Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes. These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified:d-3-phenyllactic acid and d-3-indollactic acid.
Structure of novel farnesyl transferase inhibitor, pepticinnamin E, is elucidated by NMRstudy. Pepticinnamin E is composedof five amino acids and o-pentenylcinnamic acid, having a molecular weight of 907. C-terminal glycylserine of the compounds is in the cyclized diketopiperazine form.Pepticinnamins are novel farnesyl transferase inhibitors isolated from the cultured broth of Streptomyces sp. OH-4652.1* Here, we report on the structural elucidation of pepticinnamin E (1, Fig. 2), a major componentof pepticinnamins.
Pepticinnamins A, B, C, D, E and F, a family of farnesyl-protein transferase (FPT) inhibitors were isolated from the fermentation broth of Streptomyces sp. OH-4652. These inhibitors were purified from whole broth by extraction with chloroform, followed by silica gel column chromatography, Sephadex LH-20 chromatography and reverse phase HPLC. Among these, pepticinnamin C showed the most potent inhibition (IC50~100 nM).ras proteins are localized in the inner side of the plasma membraneand has been considered that the proteins are involved in signal transduction processeslf2). The interaction of ras proteins with the plasma membranerequires post-translational modification of their carboxy-terminus3~7). All ras proteins share a sequence, knownas a CAAX box, which consists of a conserved cysteine (C), two aliphatic amino acids (AA) and a carboxy-terminal residue (X). Genetic studies have demonstrated that ras oncogenes require an intact CAAX box, and therefore proper post-translational processing, to induce malignant transformation8~10).Recent studies have unveiled the biochemical nature of the post-translational modifications within the CAAXmotif of ras proteins. They include; (i) farnesylation of the conserved cysteine residue (Cys186 in mammalian H-ras) by farnesyl-protein transferase (FPT); (ii) cleavage of the three carboxy-terminal amino acid residues (AAX) and (iii) methylation of the resulting carboxy-terminal farnesyl cysteine. Inhibition of such a isoprenylation would alter membranelocalization and transforming activity of ras oncogene1 1*. These findings have raised the possibility that available FPT inhibitor could block neoplastic transformation induced by ras oncogenes. During our screening for FPT inhibitors from microbial origins, we discovered pepticinnamins as fermentation products ofStreptomyces strain OH-4652.
Materials and Methods
MaterialsTritiated farnesyl pyrophosphate (555 GBq/mmol) and En3Hance spray were purchased from New England Nuclear. Cold farnesyl pyrophosphate was synthesized in Rhone-Poulenc Rorer laboratories. Recombinant Ha-ras p21 protein was produced in a bacterial expression system and purified as previously described12). The tetrapeptide* CVLScorresponding to the carboxy-terminal sequence of Ha-ras was synthesized with an Applied Biosystems model 43 1 and purified by HPLCin Rhone-Poulenc laboratories. THP-1 cells were purchased from ATCC(TIB 202).
Ras p21 proteins have been shown to be posttranslationally farnesylated on a specific carboxyterminal cysteine1^. Inhibition of this isoprenylation would alter membrane localization and activation of ras oncogene40. Therefore during our screening for farnesyl-protein transferase (FTase) inhibitors of microbial origin we have isolated
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