Mohammad Nur-e-Alam

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

Optimizing natural products by biosynthetic engineering: Discovery of nonquinone Hsp90 inhibitors

A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.

Deoxysugar Transfer during Chromomycin A3 Biosynthesis in Streptomyces griseus subsp. griseus: New Derivatives with Antitumor Activity

ABSTRACT Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-d-oliose (sugar A) and 4-O-methyl-d-oliose (sugar B), and a trisaccharide of d-olivose (sugar C), d-olivose (sugar D), and 4-O-acetyl-l-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two d-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.

Tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin A3 by Streptomyces griseus ssp. griseus

Chromomycin A3 is a member of the aureolic acid group family of antitumour drugs. Three tailoring modification steps occur during its biosynthesis affecting the sugar moieties: two O‐acetylations and one O‐methylation. The 4‐O‐methylation in the 4‐O‐methyl‐D‐oliose moiety of the disaccharide chain is catalysed by the cmmMIII gene product. Inactivation of this gene generated a chromomycin‐non‐producing mutant that accumulated three unmethylated derivatives containing all sugars but differing in the acylation pattern. Two of these compounds were shown to be substrates of the methyltransferase as determined by their bioconversion into chromomycin A2 and A3 after feeding these compounds to a Streptomyces albus strain expressing the cmmMIII gene. The same single membrane‐bound enzyme, encoded by the cmmA gene, is responsible for both acetyl transfer reactions, which convert a relatively inactive compound into the bioactive chromomycin A3. Insertional inactivation of this gene resulted in a mutant accumulating a dideacetylated chromomycin A3 derivative. This compound, lacking both acetyl groups, was converted in a two‐step reaction via the 4E‐monoacetylated intermediate into chromomycin A3 when fed to cultures of S. albus expressing the cmmA gene. This acetylation step would occur as the last step in chromomycin biosynthesis, being a very important event for self‐protection of the producing organism. It would convert a molecule with low biological activity into an active one, in a reaction catalysed by an enzyme that is predicted to be located in the cell membrane.

Biosynthesis of the angiogenesis inhibitor borrelidin: directed biosynthesis of novel analogues

We report the directed biosynthesis of borrelidin analogues and their selective anti-proliferative activity against human cancer cell lines.

Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family

ABSTRACT Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F < 4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2 > 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Elucidation of the glycosylation sequence of mithramycin biosynthesis: isolation of 3A-deolivosylpremithramycin B and its conversion to premithramycin B by glycosyltransferase MtmGII.

Mithramycin (MTM) is an aureolic acid-type polyketide produced by various soil bacteria of the genus Streptomyces including Streptomyces argillaceus (ATCC 12956). MTM has been used clinically to treat Paget’s disease and testicular carcinoma, and MTM’s hypocalcemic effect has been used to manage hypercalcemia in patients with malignancy-associated bone lesions. Mithramycin has also been shown to act as neuroprotective drug. All aureolic acid-group drugs (i.e. MTM, chromomycin A3, olivomycin A, UCH9, and durhamycin A) 10, 11] contain the same tricyclic core moiety, but differ mainly with regard to their saccharide moieties, which consist of various 2,6-dideoxysugar chains that are linked at the 2and 6-positions of the aglycon moiety. The structural variations in the glycosidic moieties are responsible for subtle differences in the DNA binding and activity profiles amongst the members of the aureolic acid group. 12–15] While extensive DNA–antibiotic interaction studies clearly revealed that the intact C-D-E trisaccharide moiety is essential for dimer formation as well as optimal DNA binding of MTM and chromomycin, the role of the disaccharide chain at C-6 is less well characterized. However, the X-ray structure of the MTM–DNA complex revealed that this disaccharide interacts with the phosphate backbone of the DNA, and suggested that modifications of this disaccharide chain may have a profound impact on the biological activity. Unfortunately, derivatives, which differ from their parent drug with respect to the sugar units of the disaccharide chain, were not available at that point, but are badly needed to further investigate the structure–activity relationships (SAR) of the aureolic acids in general, and of mithramycin in particular. Biosynthetic-pathway engineering attempts were hampered by the fact that it was not then possible to determine exactly which glycosyltransferases were responsible for the attachments of the two olivose moieties of the disaccharide chain. Here we describe experiments that led to a clear assignment of the two glycosyltransferases involved in the formation of the mithramycin disaccharide chain. It has been shown that MTM biosynthesis 19–28] proceeds through tetracyclic intermediates (premithramycins) with glycosylation steps occurring on these tetracyclic biosynthetic intermediates. Initially the C-D-E-trisaccharide chain, consisting of a d-olivose, a d-oliose, and a d-mycarose, is attached at the 12a-position of premithramycinone (which later becomes the 2-position in mithramycin), while the disaccharide moiety is attached afterwards. Generation of GT-minus mutants suggested that MtmGIV and MtmGIII are involved in the trisaccharide chain formation, while MtmGI and MtmGII share the responsibility of the attachment of the two d-olivoses that form the disaccharide at C-6. Surprisingly, the GI and the GII mutants accumulate exactly the same pattern of metabolites, including premithramycins A1, A2, A2’, A3, and A3’ (see Scheme 1), the last two possessing the complete C-D-E trisaccharide chain. Although these results made it clear that both MtmGI and MtmGII take part in disaccharide formation, it was unclear which GT catalyzes which exact step, since neither the GI nor the GII mutant accumulated a premithramycin with four sugars including the first d-olivose unit of the disaccharide, as one could have expected. To explain the results, it was speculated that one of the two GTs (GI or GII) might catalyze the formation of an NDP-activated diolivoside, while the other one attaches the diolivoside to the aglycon. Evidence for this view came from model experiments with the sugar-cosubstrate flexible glycosyltransferase ElmGT, which generated inter alia a diolivosyltetracenomycin upon its heterologous expression in S. argillaceus, and from experiments in which S. argillaceus M3DMG, a mutant in which all GTs of the MTM biosynthesis were inactivated, was transformed with cosmid 16F4 as well as with either the mtmGI or mtmGII gene. Cosmid 16F4 harbors the entire gene cluster for the 8-demethyltetracenomycin biosynthesis plus ElmGT. Expression of cosmid 16F4 alone yields 8-olivosyltetracenomycin C as well as 8-mycarosyltetracenomycin C, but not 8-diolivosyltetracenomycin C. From the further expression of either the mtmGI or mtmGII gene, we expected that the 8-diolivosyltetracenomycin C production would be reinstalled, if one of the encoded GTs were responsible for the attachment of the second olivose or for the formation of an NDP-activated diolivoside (assuming that ElmGT can transfer such a diolivoside). Unexpectedly, neither of the resulting constructs yielded 8diolivosyltetracenomycin C, and both transformed mutants showed the same product pattern as found previously for the S. argillaceus DGT mutant expressing cosmid 16F4 alone. Only upon transformation of S. argillaceus M3DMG(cos16F4) with both GT-encoding genes mtmGI and mtmGII, diolivosyltetracenomycin production was restored to some extent. Thus, these experiments were totally inconclusive regarding the exact role played by MtmGI and MtmGII in mithramycin biosynthesis. In this communication, we show strong evidence for the view that these last two glycosylation steps in mithramycin [a] Dr. M. Nur-e-Alam, Prof. Dr. J. Rohr Department of Pharmaceutical Sciences, College of Pharmacy University of Kentucky 725 Rose Street, Lexington, KY 40536-0082 (USA) Fax: (+ 1) 859-257-7564 E-mail : jrohr2@uky.edu [b] Prof. Dr. C. M ndez, Prof. Dr. J. A. Salas Departamento de Biolog a Funcional e Instituto Universitario de Oncolog a del Principado de Asturias Universidad de Oviedo 33006 Oviedo (Spain)

Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes

Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.

Structure guided design of improved anti-proliferative rapalogs through biosynthetic medicinal chemistry

A combination of molecular modelling and rational biosynthetic engineering of the rapamycin polyketide synthase was used to generate rapalogs lacking O- and C-linked methyl groups at positions 16 and 17 respectively. These rapalogs displayed enhanced inhibition of cancer cell lines and were produced at titres close to those of the parent strain. By recapitulating these experiments in higher-producing rapamycin strains, combined with the ectopic expression of gene products acting late in the biosynthetic pathway in order to minimise the accumulation of intermediates, gram-quantities of novel rapalogs bearing multiple structural changes were produced.

Sangamides, a new class of cyclophilin-inhibiting host-targeted antivirals for treatment of HCV infection

Sangamides are amide derivatives of sanglifehrin A, a cyclophilin-binding polyketide natural product which is structurally distinct from cyclosporine A. Cyclosporine A is the starting point for the synthesis of cyclophilin inhibitors such as alisporivir, currently in development for the treatment of HCV infection. We report here initial results of the optimisation program which led to identification of the sangamides, compounds that exhibit significantly improved potential for the treatment of chronic HCV infection.