Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual "glycolate" chain extension unit in ansamitocin biosynthesis

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-13C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster.     

Dual carbamoylations on the polyketide and glycosyl moiety by asm21 result in extended ansamitocin biosynthesis

Carbamoylation is one of the post-PKS modifications in ansamitocin biosynthesis. A novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety was identified from the ansamitocin producer, Actinosynnema pretiosum. Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Three new derivatives without the backbone carbamoyl group were isolated from an asm21 mutant and characterized by NMR spectroscopy. Among them, 18-O-methyl-19-chloroproansamitocin was the major product and the preferred substrate for macrolactam C-7 carbamoylation by Asm21. However, Asm21 exhibited higher catalytic efficiency toward the glucosyl moiety. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo. This work represents the first biochemical characterization of an O-carbamoyltransferase performing dual actions on both a polyketide backbone and a glycosyl moiety during ansamitocin biosynthesis.     

Amide N-glycosylation by Asm25, an N-glycosyltransferase of ansamitocins

Ansamitocins are potent antitumor maytansinoids produced by Actinosynnema pretiosum. Their biosynthesis involves the initial assembly of a macrolactam polyketide, followed by a series of postpolyketide synthase (PKS) modifications. Three ansamitocin glycosides were isolated from A. pretiosum and fully characterized structurally as novel ansamitocin derivatives, carrying a beta-D-glucosyl group attached to the macrolactam amide nitrogen in place of the N-methyl group. By gene inactivation and complementation, asm25 was identified as the N-glycosyltransferase gene responsible for the macrolactam amide N-glycosylation of ansamitocins. Soluble, enzymatically active Asm25 protein was obtained from asm25-expressing E. coli by solubilization from inclusion bodies. Its optimal reaction conditions, including temperature, pH, metal ion requirement, and Km/Kcat, were determined. Asm25 also showed broad substrate specificity toward other ansamycins and synthetic indolin-2-ones. To the best of our knowledge, this represents the first in vitro characterization of a purified antibiotic N-glycosyltransferase.     

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.     

Identification of asm19 as an acyltransferase attaching the biologically essential ester side chain of ansamitocins using N-desmethyl-4,5-desepoxymaytansinol,not maytansinol,as its substrate

The potent antitumor activity of the ansamitocins, polyketides isolated from Actinosynnema pretiosum, is absolutely dependent on a short acyl group esterified to the C-3 oxygen of the macrolactam ring. Asm19, a gene in the ansamitocin biosynthetic gene cluster with homology to macrolide O-acyltransferase genes, is thought to encode the enzyme catalyzing this esterification. A mutant carrying an inactivated asm19 no longer produced ansamitocins but accumulated N-desmethyl-4,5-desepoxymaytansinol, rather than maytansinol, indicating that the acylation is not the terminal step of the biosynthetic sequence. Bioconversion experiments and in vitro studies with recombinant Asm19, expressed in Escherichia coli, showed that the enzyme is very specific toward its alcohol substrate, converting N-desmethyl-4,5-desepoxymaytansinol (but not maytansinol) into ansamitocins, but rather promiscuous toward its acyl substrate, utilizing acetyl-, propionyl-, butyryl-, isobutyryl-, as well as isovaleryl-CoA.     

Preparation of Thermocleavable Conjugates Based on Ansamitocin and Superparamagnetic Nanostructured Particles by a Chemobiosynthetic Approach

A combination of mutasynthesis, precursor‐directed biosynthesis and semisynthesis provides access to new ansamitocin derivatives including new nanostructured particle–drug conjugates. These conjugates are based on the toxin ansamitocin and superparamagnetic iron oxide–silica core shell particles. New ansamitocin derivatives that are functionalized either with alkynyl‐ or azido groups in the ester side chain at C‐3 are attached to nanostructured iron oxide core–silica shell particles. Upon exposure to an oscillating electromagnetic field these conjugates heat up and the ansamitocin derivatives are released by a retro‐Diels–Alder reaction. For example, one ansamitocin derivative exerts strong antiproliferative activity against various cancer cell lines in the lower nanomolar range while the corresponding nanostructured particle‐drug conjugate is not toxic. Therefore, these new conjugates can serve as dormant toxins that can be employed simultaneously in hyperthermia and chemotherapy when external inductive heating is applied.     

Metabolism studies of the anti-tumor agent maytansine and its analog ansamitocin P-3 using liquid chromatography/tandem mass spectrometry

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.     

Chemoenzymatic approaches toward dechloroansamitocin P-3

[reaction: see text] The enantioselective total synthesis of proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin P-3, is described which bears a diene-ene RCM as the key macrocyclization step. Feeding of proansamitocin to an AHBA block mutant Actinosynnema pretiosum (HGF073) yielded ansamitocin P-3 as well as dechloroansamitocin P-3, the latter also being formed upon fermentation in the presence of 3-amino-5-methoxybenzoic acid.     

Combined muta- and semisynthesis: a powerful synthetic hybrid approach to access target specific antitumor agents based on ansamitocin P3

Access of four new tumor specific folic acid/ansamitocin conjugates is reported that relies on a synthetic strategy based on the combination of mutasynthesis and semisynthesis. Two bromo‐ansamitocin derivatives were prepared by mutasynthesis or by a modified fermentation protocol, respectively, that served as starting point for the semisynthetic introduction of an allyl amine linker under Stille conditions. A sequence of standard coupling steps introduced the pteroic acid/glutamic acid/cysteine unit to the modified ansamitocins. All new derivatives, including those that are expected to be generated after internalization of the folic acid/ansamitocin conjugates into the cancer cell and reductive cleavage of the disulfide linkage showed good to strong antiproliferative activity (IC₅₀ <10 nM ) for different cancer cell lines. Finally, the four conjugates were exposed to two cancer cell lines [cervix carcinoma, KB‐3‐1 (FR+) and lung carcinoma, A‐459 (FR?)], the latter devoid of the membrane‐bound folic acid receptor (FR?). All four conjugates showed strong antiproliferative activity for the FR+ cancer cell line but were inactive against the FR? cell line. The synthetic strategy pursued is based on the combination of mutasynthesis and semisynthesis and proved to be powerful for accessing new ansamitocin derivatives that are difficult to prepare by total synthesis.     

On the biosynthetic origin of methoxymalonyl-acyl carrier protein, the substrate for incorporation of "glycolate" units into ansamitocin and soraphen A

Feeding experiments with isotope-labeled precursors rule out hydroxypyruvate and TCA cycle intermediates as the metabolic source of methoxymalonyl-ACP, the substrate for incorporation of "glycolate" units into ansamitocin P-3, soraphen A, and other antibiotics. They point to 1,3-bisphosphoglycerate as the source of the methoxymalonyl moiety and show that its C-1 gives rise to the thioester carbonyl group (and hence C-1 of the "glycolate" unit), and its C-3 becomes the free carboxyl group of methoxymalonyl-ACP, which is lost in the subsequent Claisen condensation on the type I modular polyketide synthases (PKS). d-[1,2-(13)C(2)]Glycerate is also incorporated specifically into the "glycolate" units of soraphen A, but not of ansamitocin P-3, suggesting differences in the ability of the producing organisms to activate glycerate. A biosynthetic pathway from 1,3-bisphosphoglycerate to methoxymalonyl-ACP is proposed. Two new syntheses of R- and S-[1,2-(13)C(2)]glycerol were developed as part of this work.     

Preparative isolation and purification of anti‐tumor agent ansamitocin P‐3 from fermentation broth of Actinosynnema pretiosum using high‐performance counter‐current chromatography

Ansamitocin P‐3 is a potent anti‐tumor maytansinoid found in Actinosynnema pretiosum. However, due to the complexity of the fermentation broth of Actinomycete, how to effectively separate ansamitocin P‐3 is still a challenge. In this study, both analytical and preparative high‐performance counter‐current chromatography were successfully used to separate and purify ansamitocin P‐3 from fermentation broth. A total of 28.8 mg ansamitocin P‐3 with purity of 98.4% was separated from 160 mg crude sample of fermentation broth in less than 80 min with the two‐phase solvent system of hexane–ethyl acetate–methanol–water (0.6:1:0.6:1, v/v/v/v). The purity and structural identification were determined by HPLC, ¹H NMR, ¹³C NMR and mass spectroscopy.     

Synthesis of the N-acetylcysteamine thioester of seco-proansamitocin

[structure: see text] The enantioselective total synthesis of the N-acetylcysteamine thioester of seco-proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin, is described, which twice utilizes the Nagao acetate aldol reaction, as well as an indium-mediated alkynylation of a benzyl bromide followed by carboalumination. The key step is a Heck reaction between two terminal alkenes for merging the two major fragments.     

An isolable acyclic hemiacetal of ansamitocin P‐3

During impurity analysis of maytansinol (2), produced from the reduction of ansamitocin P‐3 (AP‐3, 1), a surprisingly stable acyclic hemiacetal (4) was isolated. A combination of 1D and 2D NMR experiments, along with liquid chromatography–mass spectrometry data was used to confirm the structure. Comparison of NMR data to the previously reported bridged acetal (3), a by‐product of AP‐3 reduction, supports reassignment of the latter to the former. Additionally, ROESY data, in conjunction with minimum energy calculations, support intramolecular hydrogen bonding that is involved in stabilizing the hemiacetal. This report adds another example to the very short list of isolable acyclic hemiacetals. Copyright © 2012 John Wiley & Sons, Ltd.     

Gemini surfactants: new synthetic vectors for gene transfection

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.     

Broad substrate specificity of the amide synthase in S. hygroscopicus--new 20-membered macrolactones derived from geldanamycin

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamitocins. This was demonstrated when blocked mutants of both strains incapable of biosynthesizing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide synthase starter unit of both natural products, were supplemented with 3-amino-5-hydroxymethylbenzoic acid instead. Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin derivatives, which matched well with a competition assay for Hsp90α.     

Preliminary molecular replacement results for a crystalline gene 4 protein-deoxyoligonucleotide complex.

Complexes of the gene 5 protein from bacteriophage fd with a variety of oligodeoxynucleotides, ranging in length from two to eight and comprised of several different sequences, have been formed and crystallized for X-ray diffraction analysis. The crystallographic parameters of four different unit cells, all of which are based on hexagonal packing arrangements, indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end-to-end closure of a linear array of six dimers. From our results we have proposed a double-helix model for the gene 5 protein-DNA complex in which the protein forms a spindle or core around which the DNA is spooled. Currently 5.0-A X-ray diffraction data from one of the crystalline complexes is being analyzed by molecular replacement techniques to obtain a direct image of the protein-nucleic acid complex.     

Trifunctional 99mTc based radiopharmaceuticals: metal-mediated conjugation of a peptide with a nucleus targeting intercalator

The development of molecular imaging agents with multiple functions has become a major trend in radiopharmaceutical chemistry. We present herein the syntheses of trifunctional compounds, combining an acridine orange (AO) based intercalator with a GRP receptor specific bombesin like peptide (BBN). Metal-mediated conjugation of these two functions via the [2 + 1] approach to the third function, the [M(CO)(3)](+) (M = (99m)Tc, Re) moiety, yielded the final trifunctional molecules. The strongly fluorescent acridine orange, a nuclear targeting agent, has been derivatised with 4-imidazolecarboxylate as a bidentate ligand and bombesin with an isonitrile group as a monodentate ligand. For cell and nuclear uptake studies, [Re(L(1)-BBN)(L(2)-Ical)(CO)(3)] type complexes were synthesized and characterized. For radiopharmaceutical purposes, the (99m)Tc analogues have been prepared in a stepwise synthesis. Fluorescence microscopy studies on PC-3 cells, bearing the BBN receptor, showed high and rapid uptake into the cytoplasm. For the bifunctional molecule, lacking the BBN peptide, no internalization was observed.     

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.     

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.     

Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition

Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.