A Continuous Fluorometric Assay of Myristoyl-Coenzyme A:Protein N-Myristoyltransferase

Current Advances in Antifungal Targets and Drug Development

“…It also plays role in diverse biological processes including signal transduction cascades and apoptosis [85][86][87]. The knowledge of crystal structure of fungal NMT [88,89] and recent advances in high throughput screening assays [90,91] has made it possible to screen various chemical libraries effectively and efficiently for this target. During the past decade, various classes of NMT inhibitors have come into the limelight.…”

A new, robust, and nonradioactive approach for exploring N-myristoylation

“…Methods for detecting NMT activity have involved radioactive and nonradioactive assays, such as use of reversephase HPLC, and the so-called "continuous assays" in which enzymatic activity is detected while the reaction is progressing. Radioactive methods are based on the quantifi cation of [ 3 H]myristate incorporation into peptides, whereas continuous assays depend on the spectrophotometric or fl uorometric detection of products released during myristoylation (e.g., HS-CoA) ( 1,4,(35)(36)(37)(38)(39)(40). A nonradioactive assay based on ELISA has been described using an antibody against N-myristoyl-glycine ( 41 ).…”

“…Nonradioactive HPLC-based methods represent a sensitive alternative to radioactive assays but are not suitable for high-throughput analysis ( 41 ). Several continuous spectrophotometric or fl uorescent procedures have been developed ( 35,38,39 ). Although they are relatively easy to perform and applicable for high throughput analysis, continuous assays depend strongly on the milieu in which the reaction occurs.…”

Analysis of Protein Acylation

“…The NMT enzymatic reaction is preceded by formation of a myristoyl‐CoA‐enzyme complex, subsequent binding of the peptide, transfer of the myristate moiety to the peptide, release of CoA, and release of the myristoylated peptide (Rudnick et al, ). Several assays have been developed for this enzyme (e.g., King and Sharma, ; Rudnick et al, ; French et al, ; Pennise et al, ; Takamune et al, ). A large number of NMT inhibitors have been identified (reviewed in Selvakumar et al, ), including proteins, histidine analogs, myristic acid analogs, and myristoyl‐CoA variants which inhibit acyl CoA synthetase and therefore block the conversion of fatty acids to acyl CoA, including 2‐hydroxymyristic acid which is converted to 2‐hydroxymyristoyl‐CoA, a potent inhibitor of NMT, and other synthetic organic compounds.…”