The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Kent S. Gates - One of the best experts on this subject based on the ideXlab platform.
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Oxidative activation of Leinamycin E1 triggers alkylation of guanine residues in double-stranded DNA
Chemical communications (Cambridge England), 2018Co-Authors: Maryam Imani Nejad, Ben Shen, Dong Yang, Kent S. GatesAbstract:It may be useful to develop prodrugs that are selectively activated by oxidative stress in cancer cells to release cell-killing reactive intermediates. However, relatively few chemical strategies exist for the activation of prodrugs under conditions of oxidative stress. Here we provide evidence for a novel process in which oxidation of a thiol residue in the natural product Leinamycin E1 by H2O2 and other byproducts of cellular oxidative stress initiates generation of an episulfonium ion that selectively alkylates guanine residues in duplex DNA.
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A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links
2016Co-Authors: Maryam Imani Nejad, Kevin M. Johnson, Nathan E. Price, Kent S. GatesAbstract:Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Monoadducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5′-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483–3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product Leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the first characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues
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Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin: Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves as an Atypical DNA Intercalator
2016Co-Authors: Mostafa I. Fekry, Jozsef Szekely, Sanjay Dutta, Leonid Breydo, Hong Zang, Kent S. GatesAbstract:Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product Leinamycin. Previous studies suggested that alkylation of duplex DNA by activated Leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because Leinamycin does not contain a classical noncovalent DNA-binding motif, such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that Leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by Leinamycin provided evidence that activated Leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M–1. Activated Leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated Leinamycin with palindromic duplexes containing 5′-CG and 5′-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of Leinamycin with respect to target guanine residues. The data allow construction of a model for the Leinamycin–DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA
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synthesis and characterization of a small analogue of the anticancer natural product Leinamycin
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Kripa Keerthi, Daekyu Sun, Anuruddha Rajapakse, Kent S. GatesAbstract:Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC(50) values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small Leinamycin analogue 19 that closely resembles the 'upper-right quadrant' of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product Leinamycin. The small Leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of Leinamycin in driving efficient DNA alkylation by the natural product.
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Cellular Responses to the DNA Damaging Natural Compound Leinamycin
Journal of Cancer Science & Therapy, 2013Co-Authors: Parvathi Sinha, Yoon Joo Shin, Allison M. Hays, Kent S. Gates, Daekyu SunAbstract:Leinamycin is a thiol dependent DNA alkylating agent which shows very potent activity against various human cancer cell lines (IC50 values in the low nanomolar range). This natural compound forms guanine adducts (N7) in DNA which are converted into abasic sites and simultaneously generates Reactive Oxygen Species (ROS), to produce DNA strand breaks in human cancer cells. Our present study shows that Leinamycin induces a group of DNA repair and transcription factor genes involved in DNA repair in a MDA-MB-231 human breast cancer cell line, which can mediate chemoresistance to Leinamycin. In addition, N-acetylcysteine decreases Leinamycin-mediated ROS production while increasing Leinamycin mediated apoptotic cell death, without affecting the induction of repair genes. These data indicate that ROS is not a crucial player in Leinamycin induced DNA damage and that a precursor of glutathione, N-acetylcysteine, can potentiate Leinamycin mediated cytotoxicity by increasing the activation of Leinamycin into its DNA reactive form
Ben Shen - One of the best experts on this subject based on the ideXlab platform.
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Challenges and opportunities for natural product discovery, production, and engineering in native producers versus heterologous hosts
Journal of Industrial Microbiology & Biotechnology, 2019Co-Authors: Christiana N. Teijaro, Ajeeth Adhikari, Ben ShenAbstract:Recent advances and emerging technologies for metabolic pathway engineering and synthetic biology have transformed the field of natural product discovery, production, and engineering. Despite these advancements, there remain many challenges in understanding how biosynthetic gene clusters are silenced or activated, including changes in the transcription of key biosynthetic and regulatory genes. This knowledge gap is highlighted by the success and failed attempts of manipulating regulatory genes within biosynthetic gene clusters in both native producers and heterologous hosts. These complexities make the choice of native producers versus heterologous hosts, fermentation medium, and supply of precursors crucial factors in achieving the production of the target natural products and engineering designer analogs. Nature continues to serve as inspiration for filling the knowledge gaps and developing new research strategies. By exploiting the evolutionary power of nature, alternative producers, with the desired genetic amenability and higher titers of the target natural products, and new strains, harboring gene clusters that encode evolutionary optimized congeners of the targeted natural product scaffolds, can be discovered. These newly identified strains can serve as an outstanding biotechnology platform for the engineered production of sufficient quantities of the target natural products and their analogs, enabling biosynthetic studies and potential therapeutic applications. These challenges and opportunities are showcased herein using fredericamycin, iso-migrastatin, platencin and platensimycin, the enediynes of C-1027, tiancimycin, and yangpumicin, and the Leinamycin family of natural products.
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P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.
Biochemistry, 2018Co-Authors: Thomas Kwong, Chunying Yang, John L Cleveland, Dong Yang, Guohui Pan, Jeremy R. Lohman, Hindra, Jeffrey D. Rudolf, Ben ShenAbstract:Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4′ positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C...
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Oxidative activation of Leinamycin E1 triggers alkylation of guanine residues in double-stranded DNA
Chemical communications (Cambridge England), 2018Co-Authors: Maryam Imani Nejad, Ben Shen, Dong Yang, Kent S. GatesAbstract:It may be useful to develop prodrugs that are selectively activated by oxidative stress in cancer cells to release cell-killing reactive intermediates. However, relatively few chemical strategies exist for the activation of prodrugs under conditions of oxidative stress. Here we provide evidence for a novel process in which oxidation of a thiol residue in the natural product Leinamycin E1 by H2O2 and other byproducts of cellular oxidative stress initiates generation of an episulfonium ion that selectively alkylates guanine residues in duplex DNA.
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P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities
2018Co-Authors: Thomas Kwong, Chunying Yang, John L Cleveland, Dong Yang, Guohui Pan, Jeremy R. Lohman, Jeffrey D. Rudolf, Ben ShenAbstract:Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4′ positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4′ hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure–activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery
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characterization of lnmo as a pathway specific crp fnr type positive regulator for Leinamycin biosynthesis in streptomyces atroolivaceus and its application for titer improvement
Applied Microbiology and Biotechnology, 2016Co-Authors: Ben Shen, Dong Yang, Yong Huang, Gong-li Tang, Guohui PanAbstract:The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.
Leonid Breydo - One of the best experts on this subject based on the ideXlab platform.
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Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin: Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves as an Atypical DNA Intercalator
2016Co-Authors: Mostafa I. Fekry, Jozsef Szekely, Sanjay Dutta, Leonid Breydo, Hong Zang, Kent S. GatesAbstract:Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product Leinamycin. Previous studies suggested that alkylation of duplex DNA by activated Leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because Leinamycin does not contain a classical noncovalent DNA-binding motif, such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that Leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by Leinamycin provided evidence that activated Leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M–1. Activated Leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated Leinamycin with palindromic duplexes containing 5′-CG and 5′-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of Leinamycin with respect to target guanine residues. The data allow construction of a model for the Leinamycin–DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA
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The macrocycle of Leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product
Bioorganic & medicinal chemistry letters, 2012Co-Authors: Santhosh Sivaramakrishnan, Leonid Breydo, Daekyu Sun, Kent S. GatesAbstract:Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of Leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of Leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property.
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Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin: Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves as an Atypical DNA Intercalator
Journal of the American Chemical Society, 2011Co-Authors: Mostafa I. Fekry, Jozsef Szekely, Sanjay Dutta, Leonid Breydo, Hong Zang, Kent S. GatesAbstract:Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product Leinamycin. Previous studies suggested that alkylation of duplex DNA by activated Leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because Leinamycin does not contain a classical noncovalent DNA-binding motif, such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that Leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by Leinamycin provided evidence that activated Leinamycin b...
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Synthesis and noncovalent DNA-binding properties of thiazole derivatives related to Leinamycin
Tetrahedron Letters, 2004Co-Authors: Leonid Breydo, Hong Zang, Kent S. GatesAbstract:A series of compounds related to the macrocyclic portion of the DNA-damaging antitumor agent Leinamycin were prepared as tools to characterize noncovalent DNA binding by this natural product. Acyclic (Z,E)-dienes were assembled via a Sonogashira coupling followed by partial hydrogenation. A Stille coupling was used in the cyclization step leading to a macrocyclic thiazole-diene analogue. Results obtained using the synthetic analogues reported here indicate that the extended p-system on the 'left-hand side' of Leinamycin is required for noncovalent association of the natural product with duplex DNA. 2004 Elsevier Ltd. All rights reserved.
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Activation of Leinamycin by thiols: a theoretical study.
The Journal of organic chemistry, 2002Co-Authors: Leonid Breydo, Kent S. GatesAbstract:Reaction of thiols with the 1,2-dithiolan-3-one 1-oxide heterocycle found in Leinamycin (1) results in the conversion of this antitumor antibiotic to a DNA-alkylating episulfonium ion (5). While the products formed in this reaction have been rationalized by a mechanism involving initial attack of thiol on the central sulfenyl sulfur (S2') of the 1,2-dithiolan-3-one 1-oxide ring, the carbonyl carbon (C3') and the sulfinyl sulfur (S1') of this heterocycle are also expected to be electrophilic. Therefore, it is important to consider whether nucleophilic attack of thiol at these sites might contribute either to destruction of the antibiotic or conversion to its episulfonium ion form. To address this question, we have used computational methods to examine the attack of methyl thiolate on each of the three electrophilic centers in a simple analogue of the 1,2-dithiolan-3-one 1-oxide heterocycle found in Leinamycin. Calculations were performed at the MP2/6-311+G(3df,p)//B3LYP/6-31G level of theory with inclusion of solvent effects. The results indicate that the most reasonable mechanism for thiol-mediated activation of Leinamycin involves initial attack of thiolate at the S2'-position of the antibiotic's 1,2-dithiolan-3-one 1-oxide heterocycle, followed by conversion to the 1,2-oxathiolan-5-one intermediate (3).
Gong-li Tang - One of the best experts on this subject based on the ideXlab platform.
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characterization of lnmo as a pathway specific crp fnr type positive regulator for Leinamycin biosynthesis in streptomyces atroolivaceus and its application for titer improvement
Applied Microbiology and Biotechnology, 2016Co-Authors: Ben Shen, Dong Yang, Yong Huang, Gong-li Tang, Guohui PanAbstract:The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.
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Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for Leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement
Applied microbiology and biotechnology, 2016Co-Authors: Yong Huang, Dong Yang, Gong-li Tang, Guohui Pan, Ben ShenAbstract:The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.
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Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.
Organic letters, 2016Co-Authors: Yong Huang, Gong-li Tang, Guohui Pan, Chin-yuan Chang, Ben ShenAbstract:Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products.
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Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase–Polyketide Synthase Interface for Leinamycin Biosynthesis
2016Co-Authors: Yong Huang, Gong-li Tang, Guohui Pan, Chin-yuan Chang, Ben ShenAbstract:Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products
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Leinamycin E1 acting as an anticancer prodrug activated by reactive oxygen species
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Sheng-xiong Huang, Dong Yang, Yong Huang, Bong Sik Yun, Hirak S. Basu, Dawn R. Church, Gudrun Ingenhorst, Jeremy R. Lohman, Gong-li TangAbstract:Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of Leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide–polyketide biosynthetic intermediate into LNM.
Yong Huang - One of the best experts on this subject based on the ideXlab platform.
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Discovery of the Leinamycin family of natural products by mining actinobacterial genomes.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Guohui Pan, Dong Yang, Hindra, Zhikai Guo, Hao Zhou, Yannick Gansemans, Xiangcheng Zhu, Yong HuangAbstract:Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the Leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.
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Characterization of LnmO as a pathway-specific Crp/Fnr-type positive regulator for Leinamycin biosynthesis in Streptomyces atroolivaceus and its application for titer improvement
Applied microbiology and biotechnology, 2016Co-Authors: Yong Huang, Dong Yang, Gong-li Tang, Guohui Pan, Ben ShenAbstract:The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.
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characterization of lnmo as a pathway specific crp fnr type positive regulator for Leinamycin biosynthesis in streptomyces atroolivaceus and its application for titer improvement
Applied Microbiology and Biotechnology, 2016Co-Authors: Ben Shen, Dong Yang, Yong Huang, Gong-li Tang, Guohui PanAbstract:The cyclic adenosine monophosphate (cAMP) receptor protein/fumarate and nitrate reductase regulatory protein (Crp/Fnr) family of transcriptional regulators are pleiotropic transcriptional regulators that control a broad range of cellular functions. Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. We previously cloned and characterized the lnm biosynthetic gene cluster from S. atroolivaceus S-140. We here report inactivation of lnmO in S. atroolivaceus S-140 and overexpression of lnmO in the S. atroolivaceus S-140 wild-type and ∆lnmE mutant SB3033 to investigate its role in LNM biosynthesis. Bioinformatics analysis revealed LnmO as the only regulator within the lnm gene cluster, exhibiting high sequence similarity to known Crp/Fnr family regulators. The inactivation of lnmO in S. atroolivaceus S-140 completely abolished LNM production but caused no apparent morphological changes, supporting that LnmO is indispensable and specific to LNM biosynthesis. Overexpression of lnmO in S. atroolivaceus S-140 and SB3033 resulted in three- and fourfold increase in LNM and LNM E1 production, respectively, supporting that LnmO acts as a positive regulator. While all of the Crp/Fnr family regulators studied to date appeared to be pleiotropic, our results support LnmO as the first Crp/Fnr family regulator that is pathway-specific. LnmO joins the growing list of regulators that could be exploited to improve secondary metabolite production in Streptomyces. Engineered strains overproducing LNM and LNM E1 will facilitate further mechanistic studies and clinical evaluation of LNM and LNM E1 as novel anticancer drugs.
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Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.
Organic letters, 2016Co-Authors: Yong Huang, Gong-li Tang, Guohui Pan, Chin-yuan Chang, Ben ShenAbstract:Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products.
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Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase–Polyketide Synthase Interface for Leinamycin Biosynthesis
2016Co-Authors: Yong Huang, Gong-li Tang, Guohui Pan, Chin-yuan Chang, Ben ShenAbstract:Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products