Mamoun M. Alhamadsheh

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-l-methionine

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-l-methionine (SAM) is converted to 5′-chloro-5′-deoxyadenosine (5′-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5′-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

Antimalarial Activity of Natural and Synthetic Prodiginines

Prodiginines are a family of linear and cyclic oligopyrrole red-pigmented compounds. Herein we describe the in vitro antimalarial activity of four natural (IC(50) = 1.7-8.0 nM) and three sets of synthetic prodiginines against Plasmodium falciparum. Set 1 compounds replaced the terminal nonalkylated pyrrole ring of natural prodiginines and had diminished activity (IC(50) > 2920 nM). Set 2 and set 3 prodiginines were monosubstituted or disubstituted at either the 3 or 5 position of the right-hand terminal pyrrole, respectively. Potent in vitro activity (IC(50) = 0.9-16.0 nM) was observed using alkyl or aryl substituents. Metacycloprodiginine and more potent synthetic analogues were evaluated in a P. yoelii murine patent infection using oral administration. Each analogue reduced parasitemia by more than 90% after 25 (mg/kg)/day dosing and in some cases provided a cure. The most favorable profile was 92% parasite reduction at 5 (mg/kg)/day, and 100% reduction at 25 (mg/kg)/day without any evident weight loses or clinical overt toxicity.

Elucidation of the Streptomyces coelicolor Pathway to 2-Undecylpyrrole, a Key Intermediate in Undecylprodiginine and Streptorubin B Biosynthesis

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

The cis-Δ2,3-double bond of phoslactomycins is generated by a post-PKS tailoring enzyme

The antifungal phoslactomycins (PLM A-F), produced by Streptomyces sp. HK803, are structurally unusual in that three of their four double bonds are in the cis form (Delta12,13, Delta14,15, Delta2,3). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1, 2, and 5 required for establishing two of these cis double bonds (Delta12,13, Delta14,15), as well as the only trans Delta6,7 double bond. By contrast, the formation of the cis Delta2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Delta2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained are made in plmT2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Delta2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT2 deletion mutant can be accomplished by plasmid based expression of plmT2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydratase-independent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated.

Synthesis and Biological Evaluation of Novel Sulfonyl-Naphthalene-1,4-diols as FabH Inhibitors

A series of analogs of 2-tosylnaphthalene-1,4-diol were prepared and were found to be potent 10-20 nM reversible inhibitors of the Escherichia coli FabH enzyme. The inhibitors were also effective but to a lesser degree (30 nM-5 microM), against the Mycobacterium tuberculosis and Plasmodium falciparum FabH enzymes. Preliminary SAR studies demonstrated that the sulfonyl group and naphthalene-1,4 diol were required for activity against all enzymes but the toluene portion could be significantly altered and leads to either modest increases or decreases in activity against the three enzymes. The in vitro activity of the analogs against E. coli FabH parallel the in vivo activity against E. coli TolC strain and many of the compounds were also shown to have antimalarial activity against P. falciparum.

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

Unsymmetric aryl–alkyl disulfide growth inhibitors of methicillin-resistant Staphylococcus aureus and Bacillus anthracis

This study describes the antibacterial properties of synthetically produced mixed aryl-alkyl disulfide compounds as a means to control the growth of Staphylococcus aureus and Bacillus anthracis. Some of these compounds exerted strong in vitro bioactivity. Our results indicate that among the 12 different aryl substituents examined, nitrophenyl derivatives provide the strongest antibiotic activities. This may be the result of electronic activation of the arylthio moiety as a leaving group for nucleophilic attack on the disulfide bond. Small alkyl residues on the other sulfur provide the best activity as well, which for different bacteria appears to be somewhat dependent on the nature of the alkyl moiety. The mechanism of action of these lipophilic disulfides is likely similar to that of previously reported N-thiolated beta-lactams, which have been shown to produce alkyl-CoA disulfides through a thiol-disulfide exchange within the cytoplasm, ultimately inhibiting type II fatty acid synthesis. However, the mixed alkyl-CoA disulfides themselves show no antibacterial activity, presumably due to the inability of the highly polar compounds to cross the bacterial cell membrane. These structurally simple disulfides have been found to inhibit beta-ketoacyl-acyl carrier protein synthase III, or FabH, a key enzyme in type II fatty acid biosynthesis, and thus may serve as new leads to the development of effective antibacterials for MRSA and anthrax infections.