AllisonFoster_SRFPoster_Fa14 (2)
- 1. Mithramycin (MTM) is an anticancer agent, produced by bacteria of the genus Streptomyces. MTM was popular in the 1960s to aid in the treatment of testicular carcinoma, chronic and acute myeloid leukemias, and hyperkalemia. MTMs anti-cancer potency lies in the ability to shut down the transcription of the proto-oncogenes through cross-linking GC-rich DNA. Unfortunately the large number of side effects caused the use of MTM to be discontinued. In recent years, MTM has been revisited as a potential anticancer agent because of the potential for semi- synthetic manipulation and development of biosynthetic analogues. One example of such work is demonstrated by inactivating the mtmW gene. This gene encodes for the enzyme responsible for the final step in the MTM biosynthetic pathway, but once deactivated, 3 new analogues are formed: MTM SA, MTM SK, and MTM SDK. MTM SK and MTM SDK improved the anti-cancer ability of the drug but, due in part to its negative charge at physiological pH, MTM SA shows reduced anticancer capabilities. Current research has shown that new derivatives of the MTM SA analogue produced by manipulating the side chain ending in the carboxylic acid have resulted in molecules with greater anti-cancer abilities than those of even MTM SK and MTM SDK. Further work was focused on optimizing the production and isolation of MTM-SA followed by further work to rationally design new drug molecules through modification of the terminal carboxylic acid side chain of MTM SA. Once developed the toxicity of the new compounds are investigated using the Jurkat human leukemia cell line. Improved Isolation, Derivatization, and Characterization of New Mithramycin Analogues Allison Foster, Sterling Brooks, Daniel Scott Department of Chemistry and Biochemistry, DePauw University, Greencastle, IN 46135, USA Summer 2014 Introduction Acknowledgments Mithramycin Analogue Biosynthesis Bacteria Growth and Drug Production Conclusions and Future Work Column 52666, a strong anion exchange column with a quaternary amine, successfully isolated SA SA isolation final protocol: equilibrate 5 times with pH7 potassium phosphate monobasic, load drug, wash 5 times with 90% 1mM NaCl/10% ACN, elute 3 times with 75% PBS pH 2/25% ACN An amino side chain is attached through a reaction composed of DCM, PyBOP, DIPEA, and a desired side chain Synthesized possible SA derivatives from phenylalanine with a successful reaction Will continue testing different side chains on the SA analogs Will complete cytotoxicity assays on Jurkat leukemia cell line, followed by a lung cancer cell line using isolated SK as a control for each The MTM analogues SA, SK, and SDK are are produced by the inactivation of the mtmW gene responsible for the last enzyme in the biosynthetic pathway of MTM. Isolation of SA The SA analogue has a caroboxylic acid that that allows for a negative charge at physiological pH. An anion exchange column was used to take advantage of the negative charge and separate this analogue from MTM SK and MTM SDK. The quaternary amine was the strongest and most effective anion exchange column S. Argillaceus is grown on R5A solid media for 5 days. A portion is cut out and transferred to liquid TSB media for two days of further promoted growth. A sample of this is transferred to R5A liquid media for 5 or more days in order to promote drug production. R5A Solid Media TSB Liquid Media R5A Liquid Media Jeff Hansen Wendy Tomamichel Dave Roberts DePauw University Science Research Fellows (SRF) Column characteristics for isolation of SA. Column 52667, a strong anion exchange column with quaternary amines, was able to separate SA from other analogues. The first attempt at isolating MTM SA involved equilibrating the column twice with pH 7 phosphate buffer, loading the drug, washing with the pH7 buffer twice, and eluting with pH2 buffer/25% ACN. All three analogues came off in the elutions. 4 9 14 19 24 29 4 6 8 10 12 14 16 18 20 SA SK SDK 5 10 15 20 25 30 35 10 11 12 13 14 15 16 17 18 19 20 Absorbanceat410nm(mAU) Time (min) Flow Through wash 1 elution 1 elution 2 0 5 10 15 20 25 30 35 40 12 13 14 15 16 17 18 19 20 Absorbanceat410nm(mAU) Time (min) Flow through Wash 1 Wash 2 Elution 1 Elution 2 Derivatization of SA A coupling agent, PyBOP, is used with DIPEA, DCM, and a desired side chain addition in order to form an SA-side chain derivative. We used tryptophan, histidine, and phenylalanine as side chain additions. An example reaction with tryptophan is shown below. HPLC spectral analysis revealed a new peak in the MTM-SA Trp compound compared to SA. NMR and MS will later reveal the compounds structure. Histidine and phenylalanine were analyzed as well, but several new peaks appeared. Further testing to be done. The Cl- ion is a counter ion for the anion exchange column, so the new washes included NaCl to wash off the MTM SK and MTM SDK in the washes. After the drug was loaded, the drug was washed with 10mM NaCl and eluted 3 times with 75% pH2 buffer/25% ACN. This time all three analogues came off in the washes. -5 0 5 10 15 20 25 30 35 40 45 14 14.5 15 15.5 16 16.5 17 17.5 18 Absorbanceat410nm(mAU) Time (min) Wash 1 Wash 2 Elution 1 Elution 2 In order to separate the MTM SA from the MTM SK and MTM SDK, ACN was used in the washes. After the drug was loaded, it was washed 5 times with 90% pH 7/10% ACN and eluted 3 times with with 75% pH2 buffer/25% ACN. The washes contained mainly MTM SK and MTM SDK, but some MTM SA did come out. 0 20 40 60 80 100 120 12 12.5 13 13.5 14 14.5 15 15.5 16 Absorbanceat410nm(mAU) Time (min) Flow Through Wash 1 Wash 2 Elution 1 Elution 2 The wash for the final protocol included ACN and NaCl. After the drug was loaded, it was washed 5 times with 1mM NaCl/10% ACN, and eluted with 75% pH2 buffer/25% ACN. MTM SK MTM SDK MTM SA Anion exchange quaternary amine The three analogues are eluted off the HPLC at different times: MTM SA at 15.7 min, MTM SK at 15.9, and MTM SDK at 16.6. This will show which of the analogs are in a sample. -8 -3 2 7 12 17 12 13 14 15 16 17 18 19 20 Phenyl Reaction MTM SA The phenyl reaction was successful as demonstrated by the peak representing SA decreased and a new peak, the SA-side chain derivative, was formed. MTM SK MTM SDK MTM SA PyBOP DIPEA