Purine Salvage Drives the Efficacy of an Adenosine Analog Inhibitor of Leishmania RNA Virus 1 (LRV1)

Leishmania is a genus of single‐cell eukaryotic protozoan parasites that cause a range of clinical presentations, from cutaneous lesions to lethal visceral disease. An estimated 1.7 billion people are at risk, with 12 million manifesting active disease and at least 10‐fold more with asymptomatic infections. Many New World parasite strains are chronically infected with Leishmania RNA virus 1 (LRV1), a member of the Totiviridae family of encapsidated dsRNA viruses. The presence of LRV1 has been shown to increase pathology and parasitemia in rodent leishmaniasis models, and to promote treatment failure and relapse in human patients infected with Leishmania guyanensis or L. braziliensis. This suggests that inhibiting LRV1 could provide therapeutic benefits either alone or in combination with existing leishmaniasis treatments. From screening a small library of known antivirals we identified several agents inhibiting Leishmania growth or showing selective inhibition of LRV1. Compounds showing both growth and LRV1 inhibition were not observed, probably because cell division is required for LRV1 loss. 2′‐C‐methyl adenosine (2CMA) was able to selectively inhibit LRV1 with an EC 50 of ~3 μM and only minimal effects on L. guyanensis growth (EC 50 > 100 μM) (Kuhlmann et al. , submitted). Here, we tested the hypothesis that 2CMA inhibits the viral RNA‐dependent RNA polymerase (RDRP) activity following activation to its triphosphate form. LRV1 virions were purified by density gradient centrifugation and fractionated into low, medium, and high density fractions, roughly corresponding to empty, ssRNA and dsRNA‐bearing particles. No inhibition of RDRP activity was seen in any fraction with up to 1 mM 2CMA. With 2CMA triphosphate (2CMA‐TP), however, IC 50 s of 130–1000 μM were obtained, depending on the particle density and quantitation of the synthesis of abortive or full length transcripts. In contrast, an IC 50 of >1mM was obtained with dTTP and dATP had no measurable effect. Thus, 2CMA acts in its triphosphate form to specifically inhibit the LRV1 RDRP, albeit with relatively low potency. Since the 2CMA‐TP IC 50 s greatly exceeded the 2CMA EC 50 for LRV1 within live cells, we hypothesized that the ability of Leishmania to salvage and convert exogenous nucleobases and nucleosides would lead to accumulation of high levels of 2CMA‐TP. We first quantitated the intracellular volume of L. guyanensis promastigotes by confocal microscopy volumetric measurements and flow cytometry, and validated HPLC methods for identifying and quantitating intracellular 2CMA‐TP. These studies showed dose‐dependent 2CMA‐TP accumulation, reaching 400 μM at an external concentration of 10 μM 2CMA. Thus, scavenging and activating nucleosides results in accumulation of 2CMA‐TP to high levels, overcoming its relatively low potency. These data suggest that efforts are warranted to identify more potent RDRP inhibitors similarly accumulating to high intracellular levels, which would facilitate testing the role of LRV1 inhibition in ameliorating disease severity. Support or Funding Information NIH grants RO1AI029646 and R56AI099364 (SMB), the Sigma‐Aldrich Predoctoral Fellowship and the Sondra Schlesinger Graduate Student Fellowship (JIR), and the Division of Infectious Diseases (FMK)