Project Details

Description

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.
StatusFinished
Effective start/end date4/1/9412/31/98

Funding

  • National Institutes of Health: $233,608.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $214,535.00
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)

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