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 (K_d = 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.