Abstract
Thiolactomycin (TLM) is a natural product inhibitor of KasA, the β-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy.
Highlights
Potent inhibitors of the tuberculosis drug target ketoacyl-AcpM synthase (KasA) are needed
Thiolactomycin (TLM) is a natural product inhibitor of KasA, the -ketoacyl synthase A from Mycobacterium tuberculosis
We demonstrate the use of this technique for detecting Interligand NOEs (ILOEs) between two ligands bound to KasA
Summary
Potent inhibitors of the tuberculosis drug target KasA are needed. Results: Three-position analogs of the natural product thiolactomycin (TLM) were designed based on transient one-dimensional NOEs that reveal the relative orientation of TLM and a pantetheine analog bound simultaneously to KasA. ILOEs between small molecules can be very weak and difficult to detect and differentiate because of chemical shift overlaps and background issues typical of two-dimensional NOESY experiments (16) Such issues can adversely limit the application of the method and the ability to obtain and interpret NOE data. Because malonyl-AcpM and TLM interact preferentially with the KasA acyl-enzyme, the C171Q KasA mutant was used for many of the experiments because this mutation has previously been shown to lead to structural changes in the active site that mimic acylation of Cys-171 (3, 20) Based on these studies, we synthesized TLM analogs that have higher affinity for KasA than the parent compound. We synthesized a pantetheine analog (PK940) and used ILOE NMR spectroscopy to analyze
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