Identification of 4-Amino-7-chloroquinoline Derivative with In Vitro Activity Against Chikungunya Virus

Pyridin-3-carbohydrazide strongly impairs Mayaro and Chikungunya virus replication in vitro

Alphaviruses, including Mayaro virus (MAYV) and Chikungunya virus (CHIKV), pose a growing public health threat, with increasing reports worldwide and no licensed antiviral treatments available. This study evaluated the antiviral activity of pyridine-3-carbohydrazide (P-3-C) against MAYV (strains BeAr and TRVL) and CHIKV (strain La Reunion, LR). Vero E6 or U-2OS cells were infected and treated with P-3-C for 24 h, and cell viability and replication levels were assessed. The 50% cytotoxic concentration (CC50), 50% effective concentration (EC50), and selective index (SI) were calculated. P-3-C showed concentration-dependent activity, strongly inhibiting post-entry stages of MAYV BeAr replication. Treatment reduced intracellular viral RNA levels by 2 log10 compared with controls and effectively decreased viral RNA release. P-3-C also inhibited MAYV TRVL (SI > 29.4) and CHIKV LR (SI > 48.8) in Vero E6 cells and retained selectivity in U-2OS cells (SI = 3.9 and 6.4, respectively). In a CHIKV U-2OS subgenomic replicon, an SI of 4.1 was found. Additionally, Fluorometric FRET-Based and MicroScale Thermophoresis (MST) assays indicated that P-3-C inhibited the nsP2 protease activity (IC50 = 12 µM ± 2) and exhibited binding affinity for nsP4 polymerase (Kd = 54 µM ± 1) from CHIKV, thereby suggesting that these non-structural proteins are potential molecular targets. Molecular docking positioned P-3-C within the catalytic pockets of nsP2 and nsP4 of both viruses. Overall, the data suggest that P-3-C exhibits a potential multitarget mechanism of action and highlight its antiviral activity against different alphavirus strains; however, this efficacy could be dependent on the cell line used.

Antiviral Activity of Synthetic Bis-Naphthoquinone (H-5) Against Chikungunya Virus Replication

Design, synthesis, and anti-influenza A virus evaluation of 2-aminoquinoline derivatives.

The rapid emergence of drug-resistant influenza A virus (IAV) strains has severely limited the efficacy of current antiviral therapies, highlighting an urgent need for novel agents with distinct mechanisms of action. In this study, a series of 2-aminoquinoline derivatives were synthesized via a trimethylsilyl trifluoromethanesulfonate (TMSOTf) -catalyzed annulation strategy. Evaluation of their anti-influenza virus activity revealed that derivative 3g exhibited potent antiviral efficacy, low cytotoxicity, and a high selectivity index(SI), making it the most promising candidate in this series. In vitro investigations revealed that 3g primarily acted during the early-to-mid stages of viral replication, significantly suppressing the transcription and translation of viral nucleoprotein (NP) and matrix protein 2 (M2), thereby effectively blocking viral replication and protein synthesis. Furthermore, 3g inhibited virus-induced apoptosis, reduced excessive production of reactive oxygen species (ROS) and nitric oxide (NO) triggered by viral infection, and markedly attenuated cytokine storm responses by suppressing the retinoic acid-inducible gene I (RIG-I)/Toll-like receptor 3 (TLR3)-mediated signaling pathways. In vivo experiments confirmed that 3g significantly reduced viral loads in the lungs of infected mice, alleviated pulmonary histopathological damage, and downregulated inflammatory factor levels, while exhibiting good biosafety. Collectively, these results position derivative 3g as a promising compound for the development of novel anti-influenza therapies.

Eugenol‐Derived Piperazine Mannich Bases as Selective Inhibitors of Mayaro Virus: In Vitro Antiviral Evaluation and Mechanistic Insights

Arboviruses represent an ongoing global health challenge, particularly in tropical regions where diseases caused by Chikungunya virus (CHIKV) and Mayaro virus (MAYV) continue to emerge. Given the absence of licensed antiviral therapies, the identification of effective small molecules remains critical. This study evaluated sixteen eugenol‐ and dihydroeugenol‐derived piperazine Mannich bases for antiviral activity against CHIKV, MAYV, and Zika virus (ZIKV) in Vero cells. Cytotoxicity was assessed using the MTT assay, and antiviral activity was quantified through median effective concentration (EC50) and selectivity index (SI) values. Time‐dependent cytopathic effect inhibition, plaque reduction assays, virucidal assays and docking were performed to characterize the mechanism of action. Three compounds (D3, D7, and E7) exhibited selective antiviral activity. Notably, compound D7 demonstrated low‐micromolar anti‐MAYV inhibition (EC50 = 14.43 μM; SI > 16.6), surpassing ribavirin by more than thirty‐fold in relative potency. Time‐dependent assays revealed progressive viral titer reductions of up to 3 log10 PFU/mL over 48 h. Virucidal assays confirmed that D7 does not directly inactivate viral particles. These findings identify D7 as a promising antiviral lead compound and provide a foundation for further mechanistic, optimization, and in vivo studies aimed at developing anti‐alphavirus therapeutics.

Development of Covalent Inhibitors of Chikungunya Virus nsP2 Cysteine Protease Enabled by Direct-to-Biology Synthesis and Screening

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.

Solanaceae-Derived Spirostane Saponins Inhibit Dengue Virus Replication and Viral Protein Expression and Exhibit Activity Against Multiple Arboviruses.

Mosquito-borne arboviruses, including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), represent a growing global health burden, yet no specific antiviral therapies are currently available for most of these infections. This unmet need highlights the importance of identifying novel antiviral compounds from biologically relevant sources such as plant-derived natural products. In this study, a bioactivity-guided approach was applied to methanolic extracts from eight Solanaceae species to identify compounds with antiviral activity against DENV-2 in Vero cells. Five extracts significantly reduced viral infectivity, with Solanum ovalifolium and Cestrum sp. achieving complete inhibition at non-cytotoxic concentrations. Subsequent fractionation and purification led to the isolation of four spirostane-type steroidal saponins, structurally characterized by UPLC-DAD-MS and 1D/2D NMR spectroscopy. Functional assays demonstrated that these compounds inhibit DENV replication, as evidenced by marked reductions in viral RNA levels, NS1 and NS3 protein expression, and infectious viral titers. Stage-specific analyses revealed that their antiviral activity is primarily associated with intracellular phases of the viral life cycle, consistent with inhibition of viral replication and/or protein processing. Molecular docking analyses supported these findings by predicting favorable interactions with key viral enzymes, particularly NS5 RNA-dependent RNA polymerase and NS3 helicase/protease. Importantly, the isolated compounds retained antiviral activity against ZIKV and CHIKV, indicating cross-arboviral efficacy. Together, these findings identify spirostane saponins as promising antiviral scaffolds and support their further development as broad-spectrum antiviral agents.