The reverse transcriptase enzyme of HIV-1 remains one of the most important targets for therapeutic treatment of AIDS. One of the most difficult obstacles for successful treatment is the development of resistance by the enzyme to both nucleoside and nonnucleoside inhibitors. In order to gain insights into the molecular mechanisms of drug resistance, the three-dimensional structures of drug-resistant mutants of HIV-1 reverse transcriptase will be determined by the techniques of X-ray crystallography. Each of the mutants described has been cloned, and several of the mutants have already been crystallized. The laboratories of Dr. Edward Arnold (CABM/Rutgers) and Dr. Stephen Hughes (NCI-Frederick) have solved the structure of a ternary complex of HIV-1 RT, a double-stranded DNA template-primer, and a monoclonal antibody Fab fragment at 3.0 A resolution, and the structure is being refined at 2.8 A resolution. Most of the mutant structures will be determined using this crystal form and convenient difference Fourier techniques. Where possible, the structures of nucleoside-resistant mutations will also be studied with bound triphosphate forms of nucleoside inhibitors. Some of the studies with nonnucleoside-resistant mutations will utilize a crystal form containing HIV-1 RT with bound nonnucleoside inhibitors that diffracts X-rays to a resolution of 2.6 A. Specifically, the structures of mutants resistant to AZT (with changes at residues 41, 67, 70, 215, 219), ddI (74 and 184), ddGTP (89), and a variety of nonnucleoside inhibitors (100, 103, 106, 181, 188, 236) will be determined. We will study mutations that, when added to an AZT- resistant genotype, lead to a return of AZT sensitivity (such as 74 added to the five AZT-resistant mutations, and 181 added to 215). We are prepared to study additional combinations that become identified as being clinically significant. Finally, we propose to determine the structure of a complex of a TIBO nonnucleoside inhibitor with a mutant (P236L) that is BHAP-resistant but hypersensitive to TIBO and other nonnucleoside RT inhibitors. These studies should provide a three-dimensional basis for understanding the molecular mechanisms of HIV-1 RT drug resistance. The known structure of HIV-1 RT in complex with template-primer has permitted placement of drug-resistant mutations in a three-dimensional context, but in order to fully interpret RT drug resistance, it will be valuable to know the precise changes caused by the amino acid changes (loss of contact, steric conflict, conformational changes of protein, nucleic acids, or both, etc). It is hoped that this information may contribute to the development of inhibitors or strategies that can slow or even overcome the development of clinical resistance to antiviral treatments of AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI036144-04
Application #
2004121
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1994-04-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Miscellaneous
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Hsiou, Y; Ding, J; Das, K et al. (2001) The Lys103Asn mutation of HIV-1 RT: a novel mechanism of drug resistance. J Mol Biol 309:437-45
Sarafianos, S G; Das, K; Clark Jr, A D et al. (1999) Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl Acad Sci U S A 96:10027-32
Hsiou, Y; Das, K; Ding, J et al. (1998) Structures of Tyr188Leu mutant and wild-type HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor HBY 097: inhibitor flexibility is a useful design feature for reducing drug resistance. J Mol Biol 284:313-23
Arnold, E; Das, K; Ding, J et al. (1996) Targeting HIV reverse transcriptase for anti-AIDS drug design: structural and biological considerations for chemotherapeutic strategies. Drug Des Discov 13:29-47
Kleim, J P; Rosner, M; Winkler, I et al. (1996) Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Le Proc Natl Acad Sci U S A 93:34-8
Das, K; Ding, J; Hsiou, Y et al. (1996) Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. J Mol Biol 264:1085-100
Hsiou, Y; Ding, J; Das, K et al. (1996) Structure of unliganded HIV-1 reverse transcriptase at 2.7 A resolution: implications of conformational changes for polymerization and inhibition mechanisms. Structure 4:853-60
Kroeger Smith, M B; Rouzer, C A; Taneyhill, L A et al. (1995) Molecular modeling studies of HIV-1 reverse transcriptase nonnucleoside inhibitors: total energy of complexation as a predictor of drug placement and activity. Protein Sci 4:2203-22
Ding, J; Das, K; Moereels, H et al. (1995) Structure of HIV-1 RT/TIBO R 86183 complex reveals similarity in the binding of diverse nonnucleoside inhibitors. Nat Struct Biol 2:407-15
Patel, P H; Jacobo-Molina, A; Ding, J et al. (1995) Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. Biochemistry 34:5351-63

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