Clickable analogue of cerulenin as chemical probe to explore protein palmitoylation.

Dynamic palmitoylation is an important post-translational modification regulating protein localization, trafficking, and signaling activities. The Asp-His-His-Cys (DHHC) domain containing enzymes are evolutionarily conserved palmitoyl acyltransferases (PATs) mediating diverse protein S-palmitoylation. Cerulenin is a natural product inhibitor of fatty acid biosynthesis and protein palmitoylation, through irreversible alkylation of the cysteine residues in the enzymes. Here, we report the synthesis and characterization of a "clickable" and long alkyl chain analogue of cerulenin as a chemical probe to investigate its cellular targets and to label and profile PATs in vitro and in live cells. Our results showed that the probe could stably label the DHHC-family PATs and enable mass spectrometry studies of PATs and other target proteins in the cellular proteome. Such probe provides a new chemical tool to dissect the functions of palmitoylating enzymes in cell signaling and diseases and reveals new cellular targets of the natural product cerulenin.

Striatal dopamine in the response of brain and liver unsaturated and saturated free fatty acids following administration of C75 in CD-1 mice.

In order to clarify the mechanism of action of cerulenin analog, C75, known to suppress feeding behavior, food intake was measured in adult CD-1 male mice n=5 per group, treated i.p. with 10 and 20mg/kg of C75. Animals in both treatment groups had significantly lower 24h food consumption rate relative to the control group injected with vehicle. Striatal monoamine neurotransmitters and striatal as well as liver long chain free fatty acids concentrations were subsequently evaluated in another group treated i.p. with 20mg/kg C75. Acute exposure to C75 at 20mg/kg led to approximately 50% increase in the striatal dopamine levels and a decrease in dopamine turnover for up to 24h following the injection. The concentration of serotonin remained unchanged. Concentration of saturated fatty acids in the liver and striatum did not change, while striatal unsaturated myristoleic acid (cis-9-tetradecenoic acid) levels were significantly higher as early as 2h post-injection and remained elevated at 24h post-injection. These preliminary data suggest a central regulatory role of unsaturated fatty acids under dopaminergic control in the C75-induced anorexia. Pharmacological alterations in fatty acid metabolism may prove beneficial in the treatment of obesity.

Cerulenin analogues as inhibitors of efflux pumps in drug-resistant Candida albicans.

Overexpression of the ABC transporters Cdr1 and Cdr2 or the major facilitator Mdr1 causes multidrug resistance in the human fungal pathogen Candida albicans. The fatty acid synthesis inhibitor cerulenin and the structurally unrelated Golgi transport inhibitor brefeldin A are substrates for both types of efflux pumps in Candida albicans. In an effort to overcome efflux pump-mediated drug resistance in Candida albicans, cerulenin analogues were generated using a variety of synthesis pathways. The so obtained cerulenin derivatives were tested on multidrug-resistant Candida albicans isolates which constitutively overexpress either Mdr1 or Cdr1 and Cdr2. Some of these compounds were found to decrease Mdr1-mediated resistance to brefeldin A up to eightfold compared to the control.

Cellular pharmacology of cerulenin analogs that inhibit protein palmitoylation.

S-palmitoylation is a dynamic post-translational modification of certain proteins, which helps determine membrane association and may function to enhance the interactions of signaling molecules with their activated receptors and effector systems. Unlike enzymes that catalyze other protein lipidation reactions, e.g. farnesylation and N-myristoylation, protein palmitoyltransferase is virtually uncharacterized biochemically. We have described previously the synthesis of cerulenin analogs including cis-2,3-epoxy-4-oxononadecanamide (16C) and cis-2,3-epoxy-4-oxododecanamide (9C) that inhibit protein palmitoylation (Lawrence et al., J Med Chem 1999;42:4932-41), most likely through covalent alkylation of protein palmitoyltransferase. [3H]9C and [3H]16C were prepared by catalytic incorporation of 3H2 into unsaturated precursors for further study of their cellular pharmacology. After 4 hr, T24 bladder carcinoma cells in the absence of serum accumulated a 4-fold higher intracellular level of [3H]16C than of [3H]9C. Uptake of [3H]9C and [3H]16C was reduced by the presence of serum in the medium, suggesting their binding to serum proteins. [3H]9C and [3H]16C alkylated unique patterns of proteins in T24 cells, with proteins of approximately 80 and 31 kDa being labeled by each compound. A panel of human tumor cell lines demonstrated half-maximal proliferation inhibition at concentrations of 7-30, 4-16, and 8-36 microM, for cerulenin, 9C, and 16C, respectively, indicating that the cell lines have approximately equal sensitivity to these compounds. Different cell lines have similar patterns of protein alkylation by [3H]9C or [3H]16C, with labeling intensity related to cytotoxicity of the compounds. Since both 9C and 16C inhibit palmitoylation, the commonly labeled proteins are candidates for human protein palmitoyltransferase.

Structure-activity studies of cerulenin analogues as protein palmitoylation inhibitors.

Activation of ras oncogenes occurs in a high percentage of tumors, making the enzymes involved in the posttranslational processing of their encoded proteins (p21s) attractive targets for the development of new drugs. Although most effort has focused on farnesyl transferase, which catalyzes the first processing step, attachment of palmitate to p21 is required for optimal transformation by H-ras and N-ras. We have demonstrated that the natural product cerulenin ([2R,3S]-2,3-epoxy-4-oxo-7,10-trans,trans-dodecadienamide) inhibits the palmitoylation of H-ras- and N-ras-encoded p21s in parallel with inhibition of cell proliferation. More than 30 analogues of cerulenin, both aromatic and aliphatic, with various chain lengths and amide substitutions, have been synthesized for use in SAR studies. Studies on the inhibition of T24 cell proliferation indicate that the alpha-keto-epoxy moiety is critical for cytotoxicity, while alkyl chain length had only modest effects on potency. Several compounds inhibited the incorporation of [(3)H]palmitate into p21 in intact T24 cells, with the unsubstituted carboxamides being more active than N,N-dimethyl compounds. In contrast to the effects on palmitoylation, the only compounds which inhibited fatty acid synthase contained alkyl side chains of 12 carbons or fewer. Regression analyses indicated that inhibition of palmitoylation is more closely related to inhibition of proliferation than is inhibition of fatty acid synthase. Further characterization of the molecular pharmacology of these and analogous compounds may define a new class of drugs with antitumor activity.

Specific inhibition of plant fatty acid elongation by a long-chain cerulenin analogue.

Cerulenin analogues with 16 or 18 carbon atoms inhibit both ATP-dependent and acyl-CoA-dependent fatty acid elongations. Prior incubation of microsomes with inhibitors is necessary to obtain maximal inhibition. The analogues act on the first reaction of the elongation process catalysed by the 3-ketoacyl-CoA synthase. The 18-carbon analogue has no, or little, effect on the fatty acid synthesis, while cerulenin and its 16-carbon analogue totally inhibit this synthesis. The 18-carbon analogue appears to be a specific inhibitor of the synthesis of very long-chain fatty acids, with no effect on de novo fatty acid synthesis.

Effect of side-chain structure on inhibition of yeast fatty-acid synthase by cerulenin analogues.

Yeast fatty-acid synthase (FAS) inhibition by cerulenin analogs with varying side-chain lengths was compared with that of cerulenin, tetrahydrocerulenin and iodoacetamide. Although inhibition by cerulenin was the highest, the analogs having (E,E)-delta 7,10 double bonds showed high inhibition. This strongly suggests that the (E,E)-delta 7,10 double bonds play an important role in the interaction of the inhibitors with the enzyme. It was suggested that the size of the hydrophobic cavity in the condensing enzyme terminates fatty-acid chain elongation by decreasing inhibition by the C18 analog. Like cerulenin itself, the shortest analog (C6) did not induce malonyl-CoA decarboxylase activity.

Syntheses of cerulenin and its analogs. II. Synthesis and biological activity of dl-carbacerulenin, a carbocyclic analog of cerulenin.

2,3-Epoxy-4-hydroxy-4-((E,E)-3,6-octadienyl)cyclopentanone (dl-carbacerulenin 5) was synthesized via the epoxyketones 15a and 15b as a mimic of the active form of the antibiotics cerulenin 1, a potent inhibitor of fatty acid synthetase (FAS). The monobenzyl ethers (12 and 13), synthetic intermediates of 15, were prepared by direct benzylation of the epoxycyclopentene (7). Inhibitory activity of synthesized 5 toward yeast FAS was less than that of cerulenin by a factor of 1000.

Sensitive, rapid, and specific bioassay for the determination of antilipogenic compounds.

A sensitive and rapid bioassay for the determination of the antilipogenic compounds cerulenin and CM-55 is described. The bioassay is based on the inhibitory effect of cerulenin and CM-55 on the in vivo luminescence of an aldehyde-requiring mutant of the marine bacterium Beneckea harveyi. A total quantity as low as 0.1 mug of cerulenin can be determined within 15 min with an error of +/-2%. The bioassay, as presented, is specific for compounds that are known to inhibit fatty acid biosynthesis and, as such, it might be used as a general screening method for the detection of antilipogenic compounds.

Relationship between the structures of fatty acid amide derivatives and their antimicrobial activities.

The structure-activity relationships of derivatives of the antibiotic cerulenin were investigated by chemically modifying dodecanoic acid, its skeletal back-bone. The dimethylamide derivatives were active against both gram-positive and -negative bacteria, and fungi. Among the compounds having modified groups at positions C2 and C4, the most active were those with a carbonyl group at C4 and a double bond at C2. The dimethylamide and pyrrolidine amide derivatives of this structure type were the most active. Activity against bacteria and yeast increased with the number of carbon atoms in the skeleton, with the maximum activity being observed at C=12. No significant differences in activity against fungi were observed with change in chain length.