THE HUMAN N-MYRISTOYLTRANSFERASES AS POTENTIAL PHARMACOTHERAPEUTIC TARGETS IN HIV-1 REPLICATION
Open Access
- Author:
- Seaton, Kelly Elizabeth
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 07, 2009
- Committee Members:
- Charles D Smith, Dissertation Advisor/Co-Advisor
Charles D Smith, Committee Chair/Co-Chair
Jong Kak Yun, Committee Chair/Co-Chair
Kent Eugene Vrana, Committee Member
Melvin Lee Billingsley, Committee Member
John Warren Wills, Committee Member - Keywords:
- human N-myristoyltransferases
myristoylation
HIV - Abstract:
- Human Immunodeficiency Virus and consequent Acquired Immune Deficiency Syndrome (AIDS) remain pressing global epidemics. A tremendous need exists for new therapies and treatment paradigms due to viral diversity and the high rate of viral mutation and drug resistance. A major limitation of currently available anti-retroviral agents is that these drugs target viral proteins or viral enzymes, rather than host cell components involved in viral replication and pathogenesis. Targeting viral processes and enzymes leads to a high rate of drug resistance due to the rapid mutation of viral enzymes and lack of a proofreading mechanism during reverse transcription. Novel anti-retroviral treatments focusing on inhibiting host cell processes critical for retroviral replication would provide a more robust treatment strategy, as the virus is unlikely to achieve the significant evolution needed to bypass the lost or diminished host cell function. A key host cell process involved in retroviral replication is the N-myristoylation of HIV-Nef and HIV-Gag. This process involves the transfer of a 14-carbon myristate moiety from myristoyl-Coenzyme A to the N-terminus of substrate proteins through the catalytic activity of the N-myristoyltransferase isozymes (NMT1 and NMT2 in humans). The human NMTs share a sequence homology of approximately 75-77%; however, little is known about the unique substrate specificity or function of the NMTs in normal or disease states. Previous reports in the literature suggest that the NMTs exhibit unique substrate specificity in the context of cancer systems; however, little research has been reported to date in regard to the specificity of the NMTs for viral substrate proteins. Elucidation of the specific NMT responsible for HIV-Gag or HIV-Nef would be a significant step toward development of novel anti-retroviral treatments as myristoylation of these proteins plays a key role in retroviral budding and pathogenesis. Our laboratory examined the roles of the individual NMTs in regard to HIV-Gag and HIV-Nef myristoylation using a variety of biochemical and molecular approaches. Fluorescently labeled peptides corresponding to the N-terminal myristoylation sequence of Gag and Nef were synthesized and myristoylated by recombinant human NMT1 and NMT2. Kinetic analysis revealed that NMT1 and NMT2 have 30- and 130-fold lower KMs for Nef than Gag, respectively. Values for Kcat indicate that once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than did NMT1. These experiments were confirmed by using full length recombinant Nef protein, which also indicated a lower KM for Nef myristoylation by NMT2 than by NMT1. We then validated the results of the in vitro kinetic data via siRNA knockdown of the NMTs, followed by confocal microscopy to determine consequent effects on localization of a Nef-sgGFP fusion protein in HEK293T cells. Depletion of NMT1 had minimal affect on the intracellular distribution of Nef-sgGFP; whereas, ablation of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant Nef-sgGFP. Together, these findings indicate that Gag is a poor substrate for the human NMTs, and that Nef is preferentially myristoylated vs. Gag when present in the same system. Results also indicate that Nef is a better substrate for human NMT2, suggesting that selectively inhibiting NMT2 activity may provide a novel means of reducing HIV virulence. Our laboratory performed a small molecule screen for novel inhibitors of recombinant human NMT1. Approximately 20 compounds selected from this screen were tested for their anti-HIV properties, including effects on production, release, and infectivity of viral particles released from the latently infected OM10.1 cell line. Compound 12 (4-(3,4-dimethoxyphenyl)-6-(4-methylphenyl)-2-(1-piperazinyl)pyrimidine) was selected as a lead compound due to its ability to decrease the number of viral particles (p24 capsid) released into the supernatant from infected cells. Treatment with this compound concurrently increased intracellular levels of p24, further supporting the importance of myristoylation in the retroviral life cycle. Ten structural analogs of Compound 12 were then selected and tested for anti-HIV activity. This work reports the discovery of two novel 2-(piperazin-1-yl)-4-phenyl-6-phenylpyrimidines (Compounds 103 and 104) which at non-cytotoxic doses decrease the number of infectious viral particles released from infected cells and simultaneously increase intracellular retention of p24. Parallel studies were performed to test the effects of Compounds 103, 104, and 109 on the subcellular localization of either Gag-eGFP or Nef-sgGFP constructs in HEK293T cells. All three compounds decreased Gag-eGFP localization to cellular membranes up to 70% vs. control, with a concomitant increase in cytosolic Gag-eGFP. Furthermore, Compound 104 abolished Nef-sgGFP localization to cellular membranes, making this an attractive compound for further characterization and optimization. To date, this is the first report to describe the effects of small molecules targeted toward the NMTs in the context of a fully functional viral infection. These results, as well as the kinetic studies, indicate the possibility of future development of novel anti-HIV therapeutics specifically targeting the N-myristoyltransferases for use in combination with other antiretroviral agents.