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ABSTRACTGS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitroantiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50= 316 nM). Additive to synergistic in vitroantiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitrocross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

Iron uptake by transferrin from triacetohydroxamatoFe(III) (Fe(AHA)3) in the presence of bicarbonate has been investigated between pH 7 and 8.2. The protein transits from the opened apo- to the closed holoform by several steps with the accumulation of at least three kinetic intermediates. All these steps are accompanied by proton losses, probably occurring from the protein ligands and the side-chains involved in the interdomain H-bonding nets. The minor bihydroxamatoFe(III) species Fe(AHA)2exchanges its iron with the C-site of apotransferrin in interaction with bicarbonate without detectable formation of any intermediate protein-iron-ligand mixed complex; direct second-order rate constant k1= 4.15(±0.05) × 107M−1s−1. The kinetic product loses a single proton and undergoes a modification in its conformation followed by the loss of two or three protons; first-order rate constant k2= 3.25(±0.15) s−1. This induces a new modification in the conformation; first-order rate constant k3= 5.90(±0.30) × 10−2s−1. This new modification in conformation rate controls iron uptake by the N-site of the protein and is followed by a single proton loss; K3a= 6.80 nM. Finally, the holoprotein or the monoferric transferrin in its thermodynamic equilibrated state is produced by a last modification in the conformation occurring in about 4000 seconds. But for the Fe(AHA)3dissociation and the involvement of Fe(AHA)2in the first step of iron uptake, this mechanism is identical to that reported for iron uptake from FeNAc3. This implies that the exchange of iron between a chelate and serum-transferrin occurs by a single general mechanism. The nature of the iron-providing chelate is only important for the first kinetic step of the exchange, which can be slowed to such an extent that it rate limits the exchange of iron.