Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates.

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.

Bioconversion of the anthracycline analogue desacetyladriamycin by recombinant DoxA, a P450-monooxygenase from Streptomyces sp. strain C5.

[reaction: see text] A recombinant P450-monooxygenase, DoxA, obtained from Streptomyces sp. strain C5, the producer of the anticancer compound daunorubicin, was expressed in S. lividans TK24 and therein used to catalyze the conversion of the anthracycline analogue desacetyladriamycin into the new anthracycline, 10-hydroxydesacetyladriamycin. This work establishes a new function for DoxA and demonstrates the use of a recombinant enzyme to prepare a new anthracycline analogue.

The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5.

The biosynthesis of daunorubicin and its precursors proceeds via the condensation of nine C-2 units derived from malonyl-CoA onto a propionyl starter moiety. The daunorubicin polyketide biosynthesis gene cluster of Streptomyces sp. strain C5 has two unique open reading frames, dpsC and dpsD, encoding, respectively, a fatty acid ketoacyl synthase (KAS) III homologue that is lacking an active-site cysteine and a proposed acyl-CoA:acyl carrier protein acyltransferase. The two genes are positioned directly downstream of dpsA and dpsB which encode the alpha and beta components of the type II KAS, respectively. Expression of the dpsABCDEFGdauGI genes in Streptomyces lividans resulted in the formation of aklanonic acid, the first stable chromophore of the daunorubicin biosynthesis pathway. Deletion of dpsC, but not dpsD, from this gene set resulted in the formation of desmethylaklanonic acid, derived from an acetyl-CoA starter unit, and aklanonic acid, derived from propionyl-CoA, in a 60:40 ratio. Thus, DpsC contributes to the selection of propionyl-CoA as the starter unit but does not alone dictate it. A dpsCD deletion mutant of Streptomyces sp. strain C5 (C5VR5) still produced daunorubicin but, more significantly, anthracycline and anthracyclinone derivatives resulting from the use of acetyl-CoA as an alternative starter moiety. Expression of dpsC, but not dpsD, in mutant C5VR5 restored the wild-type phenotype. Among the new compounds was the new biosynthesis product feudomycin D. These results suggest that in the absence of DpsC, the daunorubicin PKS complex behaves promiscuously, utilizing both acetyl-CoA (ca. 60% of the time) and propionyl-CoA (ca. 40%) as starter units. The fact that DpsC is not required for initiation with propionyl-CoA is significant, as the information must then lie in other components of the PKS complex. We propose to call DpsC the propionyl starter unit "fidelity factor."

Nonactin biosynthesis: the potential nonactin biosynthesis gene cluster contains type II polyketide synthase-like genes.

Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15¿ omitted¿559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.

Nonactin biosynthesis: the product of nonS catalyzes the formation of the furan ring of nonactic acid.

Nonactin is the parent compound of a group of ionophore antibiotics, known as the macrotetrolides, produced by Streptomyces griseus subsp. griseus ETH A7796. Nonactin is a significant compound because of its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells. Nonactin is also significant in that it is a highly atypical polyketide. Very little is presently known about the genes of the nonactin biosynthesis cluster. In this paper we describe our efforts to establish a connection between the product of a gene from the nonactin biosynthesis cluster and a known biochemical transformation in nonactin biosynthesis. Nonactate synthase is the enzyme which catalyzes the formation of nonactic acid from an acyclic precursor in nonactin biosynthesis. We have synthesized the substrate for this enzyme and have detected the in vitro cyclization activity of the substrate in cell-free preparations of S. griseus subsp. griseus ETH A7796. Previous studies by R. Plater and J. A. Robinson (Gene 112:117-122, 1992) had suggested, based on sequence homology, that the product of a partial open reading frame found close to the tetranactin resistance gene of S. griseus could be the nonactate synthase. We have therefore cloned, sequenced, and heterologously expressed this full gene (nonS), and we have shown that the gene product, NonS, does indeed catalyze the formation of the furan ring of nonactic acid as hypothesized.

Purification, properties, and characterization of recombinant Streptomyces sp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis.

DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter. The purified enzyme was a monomeric, soluble protein with an apparent Mr of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30 degreesC. The kcat/Km values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M-1 x s-1; for 13-dihydrodaunorubicin, 14,000 M-1 x s-1; for 13-dihydrocarminomycin, 280 M-1 x s-1; and for daunorubicin, 130 M-1 x s-1. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-O-methyl series of anthracyclines.

A free energy calculation can be used to predict K(+)-binding constants for new macrotetrolide antibiotics.

A free energy calculation technique was used to predict K+ binding constants for new macrotetrolides. The technique was validated by successfully predicting affinity constants for known, naturally produced, macrotetrolides.

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA.

We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: 3389-3395, 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the epsilon-rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin.

Studies on the biosynthesis of thiostrepton: 4-(1-hydroxyethyl)quinoline-2-carboxylate as a free intermediate on the pathway to the quinaldic acid moiety.

Specifically 13C-labeled quinoline-2-carboxylate derivatives were synthesized from quinoline and used to study the biosynthesis of thiostrepton in a strain of Streptomyces laurentii. 13C NMR analysis of thiostrepton recovered after feeding methyl (RS)-[11-13C]-4-(1-hydroxyethyl)quinoline-2-carboxylate or methyl [11-13C]-4-acetylquinoline-2-carboxylate showed conclusively that these compounds are specifically and efficiently incorporated into thiostrepton. Both compounds were also detected in cultures of the producing organism by isotope dilution analysis. The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S. laurentii. A highly specific enzyme activity was detected in cell-free extracts of S. laurentii that was capable of adenylating (12S)-4-(1-hydroxyethyl)quinoline-2-carboxylic acid. Partial purification of the enzyme was achieved. The enzyme was found to be specific for the enantiomer of the substrate which has the same absolute configuration as found in the natural antibiotic structure. The presence of one specific enzyme catalysing the adenylation process in S. laurentii was shown by photoaffinity labeling with [alpha-32P]-8-azido-ATP and subsequent SDS PAGE analysis of the labeled products. The native molecular weight of the active enzyme, determined by gel permeation chromatography, was found to be approximately 47 kDa, compared with a denatured weight of 50 kDa estimated for the photoaffinity-labeled protein. The enzyme is thus probably monomeric.

Efficient synthesis of radiolabeled propionyl-coenzyme A and acetyl-coenzyme A.

The efficient microscale synthesis of [1-14C]propionyl-CoA from commercially available sodium [1-14C]-propionate using 1,1'-carbonyldiimidazole in yields of nearly 70% is reported for the first time. A substantial improvement in the process for making [1-14C]acetyl-CoA from sodium [1-14C]acetate was also achieved. Yields of greater than 90% were consistently obtained for the latter synthesis. The salt-free CoA-thioesters were obtained in homogenous form by reverse-phase HPLC. The products were judged to be pure by 1H NMR analysis: neither iso-CoA analogs nor contaminants frequently found in commercial samples could be detected. The samples of acetyl- and propionyl-CoA were shown to be radiochemically pure by HPLC and by analysis of the products of incubations with acetyl- and propionyl-CoA carboxylase. This highly efficient synthesis is a cost-effective method for the preparation of radiolabeled CoA thioesters and can easily be adapted to the production of other acyl-CoA analogs.

Purification and catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis.

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4' of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.