The Role of Second Generation Antiretroviral Drugs in HIV-1 Subtype B and non-B Variants Harboring Natural Polymorphisms and Drug Resistance Mutations

This thesis focuses on HIV-1 drug resistance and on the antiviral activity of several non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). We have explored the mutational pathways and resistance patterns of several new NNRTIs (etravirine (ETR) and rilpivirine (RPV)) (Chapters 2 and 3). In addition, the drug resistance profile and potential of a novel protease inhibitor (PI) PL-100 is presented in Chapters 4 and 5. In the first project, we used both B and non-B subtypes of HIV-1 to select for ETR resistance and showed that ETR selected for mutations at positions V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y and M230L within 18 weeks of commencing drug pressure. Interestingly, E138K was the first mutation to emerge in most instances. Viral clones containing E138K displayed low-level phenotypic resistance to ETR (3.8-fold) and modestly impaired replication capacity (2-fold) compared to wild-type virus. We also examined resistance patterns to ETR and RPV in viruses containing NNRTI mutations at baseline. In wild-type (wt) viruses and viruses containing K103N alone, E138K or E138G mutations were observed in the presence of either ETR or RPV drug pressure followed by the appearance of other NNRTI resistance mutations. Alternatively, subtype B viruses containing Y181C generated V179I/F or A62V/A on exposure to ETR or RPV drug pressure, respectively, but not E138K. The addition of mutations at position 138 to Y181C did not significantly enhance levels of resistance to ETR or RPV. We also observed that the combination of Y181C and E138K may lead to a less fit virus compared to virus containing Y181C alone. Based on these findings, we suggest that Y181C may be antagonistic to E138K. The tissue culture drug resistance analysis of PL-100 in subtype C and CRF01_AE viruses is described in Chapter 4. PL-100 selected for PI resistance mutations along either of two distinct pathways, one of which involved resistance mutations at positions V82A and L90M while the other involved a mutation at position T80I, with other mutations being observed at positions M46I/L, I54M, K55R, L76F, P81S and I85V. An accumulation of at least three mutations in the protease flap and enzyme active sites were required in each case for high-level resistance to occur, demonstrating that PL-100 has a high genetic barrier against the development of drug resistance.  In Chapter 5, we evaluated the potential of PL-100 as a second generation HIV-1 protease inhibitor. PL-100 resistant variants emerged within 8-48 weeks while darunavir (DRV) did not select for resistance mutations over a period of 40 weeks. Structural modeling demonstrated that the high genetic barrier of DRV is due to numerous interactions with protease that include hydrogen-bonding to PR backbone oxygens at amino acid positions A28, D29 and D30 via di-tetrahydrofuran (THF) groups, while binding of PL-100 was predominantly based on polar interactions and delocalized hydrophobic interactions through its diphenyl groups. Our data suggest that hydrogen bonding contacts and the di-THF group in DRV, as well as the hydrophobic nature of PL-100, contribute to PI binding and a high genetic barrier for resistance and that redesigning the structure of PL-100 to include a di-THF group might improve it antiviral potency and drug resistance profile.

Keyphrases: Rilpivirine, PL-100, HIV-1 subtype, Drug resistance, Antiviral activity, Cross-resistance, second generation, Replication capacity, Genetic barrier, Etravirine, Rilpivirine, PL-100