Nucleosides for the treatment of respiratory RNA virus infections

Jordan, Paul C; Stevens, Sarah K; Deval, Jerome

The discovery of ALS-8112, the parent molecule of the prodrug ALS-8176 (lumicitabine), was the result of a screening campaign using a focused library of structurally diverse nucleoside and nucleotide analogs tested against RSV in an in vitro infectious assay. 158 The main scaffold identified from this screen was 2 0 difluoro-4 0 azido-cytidine. Further modifications at the 2 0 -and 4 0 -positions to improve anti-RSV potency and selectivity, led to the identification of ALS-8112 (2 0 fluoro-4 0 chloromethyl-cytidine) ( Figure 5 ). In vitro, ALS-8112 inhibits a broad panel of RSV A and B subtypes, as well as related pneumo-, paramyxo-, and rhabdoviruses. 159 In particular, we recently reported that ALS-8112 inhibits RSV and HMPV with similar in vitro potency. 160 The molecular target of ALS-8112 was determined by two independent methods. The polymerization function of the RSV L protein was identified as the target of ALS-8112 inhibition, first, by selecting and characterizing drug resistanceassociated mutations located in the L gene. When introduced into a wild-type RSV genome, four amino acid mutations (M628L, A789V, L795I, and I796V) were phenotypically associated with resistance to ALS-8112. 159 Enzymatic assays using purified recombinant RSV polymerase were critical to validate the mode of action of ALS-8112. In these assays, the 5 0 -triphosphate form of ALS-8112 (ALS-8112-TP) caused immediate chain termination of RNA synthesis and inhibition of the viral polymerization activity. This inhibitory effect was specific to RSV polymerase, since ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as HCV. The lack of inhibition against HCV was rationalized by molecular modeling, predicting steric clashing of ALS-8112-TP inside the active site of HCV polymerase. Because of the low oral bioavailability of ALS-8112, a series of 2 0 ,3 0 -diester prodrugs was evaluated for improved pharmacokinetic properties. One prodrug, ALS-8176, formed high levels of monophosphate and triphosphate in the lungs when administered orally to nonhuman primates. Because of its high oral bioavailability, ALS-8176 was evaluated for in vivo efficacy in African green monkeys infected with RSV. At the end of treatment, RSV RNA was undetectable in bronchoalveolar lavage samples from all four ALS-8176-treated animals. 159 Subsequently, a randomized, double-blind, clinical trial evaluated ALS-8176 given for 5 days to healthy adults inoculated with RSV. 161 The reduction in viral load in nasal washes associated with ALS-8176 treatment varied from 73% to 88% depending on the dose regimen. RSV RNA was undetectable 1.3 to 2.3 days after the start of ALS-8176 treatment compared with 7.2 days for placebo. Assessment of symptom scores and quantity of mucus produced also showed a clear effect on RSV-induced disease. This important result represents the first proofof-concept validation that an RSV replication inhibitor can be efficacious in humans. ALS-8176 is currently in clinical development for the treatment of RSV infection in hospitalized infants and adults (ClinicalTrials.gov identifier: NCT02202356, NCT02935673).