Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships.

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 'drug-like compounds' were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC50 values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyrimidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyrazolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC50 = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

Polyyne-producing Burkholderia suppress Globisporangium ultimum damping-off disease of Pisum sativum (pea).

Extensive crop losses are caused by oomycete and fungal damping-off diseases. Agriculture relies heavily on chemical pesticides to control disease, but due to safety concerns multiple agents have been withdrawn. Burkholderia were successfully used as commercial biopesticides because of their fungicidal activity and plant protective traits. However, their potential for opportunistic pathogenicity led to a moratorium on their registration as biopesticides. Subsequently, Burkholderia were shown to produce multiple specialised metabolites including potent antimicrobial polyynes. Cepacin A, a polyyne produced by Burkholderia ambifaria biopesticide strains was shown to be an important metabolite for the protection of germinating peas against Globisporangium ultimum (formerly Pythium) damping-off disease. Recently, there has been an expansion in bacterial polyyne discovery, with the metabolites and their biosynthetic gene pathways found in several bacterial genera including Burkholderia, Collimonas, Trinickia, and Pseudomonas. To define the efficacy of these bacterial polyyne producers as biopesticidal agents, we systematically evaluated metabolite production, in vitro microbial antagonism, and G. ultimum biocontrol across a panel of 30 strains representing four bacterial genera. In vitro polyyne production and antimicrobial activity was demonstrated for most strains, but only Burkholderia polyyne producers were protective within the in vivo G. ultimum damping-off pea protection model. B. ambifaria was the most effective cepacin-expressing biopesticide, and despite their known potential for plant pathogenicity Burkholderia gladioli and Burkholderia plantarii were uniquely shown to be protective as caryoynencin-producing biopesticides. In summary, Burkholderia are effective biopesticides due to their suite of antimicrobials, but the ability to deploy polyyne metabolites, caryoynencin and cepacin, is strain and species dependent. Graphical Abstract.

Discovery, mode of action and secretion of Burkholderia sensu lato key antimicrobial specialised metabolites.

Burkholderia sensu lato bacteria have genomes rich in biosynthetic gene clusters (BGCs) encoding for multiple bioactive specialised metabolites. Diverse classes of antimicrobial natural products have been isolated from Burkholderia, including polyynes, shikimate pathway derivatives, polyketides, non-ribosomal peptides and hybrid polyketide non-ribosomal peptides. We highlight examples of Burkholderia metabolites, overviewing their biosynthesis, bioactivity, mechanisms of action and secretion.

Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development.

Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non-pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast-adapted Burkholderia-Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 µ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR-amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non-pathogenic biocontrol strains.

Discovery of the Pseudomonas Polyyne Protegencin by a Phylogeny-Guided Study of Polyyne Biosynthetic Gene Cluster Diversity.

Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In bacteria, the basic biosynthesis of polyynes is known, but their biosynthetic gene cluster (BGC) distribution and evolutionary relationship to alkyne biosynthesis have not been addressed. Through comprehensive genomic and phylogenetic analyses, the distribution of alkyne biosynthesis gene cassettes throughout bacteria was explored, revealing evidence of multiple horizontal gene transfer events. After investigation of the evolutionary connection between alkyne and polyyne biosynthesis, a monophyletic clade was identified that possessed a conserved seven-gene cassette for polyyne biosynthesis that built upon the conserved three-gene cassette for alkyne biosynthesis. Further diversity mapping of the conserved polyyne gene cassette revealed a phylogenetic subclade for an uncharacterized polyyne BGC present in several Pseudomonas species, designated pgn. Pathway mutagenesis and high-resolution analytical chemistry showed the Pseudomonas protegens pgn BGC directed the biosynthesis of a novel polyyne, protegencin. Exploration of the biosynthetic logic behind polyyne production, through BGC mutagenesis and analytical chemistry, highlighted the essentiality of a triad of desaturase proteins and a thioesterase in both the P. protegens pgn and Trinickia caryophylli (formerly Burkholderia caryophylli) caryoynencin pathways. We have unified and expanded knowledge of polyyne diversity and uniquely demonstrated that alkyne and polyyne biosynthetic gene clusters are evolutionarily related and widely distributed within bacteria. The systematic mapping of conserved biosynthetic genes across the available bacterial genomic diversity proved to be a fruitful method for discovering new natural products and better understanding polyyne biosynthesis. IMPORTANCE Natural products bearing alkyne (triple carbon bond) or polyyne (multiple alternating single and triple carbon bonds) moieties exhibit a broad range of important biological activities. Polyyne metabolites have been implicated in important ecological roles such as cepacin mediating biological control of plant pathogens and caryoynencin protecting Lagriinae beetle eggs against pathogenic fungi. After further phylogenetic exploration of polyyne diversity, we identified a novel gene cluster in Pseudomonas bacteria with known biological control abilities and proved it was responsible for synthesizing a new polyyne metabolite, protegencin. The evolutionary analysis of polyyne pathways showed that multiple biosynthetic genes were conserved, and using mutagenesis, their essentiality was demonstrated. Our research provides a foundation for the future modification of polyyne metabolites and has identified a novel polyyne, protegencin, with potential bioactive roles of ecological and agricultural importance.

A novel colorimetric assay for α-methylacyl-CoA racemase 1A (AMACR; P504S) utilizing the elimination of 2,4-dinitrophenolate.

α-Methylacyl-CoA racemase (AMACR; P504S) regulates branched-chain fatty acid degradation, activates Ibuprofen and is a recognised cancer drug target. A novel, facile colorimetric assay was developed based on elimination of 2,4-dinitrophenolate. The assay was used to test 5 known inhibitors, determining IC50 and Ki values, reversibility and characterizing irreversible inhibition.