, but also exhibited degradation activity against 3CPro from multiple picornaviruses, indicating its potential for broad‐spectrum antiviral activity. By integrating multidisciplinary approaches, this work demonstrates that targeted protein degradation can effectively induce the degradation of 3CPro in picornaviruses, supporting the development of resistance‐resistant, broad‐spectrum antiviral agents and highlighting their potential to address evolving viral threats.
It is demonstrated that nanobodies targeting the PLpro/ISG15 interface can achieve synergistic antiviral and immunomodulatory effects, providing a proof-of-concept for a novel therapeutic approach to combat SARS-CoV-2 and potentially other emerging coronaviruses.
Chikungunya virus (CHIKV), an arthropod-borne alphavirus, has emerged as a global health threat due to its rapid transmission and the lack of effective antiviral therapies. The cysteine protease activity of the virus-encoded nonstructural protein 2 (nsP2) is critical for CHIKV replication, as it processes viral polyproteins and counteracts host antiviral defenses, establishing it as a highly attractive target for therapeutic intervention. In this study, we present a rapid drug development platform that integrates covalent docking with direct-to-biology (D2B) synthesis and screening to identify nsP2 inhibitors. Candidates prioritized by in silico docking were synthesized and directly tested in FRET enzymatic assays without purification. This approach led to the identification of several nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition, and antiviral activity. Together, these findings establish a streamlined strategy for covalent inhibitor development and provide promising leads for CHIKV antiviral development.
The global impact of SARS-CoV-2 and the continued emergence of zoonotic coronaviruses underscore the urgent need for broad-spectrum antivirals for pandemic preparedness. Herein, we report AVI8122, a covalent pan-coronaviral inhibitor that targets 19 Mpros across the α, β, γ, and δ genera, encompassing bat, human, and other animal coronaviruses. AVI8122 exhibits low nanomolar potency and favorable pharmacokinetics in mice. Structural studies reveal that AVI8122 forms a covalent bond with the catalytic cysteine and maintains conserved interactions within the active sites of these Mpros. In cellulo, AVI8122 efficiently inhibited the replication of SARS-CoV-2 and its variants of concern as well as the activity of Mpros from all four genera. Furthermore, in mouse models, AVI8122 conferred dose-dependent protection against a lethal SARS-CoV-2 infection. Our findings position AVI8122 as an early lead compound and a tractable structural starting point for the development of broad-spectrum antivirals against future coronavirus spillover threats.
The COVID‐19 pandemic caused by SARS‐CoV‐2 created a global health crisis, and the virus still circulates, mutates, and causes illness and death. Vaccination remains the primary defense, but effectiveness can be reduced in immunocompromised individuals and against new variants, and clinically approved antivirals are limited by side effects, drug interactions, and resistance. Herein, we propose a novel application of tanespimycin (17AAG), the chaperone heat shock protein (HSP)90 inhibitor, as an antiviral against the SARS‐CoV‐2 main protease (Mpro). Mpro, due to its indispensable role in viral replication and absence of expression in the host, represents a key target for drug development. Through biochemical and cell‐based assays, structural analysis, and resistance‐mutant testing, we present data showing that 17AAG covalently binds to the active‐site cysteine of Mpro, disrupts its secondary structure, and protects cells from Mpro‐induced toxicity more effectively than nirmatrelvir. We further showed that 17AAG retains its covalent binding and structure‐disrupting activity against the nirmatrelvir‐resistance M165I variant. Additionally, since HSP90 is important for viral protein stability, virion assembly, and modulation of host immune response, 17AAG is a promising, versatile drug candidate that could accelerate antiviral development for COVID‐19.
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