We have found that covalent attachment of myristate (Cl4:0) to the NH2-terminal Gly residue of HIV-1 Pr55gag by myristoylCoA:protein N-myristoyltransferase (NMT) is necessary for viral assembly. Our analysis of the mechanism of action of NMT has led to the synthesis of myristic acid analogs which are alternative substrates for acylCoA synthetase and NMT. Group 6B heteroatom substituted analogs with altered physical chemical properties (including reduced hydrophobicity) were found to be selectively transferred to subsets of cellular N-myristoylproteins (owing in part to an apparent co-operative interaction between the enzyme's acylCoA and peptide binding site). Once incorporated they produce analog-dependent and specific alterations In protein function. Incubation of one such analog - 13-oxatetradecanoic - acid with infected H9 cells results in its incorporation into HIV-1 Pr55gag and nef, inhibition of proteolytic processing of gag, and inhibition of viral replication in acutely and chronically infected H9 cells without cellular toxicity. Our group proposes to continue to use biochemical, organic chemical, genetic and biophysical methods to define the kinetic mechanisms and structure/activity relationships of S. cerevisiae and human NMTs in an attempt to better understand how to design novel analogs with altered physical properties which can be selectively targeted to viral proteins by cellular NMT and thereby disrupt the their function(s). This will involve (1) isolation of human NMT cDNA and expression of the human enzyme In E. coli; (2) systematic synthesis of fatty acid analogs with varying structural motifs to explore issues of enzyme recognition; (3) definition of the enzyme's kinetic mechanism using a recently developed continuous assay; (4) use ts mutants of the S. cerevisiae NMT1 gene to identify structural features critical for (a) catalysis; (b) the mechanisms underlying catalysis and (c) the nature of its functional Interactions with myristoylCoA generating systems; (5) NMR and x-ray studies of the interactions of NMT with its ligands (6) use of H9 cells and an E. coli-expression system to examine the effects of analogs on gag polyprotein precursor processing by viral protease and intracellular targeting; (7) characterization of the metabolic processing of radiolabeled analogs in H9 cell cultures and in mice; and (8) assessment of the efficacy of analogs in animal models so that decisions concerning compound selection for clinical trials can be made as rapidly as possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI027179-07
Application #
2063736
Study Section
Special Emphasis Panel (SRC (41))
Project Start
1988-09-30
Project End
1995-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Knoll, L J; Johnson, D R; Bryant, M L et al. (1995) Functional significance of myristoyl moiety in N-myristoyl proteins. Methods Enzymol 250:405-35
Bhatnagar, R S; Gordon, J I (1995) Thermodynamic studies of myristoyl-CoA: protein N-myristoyltransferase using isothermal titration calorimetry. Methods Enzymol 250:467-86
Knoll, L J; Schall, O F; Suzuki, I et al. (1995) Comparison of the reactivity of tetradecenoic acids, a triacsin, and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins. J Biol Chem 270:20090-7
Knoll, L J; Johnson, D R; Gordon, J I (1995) Complementation of Saccharomyces cerevisiae strains containing fatty acid activation gene (FAA) deletions with a mammalian acyl-CoA synthetase. J Biol Chem 270:10861-7
Lodge, J K; Johnson, R L; Weinberg, R A et al. (1994) Comparison of myristoyl-CoA:protein N-myristoyltransferases from three pathogenic fungi: Cryptococcus neoformans, Histoplasma capsulatum, and Candida albicans. J Biol Chem 269:2996-3009
Lu, T; Li, Q; Katoh, A et al. (1994) The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Polar probes of the enzyme's myristoyl-CoA recognition site. J Biol Chem 269:5346-57
Johnson, D R; Bhatnagar, R S; Knoll, L J et al. (1994) Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem 63:869-914
Lodge, J K; Jackson-Machelski, E; Toffaletti, D L et al. (1994) Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans. Proc Natl Acad Sci U S A 91:12008-12
Johnson, D R; Knoll, L J; Levin, D E et al. (1994) Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism. J Cell Biol 127:751-62
Johnson, D R; Cok, S J; Feldmann, H et al. (1994) Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation. Proc Natl Acad Sci U S A 91:10158-62

Showing the most recent 10 out of 48 publications