Synthesis of Myristoyl CoA Analogues and Myristoyl Peptides as Inhibitors of Myristoyl CoA:Protein WMyristoyltransferase zyxw GUO-QIANG ZHENG', XIUFENG Hu', JOHN M. CASSADY", LISA A. PAIGE~, AND ROBERT L. GEAHLEN~ Received December 16, 1992, from the zyxwvuts 'Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, and the *Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, I N 47907. Accepted for publication May 20, 1993". Abstract zyxwvutsrqp 0 To develop inhibitors of myristoyl CoA:protein Kmyris- toyltransferase (NMT), a series of myristoyl coenzyme A analogues and myristoyl peptides were synthesized, including S(2-oxopentadecyl)- CoA (I), S(2-hydroxypentadecyl)-CoA (2), S(2-oxopentadecy1)- pantetheine zyxwvutsrqp (3). Myr-KGly-(L)-Phe (4). Myr-KGly-(L)-Tyr (J), and Myr- N.Gly-(L)-Asn-Ala-Ala-Ser-Ala-Arg-(NH2) (6). Biological evaluation of these compounds in an in zyxwvutsrqp vitm NMT enzyme assay revealed that the nonhydrolyzable acyl CoA analogue 1 was the most potent inhibitor [inhibitor dissociation constant (4) zyxwvuts = 24 nM]. A preliminarystructure- activity relationship study showed that the adenosine moiety and the 2-keto group in this nonhydrolyzable analogue were necessary for inhibitory activity. A possible mechanism for the inhibition of NMT by 1 was proposed, in which 1 might block the reaction at the stage of an acyl-CoA-NMT-peptiie complex. Product analogues such as the myristoylated peptides 4-6 were poor inhibitors of NMT. A number of oncogene products and retroviral structural proteins, such as the protein-tyrosine kinase pp60v-src and human immunodeficiency virus (HIV) ~ 1 7 8 ~ 8 , require cotranslational myristoylation to function in malignant transformation and virus particle For example, myristoylation of pp60v-src is necessary for its localization to cellular membranes and for its cell-transforming potential.8~9 Nonmyristoylated variants of pp60v-src maintain tyrosine kinase activity, but lose membrane association and transforming ability.lOJ1 Myristolyation of the Moloney murine leukemia virus Pr65gaf and HIV Pr55gag are also essentialfor their membrane localizationand for viral particle formation. Mutagenesis of the virus genome results in the production of nonmyristoylated structural proteins of both viruses, which are no longer associated with plasma membranes and consequently fail to assemble into virus particles.6J2 Myristoyl CoA:proteinN-myristoyltransferase (NMT) catalyzes the transfer of myristate from myristoyl CoA to the N-terminal glycine residue of a growing polypeptide chain by forming an amide linkage.13J4 Because myristoylation plays important roles in the malignant transformation of cancer cells and in the assembly of viral particles, NMT can be selected as a novel target for the design of potential antitumor and antiviral agents. Specific inhibitors of myristoylation should prevent the asso- ciation of protein-tyrosine kinases, such as pp6WrC, with the plasma membrane and consequently block their effects on cell proliferation and transformation. Also, inhibitors of protein myristoylation may reasonably be predicted to serve as potential antiviral agents by blocking viral particle assembly and budding. In this article, we report the synthesis of a series of myristoyl CoA and peptide analogues, the evaluation of these compounds for their ability to inhibit NMT, and the preliminary study of structure-activity relationships includinga proposed mechanism for the enzymatic inhibition by nonhydrolyzable analogues. Two types of target compounds were designed including nonhydrolyzable myristoyl CoA analogues 1-3 and myristoyl peptides 4-6 (see Chart 1). S-(2-Oxopentadecyl)-CoA (1) was designed as a nonhydrolyzableanalogue of myristoylCoA, which *Abstract published in zyxwvutsrqpo Advance ACS Abstracts, December zyxwvuts 1,1993. structurally resembles the natural substrate, but would not participate in the transferase reaction. Compound 1 should be stable to enzymatic hydrolysis by insertion of a methylene group at the susceptible thioester linkage. This is based on the observations that several methylene-bridged nonhydrolyzable CoA analogues have been used as effective inhibitors of anumber of acyl-CoAutilizing enzymes.15J6 Heuckeroth and co-workers17 have proposed an ordered reaction mechanism for NMT in which fatty acyl CoA first binds NMT and then influences the subsequent binding of peptide substrate to NMT. Thus, 1 was expected to be a potent dead-end inhibitor of NMT. S-(2- Hydroxypentadecy1)-CoA (2) and S-(2-oxopentadecyl)pant- etheine (3) were proposed to examine the roles of the 2-keto group and adenosine moiety of 1 in binding to the enzyme. The myristoyl peptides 4-6 were designed as product analogues that might be direct inhibitors of NMT. It was found that the N-terminal glycine residue is essential for protein myris- toylation.57J0 N-Myristoyl glycinal diethylacetal, a myristoyl- glycine analogue, was reported as an inhibitor of the myristoyl- ation of HIV and HTLV-I gag proteins.18 The myristoyl dipeptides, myristoyl-N-Gly-(L)-Phe (4) and myristoyl-N-Gly- (L)-Tyr (5), were thus proposed to contain both the myristic acid and N-terminal glycine moieties that could be essential for the specific binding to NMT. To investigate the effects of peptide chain length on inhibitory activity, the synthesis of myristoyl heptapeptide, Myr-GNAASAR(NH2) (61, was proposed on the basis of the observation that the octapeptide GNAASARR(NH2) was reported as a potent inhibitor that bound to NMT with high affinity.lg Results and Discussion Chemistry-The synthetic approach to nonhydrolyzable myristoyl CoA analogues was based on a final alkylation of CoA as reported in a prior communication.20 The tetralithium salt of S-(2-oxopentadecyl)-CoA (1) was synthesized and obtained as a white solid, the exact mass of which was measured by high- resolution, fast-atom-bombardment mass spectroscopy (FAB- MS) in the matrix glycerol by the peak matching technique with the standard tris-(perfluorohepty1)-s-triazine. The quasi-mo- lecular ion at zyxwv mlz 1016.3679 (MH+) and 1010.3622 (MH*-Li+H) corresponded to the molecular formulas C~H6lN7017P3SLi4 (calcd 1016.3698) and C ~ ~ H G Z N ~ O ~ ~ P ~ S L ~ ~ (calcd 1010.3616), respectively. This compound showed a single UV (254 nm) quenching and phosphate-positive21 spot on the silica gel TLC [retardation factor (Rf), 0.571 and cellulose TLC (R,, 0.84) with the solvent n-Bu0H:HOAc:HzO (5:2:3). It exhibited the typical UV absorption of adenine at 259 nm from the CoA moiety and the characteristic IR bands from both CoA and long-chain alkyl ketone moieties, indicating that 1 was composed of CoA and 2-pentadecanone. This is further confirmed by the difference FT-IR spectrum obtained from subtraction of the spectrum of either moietyfrom that of 1. The completeassignment of protons and carbons in the spectra was aided by 2D COSY and HETCOR NMR as well as by comparison with those of its constituent moieties. The lH NMR data of 1 were consistent with the 0 1994, American Chemical Society and American Pharmaceutical Association 0022-3549/94/ 1200-233$04.50/0 Journal of Pharmaceutical Sciences / 233 Vol. 83, No. 2, February 1994