Tecovirimat is active against various MPXV strains, while cidofovir, brincidofovir, trifluridine, and gemcitabine have no detectable MPXV-specific antiviral activity

In treating patients with mpox, current treatment options are limited, with tecovirimat (TEC) being one of the few available. TEC has been approved by the European Medicines Agency (EMA) for treating patients with mpox and is in clinical use in Europe and Japan. However, following exposure to TEC, TEC-resistant variants such as A290V-containing variant (MPXV_R^TEC/A290V) emerge quickly. In such cases involving MPXV_R^TEC/A290V, alternative agents such as brincidofovir (BCV) have been used, although their efficacy remains controversial and their anti-MPXV activity is yet to be clearly defined. In the present work, we evaluated the anti-MPXV features of five agents (TEC; cidofovir, CDV; BCV; trifluridine, TFT; and gemcitabine, dFdC) reportedly active against various MPXV strains including MPXV^SPL2A7, MPXV^Zr-599, MPXV^Liberia and MPXV_R^TEC/A290V, employing cell-based quantitative assays using multiple target cell types such as VeroE6 cells as well as morphometric assays focusing on their cytostatic and cytotoxic natures. The EC₅₀ values of TEC against MPXV^SPL2A7, MPXV^Zr-599, and MPXV^Liberia were 0.001, 0.005, and 0.004 µM, respectively, without tangible cytotoxicity, while that against MPXV_R^TEC/A290V was ∼130-fold greater with 0.13 µM. The EC₅₀ values of CDV, BCV, TFT, and dFdC against MPXV_R^TEC/A290V were 18, 1.8, 3.8, and 0.02 µM, respectively; however, the apparent anti-MPXV activity of these four agents was highly associated with their cytotoxicity as they were examined with qualitative and quantitative cell-based-morphometric assays. The data strongly show that none the four agents examined exhibited significant anti-MPXV activity and indicate that effective anti-MPXV agents active against wild-type and drug-resistant variants are urgently needed.