ABSTRACT
We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.
Subject(s)
Chemistry, Pharmaceutical/methods , Combinatorial Chemistry Techniques/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinases , Binding Sites/drug effects , Crystallography, X-Ray , Drug Design , Ligands , Mass Spectrometry/methods , Models, Chemical , Molecular Structure , Purines/chemistry , Structure-Activity RelationshipABSTRACT
Disorazoles are polyketides produced by the myxobacterium Sorangium cellulosum So ce12. Their mode of action is to inhibit tubulin polymerization and destabilize microtubules. Using transposon mutagenesis, two mutant strains were identified that produced no disorazoles. Sequencing the DNA flanking the insertions revealed a polyketide synthase gene cluster that would encode three polypeptides, DszA, DszB, and DszC, with DszC containing both nonribosomal peptide synthetase and polyketide synthase modules. The disorazole polyketide synthase modules lack an acyltransferase domain. Instead, a separate gene, dszD, encodes an AT protein, thus revealing that the disorazole gene cluster falls into the trans-AT Type I family of PKS enzymes.
Subject(s)
Microtubules/metabolism , Myxococcales/genetics , Amino Acid Sequence , Catalytic Domain/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Dimerization , Gene Order , Models, Chemical , Molecular Sequence Data , Multigene Family/genetics , Myxococcales/drug effects , Myxococcales/metabolism , Oxazoles/chemistry , Oxazoles/metabolism , Oxazoles/toxicity , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tubulin/biosynthesisABSTRACT
We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602.
Subject(s)
Genes, Bacterial , Multigene Family , Quinones/metabolism , Streptomyces/genetics , Base Sequence , Benzoquinones , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/classification , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Lactams, Macrocyclic , Models, Molecular , Molecular Sequence Data , Quinones/isolation & purification , Quinones/pharmacology , Streptomyces/metabolismABSTRACT
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.
