Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.     

Novel desosamine-modified 14- and 15-membered macrolides without antibacterial activity

Novel modifications of the desosamine sugar of 14- and 15-membered antibacterial macrolides, in which the desosamine was fused with N-substituted-1,3-oxazolidin-2-ones, were developed in order to completely suppress antibacterial activity and make them promising agents for other biological targets. The synthesis of such bicyclic desosamine derivatives, especially 1,3-oxazolidin-2-one formation, was optimized and conducted under mild conditions without a need for protection/deprotection steps for other functional groups. A focused series of novel desosamine-modified macrolide derivatives was prepared and their antibacterial activities tested. It was shown that these macrolide derivatives do not possess any residual antibacterial activity.     

Visualizing the 16-membered ring macrolides tildipirosin and tilmicosin bound to their ribosomal site

The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as Pasteurella multocida. Tildipirosin is a derivative of the naturally occurring compound tylosin. Here, we define drug-target interactions by combining chemical footprinting with structure modeling and show that tildipirosin, tylosin, and an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyl-tylonolide, Micotil), bind to the same macrolide site within the large subunit of P. multocida and Escherichia coli ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues on the tunnel wall while its 20-piperidine is oriented into the tunnel lumen and is positioned to interfere with the growing nascent peptide.     

Novel 9a-carbamoyl- and 9a-thiocarbamoyl-3-decladinosyl-6-hydroxy and 6-methoxy derivatives of 15-membered macrolides

An efficient method for the synthesis of diverse 9a-carbamoyl- and 9a-thiocarbamoyl-3-decladinosyl-6-hydroxy and 3-decladinosyl-6-methoxy derivatives of 15-membered azalides has been developed. These derivatives bear various alkyl and aryl groups attached to macrolide scaffold through urea or thiourea moieties at 9a position. Chemical transformations of hydroxy group at position C-3 afforded range of ketolides, anhydrolides, hemiketals, cyclic ethers, and acylides. It has been shown that 6-hydroxy and 6-methoxy derivatives undergo different chemical transformations under otherwise identical reaction conditions. Antimicrobial properties of prepared compounds were evaluated.     

Synthesis and antibacterial activity of novel bifunctional macrolides

We report the discovery of a novel class of macrolide antibiotics that have improved antibacterial activity against Ery-resistant organisms.     

Gene expression profiles of human small airway epithelial cells treated with low doses of 14- and 16-membered macrolides

Although long-term treatment with low doses of 14-membered macrolides is widely applied in management of patients with chronic inflammatory diseases, e.g., diffuse panbronchiolitis, chronic bronchitis, or chronic lung damage in newborns, the physiological mechanisms underlying the action of macrolides in these conditions are unclear. To clarify the pathological basis of these diseases and also to aid in the design of novel drugs to treat them, we chose to investigate the molecular target(s) of macrolides. Our experiments involved long-term culture of human small airway epithelial cells (hSAEC) in media containing 14-membered macrolides erythromycin (EM) or clarithromycin (CAM), or a 16-membered macrolide, josamycin (JM), which lacks clinical anti-inflammatory effects. We then analyzed gene expression profiles in the treated cells using a cDNA microarray consisting of 18,432 genes. We identified nine genes whose expression was significantly altered during 22 days of culture with EM, and seven that were altered by CAM in that time. Four of those genes revealed similar behavior in cells treated with either of the 14-membered macrolides, but not JM. The products of these four genes may be candidates for mediating the ability of 14-membered macrolides to suppress chronic inflammation.     

Catenulisporidins A and B, 16-membered macrolides of the hygrolidin family produced by the chemically underexplored actinobacterium Catenulispora species

Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.     

Recent advances in the discovery and combinatorial biosynthesis of microbial 14-membered macrolides and macrolactones

Journal of Industrial Microbiology & Biotechnology - Macrolides, especially 14-membered macrolides, are a valuable group of antibiotics that originate from various microorganisms. In addition...     

Novel azalides derived from 16-membered macrolides. Part II: Isolation of the linear 9-formylcarboxylic acid and its sequential macrocyclization with an amino alcohol or an azidoamine

The design and synthesis of novel 14- to 16-membered 11-azalides starting from 16-membered macrolides are reported. A linear 9-formylcarboxylic acid was isolated via a mobile dialdehyde previously reported. Sequential macrocyclization of the formylcarboxylic acid with amino alcohol followed by deprotection afforded corresponding 14- to 16-membered azalides. On the other hand, reductive amination of the formylcarboxylic acid with an azidoamine followed by macrolactam formation with an amine generated from the azide gave 14- to 16-membered azalactams. Among these derivatives, 15-membered azalactams and 16-membered azalides exhibited characteristic in vitro antibacterial activities. Although optimization of 15-membered azalactams including demycarosyl analogues did not provide remarkably promising molecules, SAR studies of 16-membered azalides disclosed that substitution at the 15 position was very important for identification of a clinical candidate.     

Synthesis of 9-membered dilactams derived from 1,3-diaminopropionic and glutamic acids.

Previously unknown 9-membered bridged dipeptides derived from l or d isomers of 1,3-diaminopropionic acids and l-glutamic acid were synthesized using aminoacyl incorporation reaction. Key intermediates containing internal pyroglutamyl moiety were prepared via side chain to backbone cyclization of related protected dipeptide derivatives of glutamic acid.     

Synthesis of novel 4'-substituted 16-membered ring macrolide antibiotics derived from leucomycins

A series of novel 4'-substituted 16-membered ring macrolides was synthesized by the cleavage of the mycarose sugar and subsequent modification of 4'-hydroxyl group. This new class of macrolides antibiotics is acid stable. The synthetic methodology described here is expected to find application in the synthesis of new generation of macrolides that target the emerging bacterial resistance.     

Production of a hybrid 16-membered macrolide antibiotic by genetic engineering of Micromonospora sp. TPMA0041

Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, d-desosamine and d-mycinose, at the C-5 and C-21 positions, respectively. We previously engineered an expression plasmid pSETmycinose containing the d-mycinose biosynthesis genes from M. griseorubida A11725. This plasmid was introduced into Micromonospora sp. FERM BP-1076 cells, which produce the 16-membered macrolide antibiotic izenamicin. The resulting engineered strain TPMA0041 produced 23-O-mycinosyl-20-deoxy-izenamicin B₁ and 22-O-mycinosyl-izenamicin B₂. 23-O-mycinosyl-20-deoxy-izenamicin B₁ has been produced by the engineered strain M. rosaria TPMA0001 containing pSETmycinose as 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (=IZI) in our recent study, and 22-O-mycinosyl-izenamicin B₂ has previously been synthesized as a macrolide antibiotic TMC-016 with strong antibacterial activity. The production of 22-O-mycinosyl-izenamicin B₂ (=TMC-016) was increased when propionate, a precursor of methylmalonyl-CoA, was added to the culture broth.     

Synthesis of 10- and 9-membered dilactams derived from aspartic and glutamic acids.

9- and 10-membered bridged dipeptides derived from L-aspartic acid and L- or D-glutamic acid were synthesized using aminoacyl incorporation reaction. Key intermediates containing internal pyroglutamyl moiety were prepared via side chain to backbone cyclization of related protected dipeptide derivatives of glutamic acid.     

Synthesis and in vitro activity of dicationic indolyl diphenyl ethers as novel potent antibiotic agents against drug-resistant bacteria

A series of 4,4′-bis-[2-(6-N-substituted-amidino)indolyl] diphenyl ether have been synthesized and tested for their in vitro antibacterial activity including a range of Gram-positive and Gram-negative pathogens and cytotoxicity. Most of these compounds have mainly shown anti-Gram positive bacteria activities especially against drug resistant bacterial strains MRSA, MRSE and VRE. The anti-MRSA and anti-MRSE activities of compound 7a and 7j were more potent than that of the lead compound 2, levofloxacin and vancomycin. Interestingly, 7j had greatly improved anti negative bacterial activity, especially for the producing NDM-1 Klebsiella pneumonia strain and less toxic than that of the lead compound 2.     

Novel 16-membered macrolides modified at C-12 and C-13 positions of midecamycin A1 and miokamycin. Part 1: Synthesis and evaluation of 12,13-carbamate and 12-arylalkylamino-13-hydroxy analogues

Design and synthesis of 16-membered macrolides modified at the C-12 and 13 positions are described. The compounds we report here have an arylalkylamino group attached to the C-12 position of the macrolactone. Both types of derivatives, 12,13-cyclic carbamates and non-carbamate analogues, were synthesized via 12-amino-13-hydroxy intermediates derived from 12,13-epoxide that was prepared by selective epoxidation at the C-12 and C-13 positions. 4'-Hydroxyl analogues were also prepared by acidic hydrolysis of a neutral sugar. These compounds were evaluated for in vitro antibacterial activity against respiratory tract pathogens. Some of these analogues exhibited an improved activity compared with the corresponding parent compound.     

Synthesis and antibacterial activity of novel 4-aryl-[1,2,3]-triazole containing macrolides

Two series of novel triazole containing 14-member macrolides having either a cladinose or a 3-pyridyl acetate group at the 3-position of the macrolide ring were synthesized. The in vitro antibacterial activities against S. aureus, S. pneumoniae, S. pyogenes and E. faecalis were determined. Macrolide 7a and the fluoroketolide 1 (CEM-101) were evaluated in vivo in murine systemic infection models. All of the macrolide analogs were less active in vitro and in vivo than the fluoroketolide 1 (CEM-101).     

Synthesis and complexation studies of 16-membered [1+1] selenaaza- and some related macrocycles

A novel 16-membered [1+1] unsymmetrical selenaaza Schiff base macrocycle, 8,9,10,11-tetrahydo-7H-dibenzo-[d,o][1,3,7,10,13]diselenatriazacyclohexadecine (5) with N₃Se₂ donor set, has been synthesized by cyclocondensation of di(o-formylphenylseleno)methane and diethylenetriamine. Reduction of 5 with NaBH₄ afforded 6,7,8,9,10,11,12,13-octahydro-5H-dibenzo-[d,o][1,3,7,10,13]diselenatriazacyclohexadecine (6). Macrocycle, 10,11,12,13,14,15,16,26,27,28,29,30,31,32-tetradecahydrotetrabenzo[b,k,n,w][1,13,5,9,17,21]diselenatetraazacyclotetracosine (2b) was synthesized similarly from di(o-formylphenyl)selenide and 1,3-diaminopropane followed by reduction with NaBH₄. Single crystal X-ray structures of 5 and 2b have been determined. Attempted complexation reactions of 5 with Zn(II), Cd(II), Hg(II), Pd(II) and Cu(II) ions and those of 6 with Ag(I), Hg(II) and Pd(II) ions are reported. 28-Membered selenaaza macrocycle 2c on reaction with HgCl₂ and NH₄PF₆, provided a dinuclear Hg(II) complex 9. Complex 9 has been characterized by single crystal X-ray structure.     

Synthesis,antimycobacterial activity and influence on mycobacterial InhA and PknB of 12-membered cyclodepsipeptides

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at µM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa?>?5?µg/ml) and did not show any significant antiproliferative effect (HUVEC?>?5?µg/ml; K-562?>?5?µg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.     

Anti-cryptosporidial activity of macrolides in immunosuppressed rats.

Five macrolides were evaluated for anti-cryptosporidial activity in a dexamethasone-immunosuppressed rat model. All the macrolides evaluated reduced the severity of the ileal infection, but their effect on the cecal infection was variable, depending on the macrolide tested and the dose administered. The results of this study suggest that the use of some macrolides as potential anti-cryptosporidial agents warrants further investigation.