Erythronolide compounds

Details Erythronolide compounds Erythronolide compounds

1999-10-28

2002-06-11

Ponnaluri, Padmashri (Department: 1618)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C549S200000, C549S267000

Reexamination Certificate

active

06403775

ABSTRACT:

This application is related to U.S. Ser. No. 09/073,538, filed May 6, 1998, which is a continuation-in-part of U.S. Ser. No. 08/846,247, filed Apr. 30, 1997, and is related to PCT application No. U.S. Ser. No. 98/08792 and U.S. provisional application Serial No. 60/076,919, filed Mar. 5, 1998, now lapsed.
FIELD OF THE INVENTION
The present invention provides recombinant DNA compounds and host cells containing novel polyketide synthase (PKS) genes and novel polyketides. The invention relates to the fields of chemistry, medicinal chemistry, human and veterinary medicine, molecular biology, pharmacology, agriculture, and animal husbandry.
BACKGROUND OF THE INVENTION
Few molecules have captured interest in both chemotherapy and chemistry to the extent of the polyketide erythromycin and its semi-synthetic derivatives. Erythromycin and its congeners are the third most widely used class of antibiotics, with current worldwide sales exceeding US $3.5 billion. In addition, erythromycin analogs are gaining interest for their potential use in the treatment of gastrointestinal disorders (Omura, “The expanded horizon for microbial metabolites—a review,”
Gene
115, 141-149 (1992)), inflammatory diseases (Kawasaki et al., “Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro,”
Antimicrob. Agents Chemother
. 42, 1499-1502 (1998)), and as next-generation antibiotics for treatment of emerging drug-resistant strains of bacteria (Agoudiras et al., “In-vitro antibacterial activity of RU 004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens,”
Antimicrob. Agents Chemother
. 41, 2149-2158 (1997)).
The chemical challenges of erythromycin attracted the talents of R. B. Woodward and 48 colleagues who described its complete synthesis in a series of landmark publications (Woodward et al., “Asymmetric total synthesis of erythromycin. 1. Synthesis of erythronolide A secoacid derivative via asymmetric induction;” 2. Synthesis of an erythronolide A lactone system;” and 3. Total synthesis of erythromycin,”
J. Am. Chem. Soc
. 103, 3210-3217 (1981)), and of a cadre of medicinal chemists who prepared analogs leading to the important second generation of macrolide antibiotics—clarithromycin, azithromycin, and others (Chu, “Recent developments in 14- and 15-membered macrolides,”
Exp. Opin. Invest. Drugs
4, 65-94 (1995)). Although such efforts effectively saturated the chemical modifications possible at the existing functional groups of the macrolide ring, most of the ring remained inert to chemical modification.
The modular nature of polyketide biosynthesis (Cortés et al., “An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea,”
Nature
348, 176-178 (1990); and Donadio et al., “Modular organization of genes required for complex polyketide biosynthesis,”
Science
252, 675-679 (1991)) has facilitated genetic engineering strategies for the production of novel polyketides (McDaniel et al., “Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits,”
Nature
375, 549-554 (1995) and Katz, “Manipulation of modular polyketide synthases,”
Chem. Rev
. 97, 2557-2576 (1997)).
The “modular” PKSs are each encoded by a cluster of contiguous genes and have a linear, modular organization of similar catalytic domains that both build and modify the polyketide backbone. Each module contains a set of three domains—a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP)—that catalyze a 2-carbon extension of the growing polyketide chain (FIG.
1
and O'Hagan, The polyketide metabolites (E. Horwood, New York, 1991)). The choice of extender unit used by each module—acetate, propionate, or other small organic acids in the form of CoA thioesters—is determined by the specificity of the AT domain (Oliynyk et al., “A hybrid modular polyketide synthase obtained by domain swapping,”
Chem. & Biol
. 3, 833-839 (1996); Liu et al., “Biosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution,”
J. Am. Chem. Soc
. 119, 10553-10554 (1997); and Ruan et al., “Acyltransferase domain substitutions in erythromycin polyketide synthase yields novel erythromycin derivatives,”
J. Bacteriol
. 179, 6416-6425 (1997)).
With each 2-carbon chain extension, the oxidation state of the &bgr;-carbon is embedded as a ketone, hydroxyl, methenyl, or methylene group by the presence or absence of one, two, or three additional catalytic domains in the module—a ketoreductase (KR), dehydratase (DH) and/or enoyl reductase (ER). In effect, the composition of catalytic domains within a module provides a “code” for the structure of each 2-carbon unit, and the order of modules codes for the sequence of the 2-carbon units, together creating a linear template for the linear polyketide product. The remarkable structural diversity of polyketides is governed by the combinatorial possibilities of arranging catalytic domains within each module, the sequence and number of modules, and the post-polyketide synthesis cyclization and “tailoring enzymes” that accompany the PKS genes. The direct correspondence between the catalytic domains of modules in a PKS and the structure of the resulting biosynthetic product portends the possibility of modifying polyketide structure by modifying the domains of the modular PKS.
There remains a need for compounds with modifications of the chemically inert sites of polyketides such as erythromycin that can be produced by genetic engineering. Such novel macrolides could in themselves provide the basis for new pharmaceuticals or serve as scaffolds for new semi-synthetic analogs. The present invention meets this need.
SUMMARY OF THE INVENTION
The present invention provides a library of recombinant PKS genes, host cells containing those genes, and the polyketides produced by those host cells. The polyketides provided by the invention include the polyketides shown in
FIG. 2
, as well as the polyketides that can be prepared by any of the myriad possible combinations of the recombinant PKS genes of the invention.
The present invention also provides the glycosylated and hydroxylated forms of the polyketides of the invention that can be produced by contacting the polyketides described herein with host cells selected from the group consisting of
Saccharopolyspora erythraea, Streptomyces venezuelae, S. narbonensis, S. antibioticus, S. fradiae, S. thernotolerans
, and
Micromonospora megalomicea
. The invention also provides compounds derived from the foregoing by chemical modification, including the C-6 to C-9 hemiketals formed from the compounds of the invention having a C-6 hydroxyl group and a C-9 keto group by treatment with mild acid.
The present invention also provides novel polyketides in isolated and purified form, as well as in cultures of recombinant host cells. Particular polyketides provided include 5,6-dideoxy-10-norerythronolide B, 6-deoxy-12-norerythronolide B, 2, 10-bisnor-3-oxo-6-deoxy-10,11 -anhydroerythronolide B, and 2,4-bisnor-3-oxo-6-deoxyerythronolide B, as well as the glycosylated and hydroxylated forms thereof
The present invention also provides the polyketide compounds of the invention in the form of pharmaceutical compositions, and methods for using the same in the treatment of disease.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below.


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