Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 154-169).

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Class of 2007 Abstract / Objectives: To explore the effects of human immunodeficiency virus protease inhibitors (HPI) on insulin metabolism and protein degradation in HepG2 hepatocytes in vitro. Methods: To see if HIV-protease inhibitors affect insulin degradation in a dose-dependent manner, HepG2 cells were incubated with various concentrations of tipranavir, indinavir, or atazanavir. After 125I-insulin was added, its degradation was measured by precipitation with trichloroacetic acid (TCA). To see the effect of HPIs on protein degradation, HepG2 cells labeled overnight with 3H-leucine were incubated with 50 mM of an HPI, followed by another HPI incubation including concentrations of insulin ranging from 10-12 to 10-6 M. Cells were solubilized and proteins were precipitated using TCA. Degradation was quantified as percent TCA soluble, normalized, plotted, and then compared using student’s t-test or one- way ANOVA. Results: Cellular insulin degradation was inhibited only by tipranavir at the highest concentrations of 75 and 100 mM (12.06 ± 1.07%, p=0.047 and 9.35 ± 0.44%, p=0.024, respectively) when compared to the control (17.01 ± 1.37%; n=3). None of the concentrations of indinavir or atazanavir decreased insulin degradation significantly. From the protein degradation experiments, the log EC50 of the control (no HPI) insulin dose-response curve was not statistically different compared to those of the individual HPIs. Conclusions: Except for high concentrations of tipranavir, it appears that HPI does not inhibit the cellular degradation of insulin. HPIs do not appear to inhibit the role of insulin in the inhibition of protein degradation significantly.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.

Drug resistance is the most important factor that influences the successful treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1), the causative organism of the acquired immunodeficiency syndrome (AIDS). Tremendous advances in our understanding of HIV and AIDS have led to the development of Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, to combat the virus. Though HAART has been successful in reducing AIDS-related morbidity and mortality, HIV rapidly evolves resistance leading to therapy failure. Thus, a better understanding of the mechanisms of resistance will lead to improved drugs and treatment regimens. Protease inhibitors (PIs) play an important role in anti-retroviral therapy. The development of resistance mutations within the active site of the protease greatly reduces its affinity for the protease inhibitors. Frequently, these mutations reduce catalytic efficiency of the protease leading to an overall reduction in viral fitness. In order to overcome this loss in fitness the virus evolves compensatory mutations within the protease cleavage sites that allow the protease to continue to recognize and cleave its substrates while lowering affinity for the PIs. Improved knowledge of this substrate co-evolution would help better understand how HIV-1 evolves resistance and thus, lead to improved therapeutic strategies. Sequence analyses and structural studies were performed to investigate co-evolution of HIV-1 protease and its cleavage sites. Though a few studies reported the co-evolution within Gag, including the protease cleavage sites, a more extensive study was lacking, especially as drug resistance was becoming increasingly severe. In Chapter II, a small set of viral sequences from infected individuals were analyzed for mutations within the Gag cleavage sites that co-occurred with primary drug resistance mutations within the protease. These studies revealed that mutations within the p1p6 cleavage site coevolved with the nelfinavir-resistant protease mutations. As a result of increasing number of infected individuals being treated with PIs leading to the accumulation of PI resistant protease mutations, and with increasing efforts at genotypic and phenotypic resistance testing, access to a larger database of resistance information has been made possible. Thus in Chapter III, over 39,000 sequences were analyzed for mutations within NC-p1, p1-6, Autoproteolysis, and PR-RT cleavage sites and several instances of substrate co-evolution were identified. Mutations in both the NC-p1 and the p1-p6 cleavage sites were associated with at least one, if not more, primary resistance mutations in the protease. Previous studies have demonstrated that mutations within the Gag cleavage sites enhance viral fitness and/or resistance when they occur in combination with primary drug resistance mutations within the protease. In Chapter III viral fitness in the presence and absence of cleavage site mutations in combination with primary drug resistant protease mutations was analyzed to investigate the impact of the observed co-evolution. These studies showed no significant changes in viral fitness. Additionally in Chapter III, the impact of these correlating mutations on phenotypic susceptibilities to various PIs was also analyzed. Phenotypic susceptibilities to various PIs were altered significantly when cleavage site mutations occurred in combination with primary protease mutations. In order to probe the underlying mechanisms for substrate co-evolution, in Chapter IV, X-ray crystallographic studies were performed to investigate structural changes in complexes of WT and D30N/N88D protease variants and the p1p6 peptide variants. Peptide variants corresponding to p1p6 cleavage site were designed, and included mutations observed in combination with the D30N/N88D protease mutation. Structural analyses of these complexes revealed several correlating changes in van der Waals contacts and hydrogen bonding as a result of the mutations. These changes in interactions suggest a mechanism for improving viral fitness as a result of co-evolution. This thesis research successfully identified several instance of co-evolution between primary drug resistant mutations in the protease and mutations within NC-p1 and p1p6 cleavage sites. Additionally, phenotypic susceptibilities to various PIs were significantly altered as a result of these correlated mutations. The structural studies also provided insights into the mechanism underlying substrate co-evolution. These data advance our understanding of substrate co-evolution and drug resistance, and will facilitate future studies to improve therapeutic strategies.