Polyketides, the ubiquitous products of secondary metabolism in microorganisms, are made by a process resembling fatty acid biosynthesis that allows the suppression of reduction or dehydration reactions at specific biosynthetic steps, giving rise to a wide range of often medically useful products. The lovastatin biosynthesis cluster contains two type I polyketide synthase genes. Synthesis of the main nonaketide-derived skeleton was found to require the previously known iterative lovastatin nonaketide synthase (LNKS), plus at least one additional protein (LovC) that interacts with LNKS and is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L. The noniterative lovastatin diketide synthase (LDKS) enzyme specifies formation of 2-methylbutyrate and interacts closely with an additional transesterase (LovD) responsible for assembling lovastatin from this polyketide and monacolin J.

The fungal metabolite lovastatin (1) 1 and its derivatives are cholesterol-lowering drugs that act as potent inhibitors of (3S)hydroxy-3-methylglutaryl-coenzyme A reductase. 2 Although 1 and compactin 3 have attracted attention from synthetic chemists, 4 these drugs and some analogues (e.g., simvastatin, pravastatin) which are used in humans are manufactured by fermentation, either directly or with subsequent chemical or microbial modification. Studies on the biosynthesis of 1 in Aspergillus terreus indicate that it is formed by a polyketide pathway. [5][6][7] Of special interest is the proposal of an enzyme-catalyzed Diels-Alder cyclization of the intermediate hexaketide triene to generate the decalin system (Figure 1). 7,8 This idea is supported by the formation of dihydromonacolin L (2) 9 by a heterologous host, A. nidulans containing the loVB and loVC genes from A. terreus. 10,11 Corresponding heterologous expression of the loVB protein (lovastatin nonaketide synthase, LNKS) without loVC leads to truncated pyrones 3 and 4, formed due to failure of enoyl reduction at the tetraketide stage. 10 There are proposals that enzyme-catalyzed Diels Alder reactions may occur during biosynthesis of many secondary metabolites, 12 but the demonstrated ability of pure biological macromolecules to promote this process has been limited to catalytic antibodies generated from synthetic haptens 13 and to synthetic RNA fragments that bind metals. 14 There is also a report of a crude cellfree preparation from the fungus Alternaria solani that oxidizes an achiral allylic alcohol, prosolanopyrone II, to a conjugated triene aldehyde, thereby triggering intramolecular Diels-Alder cyclization to an optically active product, solanopyrone A. 15 We now report that purified LNKS catalyzes intramolecular Diels-Alder endo closure of a substrate analogue, (E,E,E)-(R)-6-methyldodecatri-2,8,10-enoic acid N-acetylcysteamine (NAC) thioester (5), to a bicyclic system with the same ring stereochemistry as 2, which is different from that obtained in nonenzymatic cyclization.

B-RAF V600E ͉ hypothemycin ͉ irreversible kinase inhibitor ͉ Michael adduct R esorcylic acid lactones (RALs) are polyketide natural products with a large macrocyclic ring fused to resorcylic acid. Some RALs contain an ␣,␤-unsaturated ketone in the macrocycle, as exemplified by the cis-enone RALs hypothemycin, 5Z-7-oxozeaenol, and L-783,277 (Fig. 1). The cis-enone RALs have been shown to inhibit mammalian cell proliferation and tumor growth in animals (1-3). Furthermore, several reports have indicated that cis-enone RALs inhibit certain protein kinases, such as mitogenactivated protein (MAP) kinase (MAPK) kinase (MEK)1 (4), TGF-␤-activated kinase 1 (TAK1) (5) and platelet-derived growth factor receptor (PDGFR) (6), but not others, such as RAF, PKA, PKC, endothelial growth factor receptor (EGFR), FGF receptor, ZAP70, MEK kinase 4, and lymphoid-specific Tyr kinase p56kk (LCK) (3, 4, 6). Where tested, targets inhibited by the cis-enone RALs were not affected by trans-enone RALs or RAL analogs lacking the ␣,␤-unsaturated ketone (4, 5).L-783,277 was shown to be a potent in vitro inhibitor of MEK1, competitive with ATP, that became even more potent upon preincubation (4). The apparent time-dependent inhibition caught our attention because it is a hallmark of affinity-directed covalent bond formation between enzyme and inhibitor, and the ␣,␤-unsaturated ketone moiety is an effective Michael acceptor of protein nucleophiles, particularly Cys thiolate.Off-target inhibition by a kinase inhibitor is usually unpredictable, and assessment of complete specificity usually requires screening of the entire kinome (7). In the present work, we identified a Cys residue that is conserved in the ATP site of kinase targets reported to be inhibited by cis-enone RALs but absent from those that are not. Furthermore, a structure-bioinformatics approach revealed that this Cys is present in a subset of some 46 protein kinases in the kinome that include such important targets as mitogen receptor Tyr kinases, MEK, and ERK. We show that hypothemycin forms stable covalent adducts with this Cys residue and is highly efficacious in inhibiting growth of cells dependent on the target kinases, in particular, cells dependent on mitogen receptor RAL targets or harboring B-RAF V600E mutations that drive the ERK pathway. ResultsBioinformatics. Sequence alignment of the kinases reported to be inhibited by cis-enone RALs revealed a conserved Cys residue (Cys-166 in human ERK2) adjacent to the completely conserved Asp that is involved in binding the Mg 2ϩ complexed to ATP; kinases that were reportedly not inhibited by a RAL had no Cys residue at that position. Interrogation of the human kinome sequence database revealed that some 46 of 510 identified kinases contained the target Cys and were therefore considered candidates for RAL inhibition (Table 1).Of the 46 Cys-containing RAL targets, 38 exist in 8 evolutionarily related clusters, 7 of which lie within 5 major branches of the human kinase tree (Fig. 2) (8). The largest branch of Ϸ90 Tyr kinases contains...

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.