Exploring Molecular Mechanisms of Drug Resistance in HIV-1 Protease through Biochemical and Biophysical Studies: A Dissertation

Abstract

The human immunodeficiency virus type-1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS) in the world. As there is no cure currently available to treat HIV-1 infections or AIDS, the major focus of drug development efforts has been to target viral replication in an effort to slow down the progression of the infection to AIDS. The aspartyl protease of HIV-1 is an important component in the viral replication cycle and thus, has been an important anti-HIV-1 drug target. Currently there are nine protease inhibitors (PIs) that are being used successfully as a part of highly active antiretroviral therapy (HAART). However, as is with all HIV-1 drug targets, the emergence of drug resistance substitutions within protease is a major obstacle in the use of PIs. Understanding how amino acid substitutions within protease confer drug resistance is key to develop new PIs that are not influenced by resistance mutations. Thus, the primary focus of my dissertation research was to understand the molecular basis for drug resistance caused by some of these resistance substitutions. Until recently, the genetic diversity of the HIV-1 genome was not considered to be important in formulating treatment strategies. However, as the prevalence of HIV-1 continues, the variability of the HIV-1 genome has now been identified as an important factor in how the virus spreads as well as how fast the infection progresses to AIDS. Clinical studies have also revealed that the pathway to protease inhibitor resistance can vary between HIV-1 clades. Therefore, in studying the molecular basis of drug resistance in HIV-1 protease, I have also attempted to understand how genetic variability in HIV-1 protease contributes to PI resistance. In Chapters II, III and Appendix 1, I have examined how clade specific amino acid variations within HIV-1 CRF01_AE and clade C protease affect enzyme structure and activity. Furthermore, I have examined how these sequence variations, which are predominantly outside the active site, contribute to inhibitor resistance in comparison to clade B protease. With the results presented in Chapter II, I was able to show that sequence variations within CRF01_AE protease resulted in structural changes within the protease that might influence enzyme activity. In Chapter III, I focused on how sequence variations in CRF01_AE influence protease activity and inhibitor binding in comparison to clade B protease. Enzyme kinetics data showed that the CRF01-AE had reduced catalytic turnover rates when compared to clade B protease. Binding data also indicated that CRF01_AE protease had an inherent weaker affinity for the PIs nelfinavir (NFV) and darunavir (DRV). In work described in Chapter III, I have also examined the different pathways to NFV resistance seen in CRF01_AE and clade B protease. Using x-ray crystallographic studies I have shown the molecular mechanism by which the two different pathways confer NFV resistance. Furthermore, I provide a rational for why different resistance pathways might emerge in the two clades. In Appendix I, I present results from a parallel study carried out on clade C protease. In Chapter IV, I have examined the role of residue 50 in HIV-1 protease in modulating inhibitor binding. Patients failing amprevavir (APV) and DRV therapy often develop the I50V substitution while the I50L substitution is often observed in patients failing atazanavir (ATV) therapy. This indicates that by making subtle changes at residue 50 the protease is able to confer differential PI resistance. With binding data presented in this chapter I have shown that substitutions at residue 50 change the susceptibility profiles of APV, DRV and ATV. Furthermore, from analyses of protease-inhibitor complexes, I have described structural insights into how substitutions at residue 50 can modulate inhibitor binding. This thesis presents results that reveal mechanistic insights into how a number of resistance substitutions within protease confer drug resistance. The results on non-B clade proteases demonstrate that clade specific sequence variations play a role in modulating enzyme activity and influence the pathway taken to confer PI resistance. Furthermore, the results provide structural insights into how amino acid substitutions outside the active site effectively alter inhibitor binding.