Inhibition of hepatitis C viral RNA-dependent RNA polymerase by α-P-boranophosphate nucleotides: Exploring a potential strategy for mechanism-based HCV drug design

► α-P-BH3 modification increased incorporation of ribose modified ATP by HCV NS5BΔ55. ► Rp-ATPαB is a better substrate of HCV NS5BΔ55 RdRP than ATP. ► The NS5BΔ55 polymerase prefers the Rp stereoisomer of boranophosphate analogs. ► Rp-α-P-BH3 modification shifted the potency of 2′-OMe ATP and 3′-dATP 5- and 21-fold. ► Both ribose-modified ATPαB analogs exhibited a competitive mode of inhibition. Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdRP) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA. Boranophosphate nucleotides contain a borane (BH3) group substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics capable of modulating the reading and writing of genetic information. We determine that HCV NS5BΔ55, being a stereospecific enzyme, incorporates the Rp isomer of both ATPαB and the two boranophosphate analogs: 2′-O-methyladenosine 5′-(α-P-borano) triphosphate (2′-OMe ATPαB, 5a) and 3′-deoxyadenosine 5′-(α-P-borano) triphosphate (3′-dATPαB, 5b). The Rp diastereomer of ATPαB (6), having no ribose modifications, was found to be a slightly better substrate than natural ATP, showing a 42% decrease in the apparent Michaelis–Menten constant (Km). The IC50 of both 2′-O-Me and 3′-deoxy ATP was decreased with the boranophosphate modification up to 16-fold. This “borano effect” was further confirmed by determining the steady-state inhibitory constant (Ki), showing a comparable potency shift (21-fold). These experiments also indicate that the boranophosphate analogs 5a and 5b inhibit HCV NS5B through a competitive mode of inhibition. This evidence, together with previous crystal structure data, further supports the idea that HCV NS5B (in a similar manner to HIV-1 RT) discriminates against the 3′-deoxy modification via lost interactions between the 3′-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3′-OH and the pyrophosphate leaving group during phosphoryl transfer. To our knowledge, these data represent the first time a phosphate modified NTP has been studied as a substrate for HCV NS5B RdRP.