Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 ami... more Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 amino acids (aa) long, from the Streptomyces griseoflavus Gc3592 gilvocarcin V (GV) gene cluster, revealed that the previously reported gilGT indeed contains two genes. These are the larger gilGT, which encodes the C-glycosyltransferase GilGT (379 aa), and the smaller gilV gene, which encodes an enzyme of unknown function (116 aa). The gene gilV is located immediately upstream of gilGT in the GV gene cluster. In-frame deletion of gilGT created a mutant that accumulated defucogilvocarcin E (defuco-GE). The result proves the function of GilGT as a Cglycosyltransferase. Deletion of gilOIII, which is located immediately downstream of gilGT, led to a mutant that accumulated gilvocarcin E (GE). This confirms that the corresponding P450 enzyme, GilOIII, is involved in the vinyl-group formation of GV. Cross-feeding experiments in which GE, defuco-GE, and defucogilvocarcin V (defuco-GV) were fed to an early blocked mutant of the GV biosynthetic pathway, showed that neither GE nor any of the defuco-compounds was an intermediate of the pathway.
Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Gö 3592 and various ot... more Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Gö 3592 and various other Streptomyces species. GV is usually produced along with its minor congeners, gilvocarcin M (3) and gilvocarcin E (5), that vary with respect to their side chain at the C8position. [1] Several analogues of GV (for example, 6-8, Scheme 1), which are collectively called the gilvocarcin group of natural products, have been isolated from different Streptomyces species. All of these analogues contain the characteristic, polyketide-derived benzo[d]naphtho[1,2-b]pyran-6-one chromophore, but different C-glycosidically linked sugar units Scheme 1). [2] Members of this group of natural products are well-known for strong antitumor activities, [3] a unique mode of action, [4] and remarkably low toxicities. [2c, 5] However, the inherent poor solubility of these molecules appears to be a ** This work was supported by grants CA102102 and CA091901 from the U.S. National Institutes of Health to J.R. The mass spectrometry and NMR facilities of the University of Kentucky are acknowledged for their services.
A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces... more A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase ␣ (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the postpolyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.
The views, opinions and/or findings contained in this report are those of the author(s) and shoul... more The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of S... more Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was overexpressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist ofthe same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results ofthe production oflandomycin E (which contains three sugars) by the LanGT3mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene overexpression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity ofthe new landomycins-compared to known ones-was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length ofthe deoxysugar side chain was deduced from the results.
This unit includes general protocols for the laboratory maintenance of Streptomyces species, incl... more This unit includes general protocols for the laboratory maintenance of Streptomyces species, including growth in liquid media, growth on solid agar, and short and long-term storage. Considerations for the handling of Streptomyces species and the morphology of the bacteria are also reviewed.
Chemoenzymatic synthesis of SAM analogs-This study highlights a broadly applicable platform for t... more Chemoenzymatic synthesis of SAM analogs-This study highlights a broadly applicable platform for the facile syntheses of SAM analogs that is directly compatible with downstream SAM utilizing enzymes. The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with rapid SAM analog decomposition and thereby opens the door to the further interrogation of a wide range of SAM utilizing enzymes. As a proof of concept for the feasibility of natural product 'alkylrandomization', the coupled strategy was used to generate a small set of indolocarbazole analogs in conjunction with the rebeccamycin O-methyltransferase RebM.
... Soc., Chem. Commun., 1983, 128 RSC Article . 9, R. Thomas and DJ Williams, J. Chem. Soc., Che... more ... Soc., Chem. Commun., 1983, 128 RSC Article . 9, R. Thomas and DJ Williams, J. Chem. Soc., Chem. ... Chem. Soc., 1987, 109, 5282 Article CAS . 64, MC Cone, PJ Seaton, KA Halley and SJ Gould, J. Antibiot., 1989, 42, 179 CAS . 65, PJ Seaton and SJ Gould, J. Am. Chem. ...
Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcrip... more Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS-FLI1 and EWS-ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS-FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS-ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues. Methods: Qualitative interactions between drug-DNA-protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time-resolved fluorescence energy transfer (TR-FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c-PARP, γ-H2AX) and phosphorylation at the C-terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. Results: Using TR-FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ-H2AX and c-PARP, specifically in cell lines containing EWS-ETS translocations in a concentration dependent manner. Conclusion: These studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS-ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co-immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS-ETS proteins and with RNAPII. Note: This abstract was not presented at the meeting. Citation Format: Reiya C. Hayden, Caixia Hou, Prithiba Mitra, Abhisek Mandal, Jurgen Rohr, Jon Thorson, Oleg Tsodikov, Markos Leggas. Mithramycin analogues disrupt ETS transcription factor DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2954.
ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and ... more Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2015-2628
Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 ami... more Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 amino acids (aa) long, from the Streptomyces griseoflavus Gc3592 gilvocarcin V (GV) gene cluster, revealed that the previously reported gilGT indeed contains two genes. These are the larger gilGT, which encodes the C-glycosyltransferase GilGT (379 aa), and the smaller gilV gene, which encodes an enzyme of unknown function (116 aa). The gene gilV is located immediately upstream of gilGT in the GV gene cluster. In-frame deletion of gilGT created a mutant that accumulated defucogilvocarcin E (defuco-GE). The result proves the function of GilGT as a Cglycosyltransferase. Deletion of gilOIII, which is located immediately downstream of gilGT, led to a mutant that accumulated gilvocarcin E (GE). This confirms that the corresponding P450 enzyme, GilOIII, is involved in the vinyl-group formation of GV. Cross-feeding experiments in which GE, defuco-GE, and defucogilvocarcin V (defuco-GV) were fed to an early blocked mutant of the GV biosynthetic pathway, showed that neither GE nor any of the defuco-compounds was an intermediate of the pathway.
Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Gö 3592 and various ot... more Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Gö 3592 and various other Streptomyces species. GV is usually produced along with its minor congeners, gilvocarcin M (3) and gilvocarcin E (5), that vary with respect to their side chain at the C8position. [1] Several analogues of GV (for example, 6-8, Scheme 1), which are collectively called the gilvocarcin group of natural products, have been isolated from different Streptomyces species. All of these analogues contain the characteristic, polyketide-derived benzo[d]naphtho[1,2-b]pyran-6-one chromophore, but different C-glycosidically linked sugar units Scheme 1). [2] Members of this group of natural products are well-known for strong antitumor activities, [3] a unique mode of action, [4] and remarkably low toxicities. [2c, 5] However, the inherent poor solubility of these molecules appears to be a ** This work was supported by grants CA102102 and CA091901 from the U.S. National Institutes of Health to J.R. The mass spectrometry and NMR facilities of the University of Kentucky are acknowledged for their services.
A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces... more A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase ␣ (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the postpolyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.
The views, opinions and/or findings contained in this report are those of the author(s) and shoul... more The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of S... more Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was overexpressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist ofthe same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results ofthe production oflandomycin E (which contains three sugars) by the LanGT3mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene overexpression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity ofthe new landomycins-compared to known ones-was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length ofthe deoxysugar side chain was deduced from the results.
This unit includes general protocols for the laboratory maintenance of Streptomyces species, incl... more This unit includes general protocols for the laboratory maintenance of Streptomyces species, including growth in liquid media, growth on solid agar, and short and long-term storage. Considerations for the handling of Streptomyces species and the morphology of the bacteria are also reviewed.
Chemoenzymatic synthesis of SAM analogs-This study highlights a broadly applicable platform for t... more Chemoenzymatic synthesis of SAM analogs-This study highlights a broadly applicable platform for the facile syntheses of SAM analogs that is directly compatible with downstream SAM utilizing enzymes. The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with rapid SAM analog decomposition and thereby opens the door to the further interrogation of a wide range of SAM utilizing enzymes. As a proof of concept for the feasibility of natural product 'alkylrandomization', the coupled strategy was used to generate a small set of indolocarbazole analogs in conjunction with the rebeccamycin O-methyltransferase RebM.
... Soc., Chem. Commun., 1983, 128 RSC Article . 9, R. Thomas and DJ Williams, J. Chem. Soc., Che... more ... Soc., Chem. Commun., 1983, 128 RSC Article . 9, R. Thomas and DJ Williams, J. Chem. Soc., Chem. ... Chem. Soc., 1987, 109, 5282 Article CAS . 64, MC Cone, PJ Seaton, KA Halley and SJ Gould, J. Antibiot., 1989, 42, 179 CAS . 65, PJ Seaton and SJ Gould, J. Am. Chem. ...
Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcrip... more Introduction: Ewing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS-FLI1 and EWS-ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS-FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS-ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues. Methods: Qualitative interactions between drug-DNA-protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time-resolved fluorescence energy transfer (TR-FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c-PARP, γ-H2AX) and phosphorylation at the C-terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. Results: Using TR-FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ-H2AX and c-PARP, specifically in cell lines containing EWS-ETS translocations in a concentration dependent manner. Conclusion: These studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS-ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co-immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS-ETS proteins and with RNAPII. Note: This abstract was not presented at the meeting. Citation Format: Reiya C. Hayden, Caixia Hou, Prithiba Mitra, Abhisek Mandal, Jurgen Rohr, Jon Thorson, Oleg Tsodikov, Markos Leggas. Mithramycin analogues disrupt ETS transcription factor DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2954.
ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and ... more Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2015-2628
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