Macrolide Compound Research Articles

Identification and enhancing production of a novel macrolide compound in engineered Streptomyces peucetius.

Streptomyces peucetius produces doxorubicin and daunorubicin, which are important anticancer drugs. In this study, we activate peucemycin, a new antibacterial compound, using an OSMAC strategy. In general, bioactive compounds are produced in a higher amount at room temperature; however, in this study, we have demonstrated that a bioactive novel compound was successfully activated at a low temperature (18 °C) in S. peucetius DM07. Through LC-MS/MS, IR spectroscopy, and NMR analysis, we identified the structure of this compound as a γ-pyrone macrolide. This compound was found to be novel, thus named peucemycin. It is an unusual 14-membered macrocyclic γ-pyrone ring with cyclization. Also, peucemycin exhibits potential antibacterial activity and a suppressive effect on the viability of various cancer cell lines.

Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors

AbstractAfter the transplantation of unmodified marrow from human leukocyte antigen-matched unrelated donors receiving cyclosporine (CSP) and methotrexate (MTX), the incidence of acute graft-versus-host disease (GVHD) is greater than 75%. Tacrolimus is a macrolide compound that, in previous preclinical and clinical studies, was effective in combination with MTX for the prevention of acute GVHD. Between March 1995 and September 1996, 180 patients were randomized in a phase 3, open-label, multicenter study to determine whether tacrolimus combined with a short course of MTX (n = 90), more than CSP and a short course of MTX (n = 90), would reduce the incidence of acute GVHD after marrow transplantation from unrelated donors. There was a significant trend toward decreased severity of acute GVHD across all grades (P = .005). Based on the Kaplan-Meier estimate, the probability of grade II-IV acute GVHD in the tacrolimus group (56%) was significantly lower than in the CSP group (74%;P = .0002). Use of glucocorticoids for the management of GVHD was significantly lower with tacrolimus than with CSP (65% vs 81%, respectively; P = .019). The number of patients requiring dialysis in the first 100 days was similar (tacrolimus, 9; CSP, 8). Overall and relapse-free survival rates for the tacrolimus and CSP arms at 2 years was 54% versus 50% (P = .46) and 47% versus 42% (P = .58), respectively. The combination of tacrolimus and MTX after unrelated donor marrow transplantation significantly decreased the risk for acute GVHD than did the combination of CSP and MTX, with no significant increase in toxicity, infections, or leukemia relapse.

Two novel compounds from green walnut husks (Juglans mandshurica Maxim.)

Phytochemical investigation on the ethanol extract of green walnut husks (Juglans mandshurica Maxim.) led to the isolation of two previously unknown compounds, including a macrolide compound (13S)-8-oxo-(9E, 11E)-8-oxo-octadeca-9,11-dien-13-olide (1) and a diarylheptanoid compound 1-(3′-methoxy-4′-hydroxyphenyl)-7-(3″,4″-dimethoxyphenyl)heptan-3-one (2), together with 19 known compounds. The structures of these 21 compounds were elucidated by extensive analyses of NMR and HR-MS data, and the basis of spectroscopic analysis.

Inhibitory effect of sirolimus against white rot of Brassica juncea var. tumida

White rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, has recently become a serious threat to tuber mustard (Brassica juncea var. tumida) cultivation in Hangzhou and other mustard growing parts of Zhejiang province, China. This study was conducted to evaluate the inhibitory effect of sirolimus, a natural macrolide compound, against white rot of mustard tuber caused by S. sclerotiorum in vitro and in vivo. The results showed that sirolimus was able to reduce the mycelial growth, sclerogenesis as well as myceliogenic and carpogenic germinations of three isolates of the pathogen. Application of sirolimus markedly increased activity of β-N-acetyl hexosaminidase, endochitinase and β -1,3-glucanase in plant leaves. Moreover, a significant increase was observed in the level of polyphenoloxidase and peroxidase in plant leaves two and four days after application of sirolimus. The results of in vitro tests showed that sirolimus at the concentration of 100 nM completely inhibited disease severity. The treatment with sirolimus enhanced relative conductivity of S. sclerotiorum mycelia over time compared with control. An increase of carbohydrate leakage was observed in the pathogen mycelia incubated with sirolimus. The content of methane dicarboxylic aldehyde (MDA) markedly increased at 5 h and the highest MDA content was observed after 20 h. Due to exposure to sirolimus, a decrease percentage of catalase activity was observed in pathogen mycelia. In conclusion, this study showed that sirolimus can be considered as an effective substance against white rot of tuber mustard caused by S. sclerotiorum.

Preparation and separation of epothilones with anticancer activity.

Epothilone is a kind of macrolide compound, which has anticancer activity and strong inhibitory effect on breast cancer and rectal cancer cells. Herein, the chemical and biological synthesis of epothilones and how to improve fermentation yield and separation methods were summarized and discussed.

Thiocladospolide E and cladospamide A, novel 12-membered macrolide and macrolide lactam from mangrove endophytic fungus Cladosporium sp. SCNU-F0001

Chemical investigation of the mangrove endophytic fungus Cladosporium sp. SCNU-F0001 resulted in the isolation and identification of a new macrolide compound named thiocladospolide E (1) and a novel macrolide lactam named cladospamide A (2), along with the known cladospolide B (3). The structures were elucidated based on spectroscopic methods, and the absolute configurations were determined by X-ray diffraction and HPLC analysis after chemical derivatization. All compounds were tested for their antibacterial and cytotoxic activity.

Synthesis, anticancer activity and cytotoxicity of 7-O-β-d-galactosyl-polyethylene glycol-epothilone B.

Epothilone, the macrolide compound produced by Sorangium cellulosum, has antitumor activity. Its anti-tumor mechanism is similar to that of paclitaxel, which promotes the polymerization of tubulin and induces apoptosis. Herein, 7-O-β-d-galactosyl-polyethylene glycol-epothilone B 6 was synthesized. It showed that the toxicity of the synthesized compound was 1/182 of the epothilone B. In addition, compound 6 also had significant anticancer activity under the action of enzyme.

Biosynthesis of Quinolidomicin, the Largest Known Macrolide of Terrestrial Origin: Identification and Heterologous Expression of a Biosynthetic Gene Cluster over 200 kb.

Quinolidomicin A1 is the largest macrolide compound from terrestrial sources, consisting of a 60-membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.

Genomic analysis of a Streptomyces tsukubaensis mutant with reduced FR900525 production isolated by selection for S-(2-aminoethyl) l-cysteine resistance

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been shown to be of clinical importance and has been manufactured industrially since 1993 using mutants with high FK506 production ability. These mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production, and we previously established a mutant strain that produces reduced levels of FR900525 by selecting for S-(2-aminoethyl) l-cysteine (AEC) resistance. In this study, we conducted a genomic analysis of this strain to identify the changes associated with AEC resistance and to determine its metabolism. Three mutated genes were identified by comparing the genome sequences of the parental strain (A) and the AEC-resistant mutant (B). From the metabolite pathway, it was speculated that citric acid synthase was the most relevant to AEC resistance. To investigate the effect of the mutation in citric acid synthase, we added citric acid, an inhibitor of citric acid synthase, to strain A culture, which induced strain A to exhibit a strain B-like phenotype. We conclude that the mutation in citric acid synthase enhances the carbon flow into aspartic acid, increasing lysine synthesis and resulting in AEC resistance in strain B, as well as high production of FK506 and low production of FR900525.

Abstract 1644: Combination of MT-3724 with sirolimus reduces anti-drug antibody response and prolongs drug exposure

Abstract Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit. ETBs can induce their own internalization, route through the cell in a predictable manner, enzymatically and irreversibly destroy ribosomes to shutdown protein synthesis and induce apoptosis of tumor cells. This mechanism of action is distinct from that of other therapeutics, making ETBs an attractive treatment for patients who have become resistant to chemotherapy and other treatment modalities. MT-3724 is Molecular Templates’ first-generation ETB targeting CD20, a surface receptor that is highly expressed on malignant B cells in hematological malignancies. Pre-clinical and clinical studies have shown promising results for non-Hodgkin’s lymphoma cell lines and patients; however anti-drug antibodies are formed in experimental animals and patients after repeat dosing. Sirolimus (rapamycin) is a macrolide compound approved to prevent organ rejection and in LAM, and has been used in tolerization protocols with replacement enzymes. In pre-clinical studies, we have co-administered sirolimus in combination with MT-3724 in rodents as well as in a non-human primate model in order to reduce the anti-drug antibody response to MT-3724. These studies demonstrated that the combination of sirolimus with MT-3724 decreased anti-drug antibody response, prolonged serum exposure of MT-3724, and improved B-cell depletion as compared to MT-3724 alone. Molecular Template’s ETB technology has resulted in potent and targeted therapeutic agents that have a unique mechanism of action in the field of oncology. Our first-generation, CD20-targeted therapeutic, MT-3724, has promising clinical results in the refractory setting for non-Hodgkin’s lymphoma. The combination of sirolimus with MT-3724 is an attractive and feasible regimen which will be further explored in clinical studies. Citation Format: Jack P. Higgins, Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jensing Liu, Erin K. Willert. Combination of MT-3724 with sirolimus reduces anti-drug antibody response and prolongs drug exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1644. doi:10.1158/1538-7445.AM2017-1644

Approaches towards the enhanced production of Rapamycin by Streptomyces hygroscopicus MTCC 4003 through mutagenesis and optimization of process parameters by Taguchi orthogonal array methodology.

The present research was conducted to define the approaches for enhanced production of rapamycin (Rap) by Streptomyces hygroscopicus microbial type culture collection (MTCC) 4003. Both physical mutagenesis by ultraviolet ray (UV) and chemical mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG) have been applied successfully for the improvement of Rap production. Enhancing Rap yield by novel sequential UV mutagenesis technique followed by fermentation gives a significant difference in getting economically scalable amount of this industrially important macrolide compound. Mutant obtained through NTG mutagenesis (NTG-30-27) was found to be superior to others as it initially produced 67% higher Rap than wild type. Statistical optimization of nutritional and physiochemical parameters was carried out to find out most influential factors responsible for enhanced Rap yield by NTG-30-27 which was performed using Taguchi orthogonal array approach. Around 72% enhanced production was achieved with nutritional factors at their assigned level at 23 °C, 120rpm and pH 7.6. Results were analysed in triplicate basis where validation and purification was carried out using high performance liquid chromatography. Stability study and potency of extracted Rap was supported by turbidimetric assay taking Candida albicans MTCC 227 as test organism.

Reduction of FR900525 using an S-(2-aminoethyl) l-cysteine-resistant mutant

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S.tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis.

Bioinformatics-Inspired Isolation of Akaemycin, a New Macrolide from Marine <i>Streptomyces</i> sp.

Background: Investigation of products from marine Streptomyces sp. NPS554 led to the discovery of lorneic acids and akaeolide. Genome analysis of this strain predicted the presence of more unidentified secondary metabolites. In this study, we further examined the products of strain NPS554 to obtain novel compounds predicted by bioinfomatical screening. Methods: Culture extract of strain NPS554 was fractionated by different chromatography methods. The chemical structure was elucidated by spectroscopic analysis. The absolute configuration was further determined by PKS gene analysis. Antimicrobial assay was conducted to evaluate the biological activity of the new compound. Results & Conclusion: Akaemycin, a novel macrolide compound was isolated as a yellow amorphous solid. Its structure was elucidated by MS and NMR spectroscopic analyses. Its spectroscopically defined structure was consistent with the structure predicted from the annotation of orphan type I PKS gene clusters in the NPS554 genome. Stereochemistry of akaemycin was proposed on the basis of ROESY and bioinformatical analysis. Antimicrobial assay showed activity against Gram-positive bacteria and filamentous fungi. Keywords: macrolide, marine Streptomyces, polyketide, PKS.

Macrolide analog F806 suppresses esophageal squamous cell carcinoma (ESCC) by blocking β1 integrin activation.

The paucity of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits the treatment options. This study characterized the therapeutic efficacy and action mechanism of a novel natural macrolide compound F806 in human ESCC xenograft models and cell lines. F806 inhibited growth of ESCC, most importantly, it displayed fewer undesirable side effects on normal tissues in two human ESCC xenograft models. F806 inhibited proliferation of six ESCC cells lines, with the half maximal inhibitory concentration (IC50) ranging from 9.31 to 16.43 μM. Furthermore, F806 induced apoptosis of ESCC cells, contributing to its growth-inhibitory effect. Also, F806 inhibited cell adhesion resulting in anoikis. Mechanistic studies revealed that F806 inhibited the activation of β1 integrin in part by binding to a novel site Arg610 of β1 integrin, suppressed focal adhesion formation, decreased cell adhesion to extracellular matrix and eventually triggered apoptosis. We concluded that F806 would potentially be a well-tolerated anticancer drug by targeting β1 integrin, resulting in anoikis in ESCC cells.

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation.

BackgroundHeat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin.MethodsWe adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line.ResultsWe have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile.ConclusionsAs a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells.

MTOR signaling and its roles in normal and abnormal brain development.

Target of rapamycin (TOR) was first identified in yeast as a target molecule of rapamycin, an anti-fugal and immunosuppressant macrolide compound. In mammals, its orthologue is called mammalian TOR (mTOR). mTOR is a serine/threonine kinase that converges different extracellular stimuli, such as nutrients and growth factors, and diverges into several biochemical reactions, including translation, autophagy, transcription, and lipid synthesis among others. These biochemical reactions govern cell growth and cause cells to attain an anabolic state. Thus, the disruption of mTOR signaling is implicated in a wide array of diseases such as cancer, diabetes, and obesity. In the central nervous system, the mTOR signaling cascade is activated by nutrients, neurotrophic factors, and neurotransmitters that enhances protein (and possibly lipid) synthesis and suppresses autophagy. These processes contribute to normal neuronal growth by promoting their differentiation, neurite elongation and branching, and synaptic formation during development. Therefore, disruption of mTOR signaling may cause neuronal degeneration and abnormal neural development. While reduced mTOR signaling is associated with neurodegeneration, excess activation of mTOR signaling causes abnormal development of neurons and glia, leading to brain malformation. In this review, we first introduce the current state of molecular knowledge of mTOR complexes and signaling in general. We then describe mTOR activation in neurons, which leads to translational enhancement, and finally discuss the link between mTOR and normal/abnormal neuronal growth during development.

Establishment of the absolute configuration of the 34-membered polyol macrolide compound JBIR-129

Establishment of the absolute configuration of the 34-membered polyol macrolide compound JBIR-129

Enhancing production of a 24-membered ring macrolide compound by a marine bacterium using response surface methodology

A 24-membered ring macrolide compound, macrolactin A has potential applications in pharmaceuticals for its anti-infectious and antiviral activity. In this study, macrolactin A was produced by a marine bacterium, which was identified as Bacillus subtilis by 16S ribosomal RNA (rRNA) sequence analysis. Electrospray ionization mass spectrometry (ESI/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses were used to characterize this compound. To improve the production, response surface methodology (RSM) involving Box-Behnken design (BBD) was employed. Faeces bombycis, the main by-product in sericulture, was used as a nitrogen source in fermentation. The interactions between three significant factors, F. bombycis, soluble starch, and (NH4)2SO4 were investigated. A quadratic model was constructed to fit the production and the factors. Optimum medium composition was obtained by analysis of the model. When cultivated in the optimum medium, the production of macrolactin A was increased to 851 mg/L, 2.7 times as compared to the original. This study is also useful to find another way in utilizing F. bombycis.

The enhancement of rapamycin production using Streptomyces hygroscopicus through a simple pH-shifted control

Rapamycin, a ring macrolide compound, is recognized as a potent immunosuppressive drug which works by inhibiting T-cell activation and proliferation and is also characterized as having cancer-fighting and anti-aging functions. The pellet morphology of Streptomyces hygroscopius was suggested to have the high rapamycin production titer. The change of culturing pH could alter the morphology of actinomycetes. Therefore, the influence of pH level on rapamycin production was examined. No significant effects of initial pH on rapamycin production were observed in the pH range of 6–8 in the flask trials. Nevertheless, a two-stage pH control strategy was proposed in a bench-scale fermenter, with no pH control in the 1st stage and with the pH controlled at 5.5 in the 2nd stage after the pH rebounded from the bottom. A recorded high rapamycin level of 537±23mg/L was obtained in a simple batch operation, far higher than the 45±5 and 98±5mg/L levels in the batches with the pH maintained at a fixed level of 7.0 and without the pH controlled, respectively. The strategy of a two-stage pH control was simple to operate and potentially applied in the scaled-up production of rapamycin.

Biselyngbyaside, isolated from marine cyanobacteria, inhibits osteoclastogenesis and induces apoptosis in mature osteoclasts

The mass and function of bones depend on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An inhibitor of osteoclast differentiation and/or function is expected to be useful for treatment of bone lytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis into bone. Biselyngbyaside is a recently isolated macrolide compound from marine cyanobacteria Lyngbya sp. that shows wide-spectrum cytotoxicity toward human tumor cell lines. In this study, we investigated the effects of biselyngbyaside on osteoclast differentiation and function. Biselyngbyaside inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocytic RAW264 cells and primary bone marrow-derived macrophages at a low concentration. Similarly, biselyngbyaside suppressed osteoblastic cell-mediated osteoclast differentiation in cocultures. In the RANKL-induced signaling pathway, biselyngbyaside inhibited the expression of c-Fos and NFATc1, which are important transcription factors in osteoclast differentiation. In mature osteoclasts, biselyngbyaside decreased resorption-pit formation. Biselyngbyaside also induced apoptosis accompanied by the induction of caspase-3 activation and nuclear condensation, and these effects were negated by the pancaspase inhibitor z-VAD-FMK. Taken together, the present findings indicate that biselyngbyaside suppresses bone resorption via inhibition of osteoclastogenesis and induction of apoptosis. Thus, biselyngbyaside may be useful for the prevention of bone lytic diseases.