Biochemical, Structural, And Drug Design Studies Of Multi-Drug Resistant Hiv-1 Therapeutic Targets

BIOCHEMICAL, STRUCTURAL, AND DRUG DESIGN STUDIES OF MULTI-DRUGRESISTANT HIV-1 THERAPEUTIC TARGETS TAMARIA GRACE DEWDNEYDecember 2013Advisor: Dr. Ladislau C. KovariMajor: Biochemistry and Molecular BiologyDegree: Doctor of PhilosophyProtein point mutations acquired as a mechanism of survival against therapeutics cause structural changes that effect protein function and inhibitor binding. This work investigates the structural mechanisms that lead to multi-drug resistance to HIV-1 protease and integrase inhibitors. Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection andviral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug designfor the treatment of HIV-1 infected patients, thus the development of drug resistance has arisen as a major therapeutic and drug design challenge. To understand the molecular mechanisms leading to drug resistance we selected and characterized three multi-drug resistant HIV-1 protease patient isolates, identifying a previously unreported structural role for V32I, I47V, I54M and L90M in protease dynamics.To examine the role of the P1 and P1’ positions of the substrate in inhibitory efficacy of multi-drugresistant HIV-1 protease 769 (MDR 769), we performed a structure-function studies. We designed aseries of ligands and evaluated them using a combination of computational and experimental methods. Our results suggest two important strategies for rational drug design of protease inhibitors: (1) thepresence of fluorinated P1 or P1’ groups enhance the binding affinities in both wild-type and MDR PRvariants and (2) non-identical P1/P1’ residues play an important role in binding to multi-drug resistantHIV-1 protease. HIV-1 integrase is an essential enzyme necessary for the replication of the HIV virus as itcatalyzes the insertion of the viral genome into the host chromosome. Raltegravir was the first integraseinhibitor approved by the FDA for treatment of HIV-1 infection. HIV patients on raltegravir containingregimens may develop drug resistance mutations at residue 140 and 148 in the catalytic 140’s loopresulting in a 5-10 fold decrease in susceptibility to raltegravir. To understand the molecular mechanismsof drug resistance in HIV-1 integrase we performed molecular dynamics studies on the catalytic coredomain of raltegravir resistant HIV-1 protease in complex with raltegravir. These experiments suggest a gating function of the catalytic 140’s loop in active site accessibility as well as reduced flexibility as amechanism of raltegravir resistance. In addition, we developed a model for the full length HIV-1integrase and performed molecular dynamics of our model in complex with raltegravir and the viral DNA ends, identifying unique alterations in non-bonded interactions between the protein, DNA, and raltegravir as a result of the drug resistant mutations.The results of this work provide detailed structural information on HIV-1 protease and integrase in order to aid in the development of new therapeutics against these targets. Our work has implicationsnot only on HIV-1 therapy but the techniques described here can be used to study other proteins, design new compounds, and help aid in our understanding of protein structure. AUTOBIOGRAPHICAL STATEMENT Education09/2009-12/2013 Ph.D. in Biochemistry and Molecular BiologyDepartment of Biochemistry and Molecular BiologyWayne State University School of Medicine09/200408/2009 B.S. in Biological SciencesDepartment of Biological SciencesWayne State University PublicationsDewdney, T.G., Wang, Y., Reiter, S.J., Brunzelle, J., Kovari, I.A., Kovari L.C. 2013 LigandModifications to reduce the relative resistance of multidrug resistance HIV-1 protease. Biorg Med Chem21(23): 7430-7434 Dewdney, T.G., Wang, Y., Reiter, S.J., Kovari, I.A., Kovari L.C. 2013. Reduced HIV-1 integraseflexibility as a mechanism for raltegravir resistance. J Struct Biol S1047-8477(13)00188-3.10.1016/j.jsb.2013.07.008 Liu, Z., Yedidi, R.., Wang, Y., Dewdney, T.G., Reiter, S.J., Brunzelle, J.S., Kovari, I.A., Kovari, L.C.2013. Crystallographic study of multi-drug resistant HIV-1 protease lopinavir complex: Mechanism ofdrug recognition and resistance. Biochem Biophys Res Commun 437(2):199-204Liu, Z., Yedidi, R.., Wang, Y., Dewdney, T.G., Reiter, S.J., Brunzelle, J.S., Kovari, I.A., Kovari, L.C.2013. Insights into the mechanism of drug resistance: X-ray structure analysis of multi-drug resistantHIV-1 protease Ritonavir complex. Biochem Biophys Res Commun 431(2): 232-8Liu, Z., Wang, Y., Yedidi, R., Dewdney, T.G., Reiter, S.J., Brunzelle, J.S., Kovari, I.A., Kovari, L.C.2013. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes.Biochem Biophys Res Commun 430 (3): 1022-7 Wang, Y., Dewdney, T.G., Liu, Z., Reiter, S.J., Brunzelle, J., Kovari, I.A., Kovari L.C. 2012. Higherdesolvation energy reduces molecular recognition in multi-drug resistant HIV-1 protease. Biology 1(1),81-93Wang, Y., Dewdney, T.G., Liu, Z., Reiter S.J., Brunzelle, J.S., Kovari, I.A., Kovari L.C. 2011 X-raycrystal structure and dynamics reveal HIV-1 protease drug interactions. Studia UBB ChemiaWang, Y., Liu, Z., Brunzelle, J.S., Kovari, I.A., Dewdney, T.G. Reiter S.J., Kovari L.C. 2011 The higherbarrier of darunavir and tipranavir resistance for HIV-1 protease. Biochem Biophys Res Commun. 412(4):737-42