In summary P P macrocyclization proved to be an effective

In summary, P2-P4 macrocyclization proved to be an effective strategy to drive activity in this series of HCV NS3 protease inhibitors while microsomal stability was enhanced by the introduction of steric bulk along the tether. This modification augmented the previously-described tactic of improving microsomal stability by converting the P1 cyclopropyl vinyl moiety into the corresponding -cyclopropyl moiety. Compound , which incorporated both structural elements (P1 -cyclopropyl moiety and steric bulk along the tether) exhibited an excellent PK profile in rats where the oral bioavailability was 100%. However, the CV profiles of and were not acceptable as a significant effect on cardiac function was observed in the isolated rabbit heart model. This result, while disappointing, was enlightening since it suggested that restricting the conformation between P2∗ and P4 was not an effective strategy to mitigate the CV toxicology signal noted in acyclic analogues such as . This conclusion led to a discontinuation of efforts in this chemical series. Acknowledgments
Introduction Direct Acting antivirals (DAAs) have revolutionized treatment of chronic HCV infection (Lange et al., 2014, Polenakovik, 2015, Zhang et al., 2016). In 2003 the first anti-HCV DAA was described (BILN 2061, also known as Ciluprevir), providing proof of concept that targeting the HCV protease represents a potent therapeutic option (Lamarre et al., 2003). The non-structural protein 3 (NS3) is a multifunctional protein that contains a serine protease domain at its N-terminus. Inhibition of the NS3 protease supports viral clearance via both inhibition of viral polyprotein cleavage, reducing viral replication, in addition to restoration of MAVS and TRIF mediated innate immune signaling (Meylan et al., 2005). IFN-free DAA combination therapies including second generation NS3 PIs can achieve high clearance rates in hard-to-treat patient groups (Buti et al., 2016, Zeuzem et al., 2015). However, despite the NS3 protease domain's importance as a therapeutic target, genetic variability in viral NS3 protease populations remains poorly characterized in HCV infection at different stages of the viral life-cycle. Studying HCV transmission in a natural setting is challenging due to the often asymptomatic nature of infection (Irving and Brown, 2010), with limited numbers of studies characterizing samples from both donor and recipient(s). To circumvent the low availability of samples from donor/recipient pairs, permissive animal models can be used to model such scenarios (Vercauteren et al., 2015b), yielding novel insights into the transmission process (Brown et al., 2012). HCV replication in infected hepatocytes is characterized by a high rate of virus production and a correspondingly high level of genetic Cholic acid in circulating viruses (Simmonds, 2004, Tarr et al., 2015). This is due to the lack of efficient proofreading by the HCV RNA-dependent RNA polymerase. As a result, HCV exists as a dynamic swarm of genetically related genomes within infected patients rather than a clonal population (Martell et al., 1992, Pawlotsky, 2006). While chimpanzees are permissive to infection with non-laboratory strains of HCV (Bukh, 2012) ethical concerns have resulted in research animals being retired in both the US and the EU. Consequently, the current state-of-the-art animal model for heterologous challenge with clinically relevant strains is the human-liver chimeric mouse model: the liver of Alb-uPA transgenic SCID mice can be engrafted with human adult hepatocytes rendering them permissive to HCV infection (Vercauteren et al., 2015b, Vercauteren et al., 2014).
Material and methods
Results
Discussion Previous studies which have tracked HCV transmission events from donor to recipient often focus on the envelope glycoproteins E1 and E2, which are located on the virion surface and represent the primary determinants of HCV entry (Tarr et al., 2015). These data indicate that, in specific instances, a genetic bottleneck is observed upon transmission due to initial infection with a limited number of founder viruses (D'Arienzo et al., 2013, Li et al., 2016, Li et al., 2012, Wang et al., 2010). However, current antiviral therapies target the non-structural proteins and the effect of transmission on these important coding-regions was previously poorly defined. To address this shortfall, we utilized NGS to track the effect of HCV transmission on NS3 protease populations in vivo. In some scenarios of HCV transmission, such as via needle stick injury or intravenous drug use, a limited number of founder strains initiate productive infection (D'Arienzo et al., 2013, Li et al., 2016, Li et al., 2012, Wang et al., 2010). These scenarios are not accurately modeled in our study as a relatively high multiplicity of infection was used to inoculate recipient animals. However, in instances of vertical transmission from mother-to-child (Honegger et al., 2013) or graft re-infection post orthotopic liver transplantation (Fafi-Kremer et al., 2010), a high multiplicity of strains can potentially initiate infection. Our study is likely to recapitulate the viral population complexity transmitted in these scenarios. Furthermore, our analyses confirm that diverse viral populations, similar to those observed in natural infection, can be transmitted in animal challenge studies for studying preclinical effectiveness of vaccine candidates and novel therapeutic regimens. However, in addition to viral load, the diversity of transmitted populations differs between individual recipients and this should be monitored and taken into consideration when interpreting results.