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Structure-Activity-Resistance Relationships of Novel Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors

Herman, Brian (2012) Structure-Activity-Resistance Relationships of Novel Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Nucleos(t)ide reverse transcriptase inhibitors (N(t)RTI) are essential components of combination antiretroviral therapy for treatment of human immunodeficiency virus type-1 (HIV-1) infection. N(t)RTI are analogs of natural 2’-deoxyribonucleos(t)ides that lack a 3’-hydroxyl. Once metabolized by host kinases to the active form, their incorporation into viral DNA by HIV-1 reverse transcriptase (RT) results in chain termination of DNA synthesis. N(t)RTI efficacy is undermined primarily by rapid selection of resistant/cross-resistant HIV-1 variants. Consequently, the development of novel N(t)RTI with activity against a broad range of N(t)RTI-resistant HIV-1 is of critical importance. Rational design of novel N(t)RTI with knowledge of analog structure-activity-resistance relationships with the RT target enzyme is the most promising approach. We hypothesized that uncovering knowledge of how N(t)RTI base, sugar, and phosphate structures influence activity and resistance phenotypes would aid in the rational design of new N(t)RTI with improved activity and resistance profiles. Therefore, a combination of biochemical, antiviral, molecular modeling, and cellular pharmacology analyses provided a detailed characterization of structure-activity-resistance relationships for inhibition of wild-type and NRTI-resistant HIV-1 by novel N(t)RTI. First, we studied two novel nucleoside phosphonate NtRTI, (R)-6-[2-phosphonylmethoxy]propoxy]-2,4-diaminopyrimidine (PMEO-DAPym) and (5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonate (GS-9148). We showed the diphosphate (-DP) form, PMEO-DAPym-DP acts as a purine mimetic that is recognized by RT as an adenosine analog and unambiguously incorporated across from thymine (DNA) or uracil (RNA). Studies indicated that PMEO-DAPym-DP and GS-9148-DP were superior to tenofovir-DP against both discrimination and excision RT resistance mechanisms. Next, we examined structure-activity-resistance relationships of 6-modified, 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) NRTI analogs. In RT-mediated DNA synthesis assays the triphosphate (-TP) form of each analog behaved as an adenosine mimetic for incorporation by HIV-1 RT. Importantly, the structure-activity relationships for incorporation and ATP-mediated excision were different, suggesting that new analogs can be designed that are efficiently incorporated but poorly excised by RT. RS-788, a 5’-monophosphate prodrug of 3’-azido-2’,3’-dideoxy-2,6-diaminopurine (3’-azido-2,6-DA-P), displayed potent activity against multi-NRTI-resistant HIV-1 and unique cellular metabolism. RS-788 was metabolized ~1:1 to both 3’-azido-2,6-DA-P and 3’-azido-ddG, thus delivering two distinct metabolites, each of which are potent RT chain-terminators that are incorporated opposite different bases, thymine and cytosine, respectively. Combinations of 3’-azido-2,6-DA-P+3’-azido-ddG synergistically inhibited multi-NRTI-resistant RT DNA synthesis.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Herman, Brianbdh9@pitt.eduBDH9
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorSluis-Cremer, Nicolasnps2@pitt.eduNPS2
Committee MemberMellors, John W.mellors@dom.pitt.eduJWM1
Committee MemberAmbrose, Zandreazaa4@pitt.eduZAA4
Committee MemberKhan, Saleemkhan@pitt.eduKHAN
Committee MemberTrakselis, Michaelmtraksel@pitt.eduMTRAKSEL
Date: 25 August 2012
Date Type: Publication
Defense Date: 9 July 2012
Approval Date: 25 August 2012
Submission Date: 20 August 2012
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 210
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Virology and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: HIV-1 Reverse Transcriptase Drug Resistance Kinetics Nucleoside Reverse Trascriptase Inhibitor Nucleotide Reverse Trascriptase Inhibitor
Date Deposited: 25 Aug 2012 15:43
Last Modified: 15 Nov 2016 14:02


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