Christopher Carreras

PURIFICATION AND IN VITRO RECONSTITUTION OF THE ESSENTIAL PROTEIN COMPONENTS OF AN AROMATIC POLYKETIDE SYNTHASE

A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

Filter binding assay for the geldanamycin–heat shock protein 90 interaction

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.

Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins

A method was developed for the large-scale bioconversion of novel 6-deoxyerythronolide B (6-dEB) analogs into erythromycin analogs. Erythromycin biosynthesis in Saccharopolyspora erythraea proceeds via the formation of a polyketide aglycone, 6-dEB, which is subsequently glycosylated, hydroxylated and methylated to yield the antibiotic erythromycin A. A modular polyketide synthase (PKS) directs 6-dEB synthesis using a dedicated set of active sites for the condensation of each of seven propionate units. Strategies based on genetic manipulation and precursor feeding are available for the efficient generation of novel 6-dEB analogs using a plasmid-based system in Streptomyces coelicolor. 6-dEB and 13-substituted 6-dEB analogs produced in this manner were fed to S. erythraea mutants which could not produce 6-dEB, yet retained their 6-dEB modification systems, and resulted in the generation of erythromycin A and 13-substituted erythromycin A analogs. Erythromycin B, C and D analogs were observed as intermediates of the process. Dissolved oxygen, temperature, the specific aglycone feed concentration, and pH were found to be important for obtaining a high yield of erythromycin A analogs. Cultivation conditions were identified which resulted in the efficient bioconversion of 6-dEB analogs into erythromycin A analogs, which this process demonstrated at the 100 l scale.

Utilization of Enzymatically Phosphopantetheinylated Acyl Carrier Proteins and Acetyl−Acyl Carrier Proteins by the Actinorhodin Polyketide Synthase†

The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogs in Streptomyces coelicolor: understanding precursor effects.

A fermentation process employing precursor‐directed biosynthesis is being developed for the manufacture of 6‐deoxyerythronolide B (6‐dEB) analogues. Through a plasmid‐based system in Streptomyces coelicolor, 6‐dEB synthesis is catalyzed by 6‐dEB synthase (DEBS). 6‐dEB synthesis is abolished by inactivation of the ketosynthase (KS) 1 domain of DEBS but can be restored by providing synthetic activated diketides. Because of its inherent catalytic flexibility, the KS1‐deficient DEBS is capable of utilizing unnatural diketides to form various 13‐substituted 6‐dEBs. Here we characterize process variables associated with diketide feeding in shake‐flask experiments. 13‐R‐6‐dEB production was found to depend strongly on diketide feed concentrations, on the growth phase of cultures at feeding time, and on the R‐group present in the diketide moiety. In all cases a major portion of the fed diketides was degraded by the cells.

Potent cytotoxic C-11 modified geldanamycin analogues.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) inhibits the activity of Hsp90, an important target for treatment of cancers. In an effort to identify analogues of geldanamycin (GDM) with properties superior to those of 17-AAG, we synthesized C-11 modified derivatives of GDM including ethers, esters, carbazates, ketones, and oximes and measured their affinity for Hsp90 and their ability to inhibit growth of human cancer cells. In accordance with crystal structures reported for complexes of GDMs with Hsp90, bulky groups attached to C-11 interfered with Hsp90 binding while smaller groups such as 11-O-methyl allowed Hsp90 binding. In addition, these analogues also showed in vitro cytotoxicity against human cancer cell lines. Esterification of the 11-OH of 17-AAG eliminated Hsp90 binding in vitro. The readily hydrolyzed esters acted as prodrugs during the measurement of cytotoxicity. Thus, during these experiments, the esters were hydrolyzed, releasing 17-AAG. Several 11-O-methyl-17-alkylaminogeldanamycin analogues were identified with improved potency relative to 17-AAG.

Structure−Activity Relationships of 9-Substituted-9-Dihydroerythromycin-Based Motilin Agonists: Optimizing for Potency and Safety

A series of 9-dihydro-9-acetamido-N-desmethyl-N-isopropyl erythromycin A analogues and related derivatives was generated as motilin agonists. The compounds were optimized for potency while showing both minimal antibacterial activity and hERG inhibition. As the substituent on the amide was increased in lipophilicity the potency and hERG inhibition increased, while polar groups lowered potency, without significantly impacting hERG inhibition. The N-methyl acetamide 7a showed the optimal in vitro profile and was probed further by varying the chain length to the macrocycle as well as changing the macrocycle scaffold. 7a remained the compound with the best in vitro properties.

9-Dihydroerythromycin ethers as motilin agonists--developing structure-activity relationships for potency and safety.

A series of derivatives of the amine of 9-dihydro-9-O-ethylamino-N-desmethyl-N-isopropyl erythromycin A derivatives were synthesized as motilin agonists. The compounds were developed for potency without showing antibacterial activity and inhibition of the hERG potassium channel. The formamide of the amide series was found to show the optimal combination of properties relative to carbamates, ureas, thioureas, and amines. This prompted an investigation of heterocyclic isosteres for the amide. In this series the triazole had the optimal combination of properties. From the study, two compounds met the criteria for detailed pharmacokinetic studies.

9-Dihydroerythromycins as non-antibiotic motilin receptor agonists

A series of 9-dihydroerythromycin A and B analogues with modification of the desosamine nitrogen have been synthesized and screened for motilin agonist activity, antibiotic activity, tachyphylaxis and hERG channel current inhibition. Small alkyl groups resulted in the potency while compounds with a primary or secondary amine resulted in the low motilin agonist potency. Several compounds were identified as non-antibiotic motilin receptor agonists with minimal tachyphylaxis and low hERG interaction.