We are investigating the post-translational protein acylation with myristic acid (i.e. N-myristoylation) and protein modification with isoprenoids (i.e. S-isoprenylation) as potential new targets for chemotherapeutic drug development. Both phenomenon have been shown to play roles in the targeting of cytoplasmic onc-kinases or retroviral gag structural proteins to the inner plasma membrane surface. In the case of N-myristoylation, site directed mutagenesis designed to block N-myristoylation prevents membrane binding and inhibits both cellular transformation and viral replication, respectively. We have purified N-myristoyl transferase (NMT) from cow brain and shown that the active enzyme is a 120 Kda dimmer made up of a mixture of 4-5 different charge and size isoenzymes. A 52 Kda NMT isoenzyme subunit has been purified sufficiently for microsequencing and antibody production which will aid in the development of probes for screening for the NMT gene in cow brain cDNA libraries. Several new compounds have been identified as in vitro NMT inhibitors: the acyl CoA analog, S-(3-epoxymethylene)- dodecanoyl)-CoA, the acyl analog, 1-bromo-2-pentadecanone, and the multisubstrate analog, N-(S-)(2-tetradecanoyl)-CoA)glycinamide. We have also found that N-myristoyl tetrazole inhibits N-myristoyl Coenzyme A synthetase in vitro as well a HIV replication in vivo. Both enzymes are important in the N-myristoylation pathway. We have also prepared cDNAs of the HIV pl7gag and p27nef genes which encode for N-myristoylated proteins. Bacterial and mammalian vectors of each cDNA have been constructed and used to express the gene proteins in E. coli, N-myristoylated in vitro with cow brain NMT, and are now being used as probes for identifying specific N-myristoyl-protein membrane """"""""acceptors"""""""". The expressed nef has been shown to be localized in cos cells to discrete cytoplasmic structures rather than the plasma membrane. In the case of S-isoprenylation, we have prepared the cDNA of a yeast RAM gene, expressed it in E. coli, and are testing its possible identity the S-farnesyl transferase required for p2lras transforming activity.
