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Jürgen Rohr - One of the best experts on this subject based on the ideXlab platform.
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Mithramycin and Analogs for Overcoming Cisplatin Resistance in Ovarian Cancer.
Biomedicines, 2021Co-Authors: David Schweer, Jürgen Rohr, J Robert Mccorkle, Oleg V Tsodikov, Frederick Ueland, Jill KolesarAbstract:Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of Mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to Mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to Mithramycin and postulates a role for the use of Mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.
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Bioanalytical method for quantitative determination of Mithramycin analogs in mouse plasma by HPLC-QTOF.
Biomedical chromatography : BMC, 2019Co-Authors: Joseph M Eckenrode, Jürgen Rohr, Prithiba Mitra, Markos LeggasAbstract:Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of Mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, Mithramycin SK (MTMSK) and Mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of
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semi synthetic Mithramycin sa derivatives with improved anticancer activity
Chemical Biology & Drug Design, 2013Co-Authors: Daniel Scott, Jhong-min Chen, Younsoo Bae, Jürgen RohrAbstract:Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of Mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved Mithramycin analogues Mithramycin SK and Mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to Mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as Mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to Mithramycin SK. This work shows the ability to transform the previously useless Mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.
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Semi‐Synthetic Mithramycin SA Derivatives with Improved AntiCancer Activity
Chemical biology & drug design, 2013Co-Authors: Daniel Scott, Jhong-min Chen, Younsoo Bae, Jürgen RohrAbstract:Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of Mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved Mithramycin analogues Mithramycin SK and Mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to Mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as Mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to Mithramycin SK. This work shows the ability to transform the previously useless Mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.
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generation of new derivatives of the antitumor antibiotic Mithramycin by altering the glycosylation pattern through combinatorial biosynthesis
ChemBioChem, 2008Co-Authors: Maria Perez, Alfredo F Brana, Jose A Salas, Jürgen Rohr, Irfan Baig, Carmen MendezAbstract:Mithramycin is an antitumor drug produced by Streptomyces argillaceus. It consists of a tricyclic aglycone and five deoxyhexoses that form a disaccharide and a trisaccharide chain, which are important for target interaction and therefore for the antitumor activity. Using a combinatorial biosynthesis approach, we have generated nine Mithramycin derivatives, seven of which are new compounds, with alterations in the glycosylation pattern. The wild-type S. argillaceus strain and the mutant S. argillaceus M7U1, which has altered D-oliose biosynthesis, were used as hosts to express various "sugar plasmids", each one directing the biosynthesis of a different deoxyhexose. The newly formed compounds were purified and characterized by MS and NMR. Compared to Mithramycin, they contained different sugar substitutions in the second (D-olivose, D-mycarose, or D-boivinose instead of D-oliose) and third (D-digitoxose instead of D-mycarose) sugar units of the trisaccharide as well as in the first (D-amicetose instead of D-olivose) sugar unit of the disaccharide. All compounds showed antitumor activity against different tumor cell lines. Structure-activity relationships are discussed on the basis of the number and type of deoxyhexoses present in these Mithramycin derivatives.
Jose A Salas - One of the best experts on this subject based on the ideXlab platform.
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heterologous reconstitution of the biosynthesis pathway for 4 demethyl preMithramycinone the aglycon of antitumor polyketide Mithramycin
Microbial Cell Factories, 2020Co-Authors: Jose A Salas, Carmen Mendez, Daniel Zabala, Lijiang Song, Yousef Dashti, Gregory L ChallisAbstract:Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of Mithramycin proceeds through the formation of the key intermediate 4-demethyl-preMithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to Mithramycin. However, the biosynthesis pathway for 4-demethyl-preMithramycinone remains unclear. Expression of cosmid cosAR7, containing a set of Mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-preMithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-preMithramycinone. Expression of different sets of Mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-preMithramycinone, assigning gene functions and establishing the order of biosynthetic steps. We established the biosynthesis pathway for 4-demethyl-preMithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-preMithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new Mithramycin analogs and improve their production titers for bioactivity investigation.
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Expanding the Chemical Diversity of the Antitumoral Compound Mithramycin by Combinatorial Biosynthesis and Biocatalysis: The Quest for Mithralogs with Improved Therapeutic Window
Planta medica, 2015Co-Authors: Carmen Mendez, Francisco Moris, Javier González-sabín, Jose A SalasAbstract:Mithramycin is an antitumor compound of the aureolic acid family produced by Streptomyces ar- gillaceus. It has been used to treat several types of cancer including testicular carcinoma, chronic and acute myeloid leukemia as well as hypercal- cemias and Pagetʼs disease. Although the use of Mithramycin in humans has been limited because its side effects, in recent years a renewed interest has arisen since new uses and activities have been ascribed to it. Chemically, Mithramycin is charac- terized by a tricyclic aglycone bearing two ali- phatic side chains attached at C3 and C7, and di- saccharide and trisaccharide units attached at po- sitions 2 and 6, respectively. The Mithramycin gene cluster has been characterized. This has al- lowed for the development of several mithramy- cin analogs ("mithralogs") by combinatorial bio- synthesis and/or biocatalysis. The combinatorial biosynthesis strategies include gene inactivation and/or the use of sugar biosynthesis plasmids for sugar modification. In addition, lipase-based bio- catalysis enabled selective modifications of the hydroxyl groups, providing further Mithramycin analogs. As a result, new Mithramycin analogs with higher antitumor activity and/or less toxicity have been generated. One, demycarosyl-3D-β-D- digitoxosyl-Mithramycin SK (EC-8042), is being tested in regulatory preclinical assays, represent- ing an opportunity to open the therapeutic win- dow of this promising molecular scaffold.
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Transcriptional regulation of Mithramycin biosynthesis in Streptomyces argillaceus: dual role as activator and repressor of the PadR-like regulator MtrY.
Microbiology, 2015Co-Authors: Ana Belén Flórez, Alfredo F Brana, Jose A Salas, Susana Álvarez, Daniel Zabala, Carmen MendezAbstract:The Mithramycin biosynthesis gene cluster of Streptomyces argillaceus ATCC 12956 contains 34 ORFs and includes two putative regulatory genes (mtmR and mtrY), which encode proteins of the SARP (Streptomyces antibiotic regulatory protein) and PadR transcriptional regulator families, respectively. MtmR was proposed to behave as a positive regulator of Mithramycin biosynthesis. Inactivation and overexpression of mtrY indicated that it is also a positive regulator of Mithramycin biosynthesis, being non-essential but required to maintain high levels of Mithramycin production in the producer strain. Transcriptional analyses by reverse transcription PCR and quantitative real-time PCR of Mithramycin genes, and promoter-probe assays in S. argillaceus polyketide synthase and regulatory mutants and the WT strain, and in the heterologous host Streptomyces albus, were carried out to analyse the role of MtmR and MtrY in the regulation of the Mithramycin gene cluster. These experiments revealed that MtmR had a positive role, activating expression of at least six polycistronic units (mtmR–mtmE, mtmQ–mtmTII, mtmX–mtmY, mtmV–mtmTIII, mtmW–mtmMI and mtmGI–mtrB) and one monocistronic unit (mtmGII) in the Mithramycin gene cluster. However, MtrY played a dual role in the Mithramycin gene cluster: (i) repressing the expression of resistance genes and its coding gene itself by controlling the activity of the mtrYp promoter that directs expression of the regulator mtrY and resistance genes, with this repression being released in the presence of Mithramycin; and (ii) enhancing the expression of Mithramycin biosynthesis genes when Mithramycin is present, by interacting with the mtmRp promoter that controls expression of the mtmR regulator, amongst others.
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the chromomycin cmma acetyltransferase a membrane bound enzyme as a tool for increasing structural diversity of the antitumour Mithramycin
Microbial Biotechnology, 2011Co-Authors: Beatriz Garcia, Luzelena Nunez, Francisco Moris, Javier Gonzalezsabin, Nuria Menendez, Alfredo F Brana, Jose A Salas, Carmen MendezAbstract:Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated Mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new Mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl‐CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
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generation of new derivatives of the antitumor antibiotic Mithramycin by altering the glycosylation pattern through combinatorial biosynthesis
ChemBioChem, 2008Co-Authors: Maria Perez, Alfredo F Brana, Jose A Salas, Jürgen Rohr, Irfan Baig, Carmen MendezAbstract:Mithramycin is an antitumor drug produced by Streptomyces argillaceus. It consists of a tricyclic aglycone and five deoxyhexoses that form a disaccharide and a trisaccharide chain, which are important for target interaction and therefore for the antitumor activity. Using a combinatorial biosynthesis approach, we have generated nine Mithramycin derivatives, seven of which are new compounds, with alterations in the glycosylation pattern. The wild-type S. argillaceus strain and the mutant S. argillaceus M7U1, which has altered D-oliose biosynthesis, were used as hosts to express various "sugar plasmids", each one directing the biosynthesis of a different deoxyhexose. The newly formed compounds were purified and characterized by MS and NMR. Compared to Mithramycin, they contained different sugar substitutions in the second (D-olivose, D-mycarose, or D-boivinose instead of D-oliose) and third (D-digitoxose instead of D-mycarose) sugar units of the trisaccharide as well as in the first (D-amicetose instead of D-olivose) sugar unit of the disaccharide. All compounds showed antitumor activity against different tumor cell lines. Structure-activity relationships are discussed on the basis of the number and type of deoxyhexoses present in these Mithramycin derivatives.
Carmen Mendez - One of the best experts on this subject based on the ideXlab platform.
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heterologous reconstitution of the biosynthesis pathway for 4 demethyl preMithramycinone the aglycon of antitumor polyketide Mithramycin
Microbial Cell Factories, 2020Co-Authors: Jose A Salas, Carmen Mendez, Daniel Zabala, Lijiang Song, Yousef Dashti, Gregory L ChallisAbstract:Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of Mithramycin proceeds through the formation of the key intermediate 4-demethyl-preMithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to Mithramycin. However, the biosynthesis pathway for 4-demethyl-preMithramycinone remains unclear. Expression of cosmid cosAR7, containing a set of Mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-preMithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-preMithramycinone. Expression of different sets of Mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-preMithramycinone, assigning gene functions and establishing the order of biosynthetic steps. We established the biosynthesis pathway for 4-demethyl-preMithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-preMithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new Mithramycin analogs and improve their production titers for bioactivity investigation.
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Expanding the Chemical Diversity of the Antitumoral Compound Mithramycin by Combinatorial Biosynthesis and Biocatalysis: The Quest for Mithralogs with Improved Therapeutic Window
Planta medica, 2015Co-Authors: Carmen Mendez, Francisco Moris, Javier González-sabín, Jose A SalasAbstract:Mithramycin is an antitumor compound of the aureolic acid family produced by Streptomyces ar- gillaceus. It has been used to treat several types of cancer including testicular carcinoma, chronic and acute myeloid leukemia as well as hypercal- cemias and Pagetʼs disease. Although the use of Mithramycin in humans has been limited because its side effects, in recent years a renewed interest has arisen since new uses and activities have been ascribed to it. Chemically, Mithramycin is charac- terized by a tricyclic aglycone bearing two ali- phatic side chains attached at C3 and C7, and di- saccharide and trisaccharide units attached at po- sitions 2 and 6, respectively. The Mithramycin gene cluster has been characterized. This has al- lowed for the development of several mithramy- cin analogs ("mithralogs") by combinatorial bio- synthesis and/or biocatalysis. The combinatorial biosynthesis strategies include gene inactivation and/or the use of sugar biosynthesis plasmids for sugar modification. In addition, lipase-based bio- catalysis enabled selective modifications of the hydroxyl groups, providing further Mithramycin analogs. As a result, new Mithramycin analogs with higher antitumor activity and/or less toxicity have been generated. One, demycarosyl-3D-β-D- digitoxosyl-Mithramycin SK (EC-8042), is being tested in regulatory preclinical assays, represent- ing an opportunity to open the therapeutic win- dow of this promising molecular scaffold.
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Transcriptional regulation of Mithramycin biosynthesis in Streptomyces argillaceus: dual role as activator and repressor of the PadR-like regulator MtrY.
Microbiology, 2015Co-Authors: Ana Belén Flórez, Alfredo F Brana, Jose A Salas, Susana Álvarez, Daniel Zabala, Carmen MendezAbstract:The Mithramycin biosynthesis gene cluster of Streptomyces argillaceus ATCC 12956 contains 34 ORFs and includes two putative regulatory genes (mtmR and mtrY), which encode proteins of the SARP (Streptomyces antibiotic regulatory protein) and PadR transcriptional regulator families, respectively. MtmR was proposed to behave as a positive regulator of Mithramycin biosynthesis. Inactivation and overexpression of mtrY indicated that it is also a positive regulator of Mithramycin biosynthesis, being non-essential but required to maintain high levels of Mithramycin production in the producer strain. Transcriptional analyses by reverse transcription PCR and quantitative real-time PCR of Mithramycin genes, and promoter-probe assays in S. argillaceus polyketide synthase and regulatory mutants and the WT strain, and in the heterologous host Streptomyces albus, were carried out to analyse the role of MtmR and MtrY in the regulation of the Mithramycin gene cluster. These experiments revealed that MtmR had a positive role, activating expression of at least six polycistronic units (mtmR–mtmE, mtmQ–mtmTII, mtmX–mtmY, mtmV–mtmTIII, mtmW–mtmMI and mtmGI–mtrB) and one monocistronic unit (mtmGII) in the Mithramycin gene cluster. However, MtrY played a dual role in the Mithramycin gene cluster: (i) repressing the expression of resistance genes and its coding gene itself by controlling the activity of the mtrYp promoter that directs expression of the regulator mtrY and resistance genes, with this repression being released in the presence of Mithramycin; and (ii) enhancing the expression of Mithramycin biosynthesis genes when Mithramycin is present, by interacting with the mtmRp promoter that controls expression of the mtmR regulator, amongst others.
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the chromomycin cmma acetyltransferase a membrane bound enzyme as a tool for increasing structural diversity of the antitumour Mithramycin
Microbial Biotechnology, 2011Co-Authors: Beatriz Garcia, Luzelena Nunez, Francisco Moris, Javier Gonzalezsabin, Nuria Menendez, Alfredo F Brana, Jose A Salas, Carmen MendezAbstract:Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated Mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new Mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl‐CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
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generation of new derivatives of the antitumor antibiotic Mithramycin by altering the glycosylation pattern through combinatorial biosynthesis
ChemBioChem, 2008Co-Authors: Maria Perez, Alfredo F Brana, Jose A Salas, Jürgen Rohr, Irfan Baig, Carmen MendezAbstract:Mithramycin is an antitumor drug produced by Streptomyces argillaceus. It consists of a tricyclic aglycone and five deoxyhexoses that form a disaccharide and a trisaccharide chain, which are important for target interaction and therefore for the antitumor activity. Using a combinatorial biosynthesis approach, we have generated nine Mithramycin derivatives, seven of which are new compounds, with alterations in the glycosylation pattern. The wild-type S. argillaceus strain and the mutant S. argillaceus M7U1, which has altered D-oliose biosynthesis, were used as hosts to express various "sugar plasmids", each one directing the biosynthesis of a different deoxyhexose. The newly formed compounds were purified and characterized by MS and NMR. Compared to Mithramycin, they contained different sugar substitutions in the second (D-olivose, D-mycarose, or D-boivinose instead of D-oliose) and third (D-digitoxose instead of D-mycarose) sugar units of the trisaccharide as well as in the first (D-amicetose instead of D-olivose) sugar unit of the disaccharide. All compounds showed antitumor activity against different tumor cell lines. Structure-activity relationships are discussed on the basis of the number and type of deoxyhexoses present in these Mithramycin derivatives.
Alfredo F Brana - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation of Mithramycin biosynthesis in Streptomyces argillaceus: dual role as activator and repressor of the PadR-like regulator MtrY.
Microbiology, 2015Co-Authors: Ana Belén Flórez, Alfredo F Brana, Jose A Salas, Susana Álvarez, Daniel Zabala, Carmen MendezAbstract:The Mithramycin biosynthesis gene cluster of Streptomyces argillaceus ATCC 12956 contains 34 ORFs and includes two putative regulatory genes (mtmR and mtrY), which encode proteins of the SARP (Streptomyces antibiotic regulatory protein) and PadR transcriptional regulator families, respectively. MtmR was proposed to behave as a positive regulator of Mithramycin biosynthesis. Inactivation and overexpression of mtrY indicated that it is also a positive regulator of Mithramycin biosynthesis, being non-essential but required to maintain high levels of Mithramycin production in the producer strain. Transcriptional analyses by reverse transcription PCR and quantitative real-time PCR of Mithramycin genes, and promoter-probe assays in S. argillaceus polyketide synthase and regulatory mutants and the WT strain, and in the heterologous host Streptomyces albus, were carried out to analyse the role of MtmR and MtrY in the regulation of the Mithramycin gene cluster. These experiments revealed that MtmR had a positive role, activating expression of at least six polycistronic units (mtmR–mtmE, mtmQ–mtmTII, mtmX–mtmY, mtmV–mtmTIII, mtmW–mtmMI and mtmGI–mtrB) and one monocistronic unit (mtmGII) in the Mithramycin gene cluster. However, MtrY played a dual role in the Mithramycin gene cluster: (i) repressing the expression of resistance genes and its coding gene itself by controlling the activity of the mtrYp promoter that directs expression of the regulator mtrY and resistance genes, with this repression being released in the presence of Mithramycin; and (ii) enhancing the expression of Mithramycin biosynthesis genes when Mithramycin is present, by interacting with the mtmRp promoter that controls expression of the mtmR regulator, amongst others.
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the chromomycin cmma acetyltransferase a membrane bound enzyme as a tool for increasing structural diversity of the antitumour Mithramycin
Microbial Biotechnology, 2011Co-Authors: Beatriz Garcia, Luzelena Nunez, Francisco Moris, Javier Gonzalezsabin, Nuria Menendez, Alfredo F Brana, Jose A Salas, Carmen MendezAbstract:Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated Mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new Mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl‐CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
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generation of new derivatives of the antitumor antibiotic Mithramycin by altering the glycosylation pattern through combinatorial biosynthesis
ChemBioChem, 2008Co-Authors: Maria Perez, Alfredo F Brana, Jose A Salas, Jürgen Rohr, Irfan Baig, Carmen MendezAbstract:Mithramycin is an antitumor drug produced by Streptomyces argillaceus. It consists of a tricyclic aglycone and five deoxyhexoses that form a disaccharide and a trisaccharide chain, which are important for target interaction and therefore for the antitumor activity. Using a combinatorial biosynthesis approach, we have generated nine Mithramycin derivatives, seven of which are new compounds, with alterations in the glycosylation pattern. The wild-type S. argillaceus strain and the mutant S. argillaceus M7U1, which has altered D-oliose biosynthesis, were used as hosts to express various "sugar plasmids", each one directing the biosynthesis of a different deoxyhexose. The newly formed compounds were purified and characterized by MS and NMR. Compared to Mithramycin, they contained different sugar substitutions in the second (D-olivose, D-mycarose, or D-boivinose instead of D-oliose) and third (D-digitoxose instead of D-mycarose) sugar units of the trisaccharide as well as in the first (D-amicetose instead of D-olivose) sugar unit of the disaccharide. All compounds showed antitumor activity against different tumor cell lines. Structure-activity relationships are discussed on the basis of the number and type of deoxyhexoses present in these Mithramycin derivatives.
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Mithramycin SK, a novel antitumor drug with improved therapeutic index, Mithramycin SA, and demycarosyl-Mithramycin SK: three new products generated in the Mithramycin producer Streptomyces argillaceus through combinatorial biosynthesis.
Journal of the American Chemical Society, 2003Co-Authors: Lily L. Remsing, Alfredo F Brana, Jose A Salas, Carmen Mendez, Ana González, Mohammad Nur-e-alam, M. José Fernández-lozano, Uwe Rix, Marcos A. Oliveira, Jürgen RohrAbstract:To gain initial structure-activity relationships regarding the highly functionalized pentyl side chain attached at C-3 of Mithramycin (MTM), we focused on a post-polyketide synthase (post-PKS) tailoring step of the MTM biosynthesis by Streptomyces argillaceus ATCC 12956, which was proposed to be catalyzed by ketoreductase (KR) MtmW. In this last step of the MTM biosynthesis, a keto group of the pentyl side chain is reduced to a secondary alcohol, and we anticipated the generation of an MTM derivative with an additional keto group in the 3-side chain. Insertional inactivation of mtmW, a gene located ca. 8 kb downstream of the Mithramycin-PKS genes, yielded an S. argillaceus mutant, which accumulated three new Mithramycin analogues, namely Mithramycin SA, demycarosyl-Mithramycin SK, and Mithramycin SK (MTM-SK). The structures of these three compounds confirmed indirectly the proposed role of MtmW in MTM biosynthesis. However, the new Mithramycin derivatives bear unexpectedly shorter 3-side chains (ethyl or butyl) than MTM, presumably caused by nonenzymatic rearrangement or cleavage reactions of the initially formed pentyl side chain with a reactive beta-dicarbonyl functional group. The major product, MTM-SK, was tested in vitro against a variety of human cancer cell lines, as well as in an in vitro toxicity assay, and showed an improved therapeutic index, in comparison to the parent drug, MTM.
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ketopreMithramycins and ketoMithramycins four new aureolic acid type compounds obtained upon inactivation of two genes involved in the biosynthesis of the deoxysugar moieties of the antitumor drug Mithramycin by streptomyces argillaceus reveal novel
Journal of the American Chemical Society, 2002Co-Authors: Lily L. Remsing, Alfredo F Brana, Jose A Salas, Carmen Mendez, Eva Künzel, Ana González, Jose Garciabernardo, Daniel W Bearden, Jürgen RohrAbstract:: Mithramycin is an aureolic acid-type antimicrobial and antitumor agent produced by Streptomyces argillaceus. Modifying post-polyketide synthase (PKS) tailoring enzymes involved in the production of Mithramycin is an effective way of gaining further information regarding the late steps of its biosynthetic pathway. In addition, new "unnatural" natural products of the aureolic acid-type class are likely to be produced. The role of two such post-PKS tailoring enzymes, encoded by mtmC and mtmTIII, was investigated, and four novel aureolic acid class drugs, two preMithramycin-type molecules and two Mithramycin derivatives, were isolated from mutant strains constructed by insertional gene inactivation of either of these two genes. From data bank comparisons, the corresponding proteins MtmC and MtmTIII were believed to act as a C-methyltransferase involved in the production of the D-mycarose (sugar E) of Mithramycin and as a ketoreductase seemingly involved in the biosynthesis of the Mithramycin aglycon, respectively. However, gene inactivation and analysis of the accumulated products revealed that both genes encode enzymes participating in the biosynthesis of the D-mycarose building block. Furthermore, the inactivation of MtmC seems to affect the ketoreductase responsible for 4-ketoreduction of sugar C, a D-olivose. Instead of obtaining preMithramycin and Mithramycin derivatives with a modified E-sugar upon inactivation of mtmC, compounds were obtained that completely lack the E-sugar moiety and that possess an unexpected 4-ketosugar moiety instead of the D-olivose at the beginning of the lower deoxysaccharide chain. The inactivation of mtmTIII led to the accumulation of 4E-ketoMithramycin, showing that this ketoreductase is responsible for the 4-ketoreduction of the D-mycarose moiety. The new compounds of the mutant strains, 4A-ketopreMithramycin A2, 4A-keto-9-demethylpreMithramycin A2, 4C-keto-demycarosylMithramycin, and 4E-ketoMithramycin, indicate surprising substrate flexibility of post-PKS enzymes of the Mithramycin biosynthetic pathway. Although the glycosyltransferase responsible for the attachment of D-mycarose cannot transfer the unmethylated sugar to the existing lower disaccharide chain, it can transfer the 4-ketoform of sugar E. In addition, the glycosyltransferase MtmGIV, which is responsible for the linkage of sugar C, is also able to transfer an activated 4-ketosugar. The oxygenase MtmOIV, normally responsible for the oxidative cleavage of the tetracyclic preMithramycin B into the tricyclic immediate precursor of Mithramycin, can act on a substrate analogue with a modified or even incomplete trisaccharide chain. The same is true for glycosyltransferases MtmGI and MtmGII, both of which partake in the formation and attachment of the A-B disaccharide in Mithramycin.
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semi synthetic Mithramycin sa derivatives with improved anticancer activity
Chemical Biology & Drug Design, 2013Co-Authors: Daniel Scott, Jhong-min Chen, Younsoo Bae, Jürgen RohrAbstract:Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of Mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved Mithramycin analogues Mithramycin SK and Mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to Mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as Mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to Mithramycin SK. This work shows the ability to transform the previously useless Mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.
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Semi‐Synthetic Mithramycin SA Derivatives with Improved AntiCancer Activity
Chemical biology & drug design, 2013Co-Authors: Daniel Scott, Jhong-min Chen, Younsoo Bae, Jürgen RohrAbstract:Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of Mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved Mithramycin analogues Mithramycin SK and Mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to Mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as Mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to Mithramycin SK. This work shows the ability to transform the previously useless Mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.