Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox

Abstract

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 μM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

Keywords: antiviral agents; chemical synthesis; chemistry; poxvirus.

FIG 1

Chemical structure of CMLDBU6128 (1) and overview of exploratory PDPM analog libraries based on variation at C3 (R¹), C7 (R²), and N2 (R³). The red numbers represent sites of potential modifications described in this study.

FIG 2

Synthesis of pyridopyrimidinones (PDPMs) used in this study via Biginelli multicomponent reaction followed by gold-catalyzed cycloisomerization (Equation 1). Variation of the input aldehyde (3) afforded a variety of PDPM analogs (1) and (10 to 27) (Equation 2). A limited number of N-alkylation reactions gave analogs (7 to 9) (Equation 3).

FIG 3

Vaccinia virus gene expression in the presence of compounds (12) and (16). Late viral reporter expression (normalized) at 24 h postinfection (hpi) following infection with vaccinia virus LV at an MOI = 0.1 in Vero E6 cells in the presence of increasing concentrations of (12) (A) or (16) (B) is shown in closed circles. Dotted lines represent cell viability (normalized to DMSO control) and the same concentrations, determined at 24-h post drug treatment. Virus gene expression at 12 hpi at MOI = 1 in the presence of increasing concentrations of (12) (A) or (16) (B) is shown in (C) and (D), respectively. Error bars represent standard deviations from triplicate (B) or quadruplicate (A, C, and D) repeated conditions. A four-point (0.01 μM to 10 μM) dilution series shows plaque reduction in triplicate conditions with both compounds compared to no treatment (Tx) (E). LV reporter virus was compared to two demonstrated anti-poxviral drugs cidofovir and molnupiravir (F) in the same dilution series of (A to D) in Vero E6 cells.

FIG 4

Ability of compounds to decrease variola virus growth and spread. (A) The ability of the compounds to halt a single round of variola virus replication was observed by infecting A549 cells with an MOI = 5 and sampling up to 24 hpi. The starting inoculum varied by 13% and 26%, when comparing the compound-treated wells to the DMSO control, for BSH74_sol and SLN68_258, respectively. (B) The ability of the compounds to decrease variola virus spread was observed by infecting A549 cells at an MOI = 0.01 and sampling up to 72 hpi. The starting inoculum varied by 25 to 26%, for BSH74_sol and SLN68_258, respectively, compared to the DMSO control. All compounds were able to decrease viral spread which is evident by percent reduction calculations consistent with a conserved mechanism of anti-poxviral activity. Experiments were conducted in duplicate by titering on BSC-40 cells to calculate the PFU/mL. Titer counts were averaged, and the standard deviation was calculated and graphed using GraphPad.