Publication Date


Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology


Biochemistry and Molecular Pharmacology

First Thesis Advisor

Celia A. Schiffer, Ph.D.


HIV Protease, HIV Protease Inhibitors, Drug Resistance, Viral


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.



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