While changes in vdW interactions of K136 with GLE were solely dependent on this residues conformation, those of 123 were caused by GT3a-specific R123T polymorphism. Although the protease structures of the different genotypes are overall very similar, analysis of distance difference plots (Figure S.5) revealed that outside the active site, these structures show extensive overall structural plasticity with many regions of the enzyme diverging between 1C1.4 ? (Physique 3B and Physique S.5) with respect to each other and some loop regions diverging up to 7 ?. permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially Hhex can be extended to other proteins. Introduction An estimated 71 million people (~3.5M in the US) are chronically infected with HCV, which is the leading cause of liver cancer and cirrhosis.1 There are seven different HCV genotypes (GTs) and multiple subtypes of diverse global distributions with GT1 accounting for ~50% and GT3 for ~30% of the global infections.2C5 Genotypes 1 and 2 have a diverse global distribution; 3 is usually endemic in South Asia, 4 in the Middle East and Central Africa, 5 in South Africa, 6 in Asia and 7 in central Africa.2C5 In the last decade the treatment of HCV infection has been revolutionized with direct-acting antivirals (DAAs) including NS3/4A protease inhibitors (PIs),6C10 but the genetic diversity among genotypes and within a viral population presented a challenge to the development H100 of efficient therapies. HCV NS3/4A is usually a bifunctional protein comprised of an N-terminal protease domain name and a C-terminal helicase domain name. The protease domain name (amino acids 1C180) is usually a serine protease requiring an 11 amino acid peptide from NS4A as a cofactor H100 for folding and activity. The protease is essential for viral maturation, responsible for cleaving the viral polyprotein at various sites (3C4A, 4A4B, 4B5A, and 5A5B). HCV NS3/4A protease sequences vary among the seven genotypes with sequence identities on amino acid level ranging from 68% to 82% (Table S.1). Alignment of the amino acid sequences (Physique S.1) highlights the high degree of conservation throughout the protein and its active side. So far, structural and most biochemical studies focused on GT1a, the only GT that allowed structural characterization. Without crystal structures of NS3/4A proteases of the other genotypes, the impact of various polymorphisms and sequence variations, especially those outside the active site, on protease structure, activity H100 or inhibition has not been investigated. Previously, we created a chimeric protease to emulate the inhibition profile of GT3a by substituting three active site polymorphisms (R123T, D168Q and I132L) into GT1a NS3/4A.11 This GT1a3a chimera largely recapitulated inhibition characteristics of GT3a, and allowed crystal structure characterization. Other than the GT1a3a chimera, no structure of non-GT1a NS3/4A has been decided before and differences among genotypes have been unexplored. HCV genotypes have varied resistance-associated substitutions (RASs), and susceptibility to DAAs. The 7 FDA approved all-oral DAA combination therapies have varied effectiveness, and especially the earlier combinations can fail against certain genotypes.6 Fortunately, the three newest oral DAA regimens, Epclusa (sofosbuvir, velpatasvir),12 Vosevi (sofosbuvir, velpatasvir, voxilaprevir),13C14 and Mavyret (pibrentasvir, glecaprevir),15C16 are effective against all HCV genotypes with improved sustained virological response (SVR) rates and good tolerance in patients. While Epclusa, which does not contain a PI, is widely used, Mavyret with the latest generation PI glecaprevir (GLE; Physique 1A) is the most recommended therapy due to its short 8-week treatment duration and pan-genotypic activity, especially for treatment-naive patients without cirrhosis.8C10 In clinical studies Mavyret had a cure rate of 98%, and treatment failures of 1% are primarily reported for patients infected with GT3a.17 The basis of improved activity of GLE is not readily apparent considering the stark similarity in chemical structure with the earlier PI grazoprevir (GZR), which had lower potency especially against GT3 and certain resistance-associated substitutions (RASs). Open in a separate window Physique 1: Structure of Glecaprevir.(A) Chemical structure.