Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide.

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability.

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC50=380 nM rat mitochondria) with favorable PK properties (F=93%, t(1/2)=13.6h, dog).

Rapamycin inhibits activation of ryanodine receptors from skeletal muscle by the fatty acyl CoA-acyl CoA binding protein complex.

We previously showed (Fulceri et al., Biochem. J. 325, 423, 1997) that the fatty acyl CoA ester palmitoyl CoA (PCoA) complexed with a molar excess of its cytosolic binding protein (ACBP) causes a discrete Ca(2+) efflux or allows Ca(2+) release by suboptimal caffeine concentrations, in the Ca(2+)-preloaded terminal cisternae fraction (TC) from rabbit skeletal muscle, by activating ryanodine receptor Ca(2+) release channels (RyRC). We show here that both effects were abolished by pretreating TC with the FKBP12 ligand rapamycin (20 microM). Moreover, rapamycin reversed the Ca(2+) release induced by combined treatment with 3 mM caffeine and the PCoA-ACBP complex. Rapamycin also reduced the Ca(2+)-releasing activity by PCoA alone. Under the above experimental conditions, rapamycin removed FKBP12 from the TC membranes, as revealed by Western blot analysis. We conclude that FKBP12 associated with RyRC in the TC membrane participates in the activation of the Ca(2+) channel by fatty acyl CoA esters.

Veratridine-induced depolarization reduces the palmitoylation of brain and myelin glycerolipids.

In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 microM) reduced the incorporation of [3H]palmitate into all brain glycerolipids by 40-50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [3H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 microM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage-gated sodium channels and involves the stimulation of the Na+/ K+ pump. Decreased levels of both ATP, due to activation of the Na+,K+-ATPase, and the precursor palmitoyl-CoA were found in the veratridine-treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage-dependent Na+ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K+]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.

Skeletal muscle mitochondrial beta-oxidation of dicarboxylates.

(1) The oxidation of [U-14C]hexadecanedionoyl-mono-CoA by rat skeletal muscle mitochondrial fractions is carnitine dependent and is inhibited by cyanide. (2) [U-14C]hexadecanedionoyl-mono-CoA was oxidised at a rate 8% of that of [U-14C]hexadecanoyl-CoA. (3) Oxidations were saturable and no substrate inhibition was observed. (4) We demonstrate the formation of dicarboxylyl-mono-CoA esters and the corresponding carnitine derivatives. (5) We conclude that, although skeletal muscle mitochondria are capable of the beta-oxidation of dicarboxylic acids, this is unlikely to be of great physiological significance.