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Leveraging Microbial Therapeutics: Challenges and Innovations in Drug Development

This presentation discusses the potential of microbes in developing therapeutics, specifically focusing on antibiotics, antimalarials, and anticancer agents derived from sources like Penicillium chrysogenum and Artemisia annua. It highlights the market dynamics of antibacterial products and the urgent need for new chemical entities to counteract antibiotic resistance. The challenges of extracting complex natural products and advancements in biosynthesis optimization are addressed, showcasing opportunities for harnessing microbial potential in drug production and speeding up therapeutic accessibility.

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Leveraging Microbial Therapeutics: Challenges and Innovations in Drug Development

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  1. Therapeutics from Microbes: Pathways and Specific Examples Blaine Pfeifer Department of Chemical & Biological Engineering Tufts University Medford, MA 02155, U.S.A. FMM Industry Day February 23, 2011

  2. Motivation I – Range of therapeutics and activities Penicilliumchrysogenum Artemisia annua Salinosporatropica Penicillin Artemisinin Lomaiviticin Antibiotic Antimalarial Anticancer

  3. Motivation II – Market value and emerging drug resistance • NCE’s between 1981 and 2006 • 34% were natural products or semi-synthetic derivatives • Global antibacterial market generated $42 billion in sales in 2009 • ~4% annual growth over last five years • MRSA, VRSA, other antibiotic resistant microbes in community and hospital environments • Now, MRSA kills more U.S. citizens each year than AIDS • Infectious Diseases Society of America “10 by ‘20” initiative Pollack A. New York Times. 2010 Nov 10. Clin Infect Dis. 2010 Apr 15;50(8):1081-3. Hasmad B. Nat Rev Drug Discov. 2010 Sep;9(9):675-6. Newman DJ, Cragg GM. J Nat Prod. 2007 Mar;70(3):461-77.

  4. Challenges I –Intractable natural sources Penicilliumchrysogenum Artemisia annua Salinosporatropica Penicillin Artemisinin Lomaiviticin Antibiotic Antimalarial Anticancer

  5. Challenges II – Molecular complexity Penicilliumchrysogenum Artemisia annua Salinosporatropica Penicillin Artemisinin Lomaiviticin Antibiotic Antimalarial Anticancer

  6. Approach I –Heterologous Biosynthesis Heterologous Expression Identification & Isolation GeneA GeneA GeneB GeneC Escherichia coli GeneB GeneC • Scientific Motivation: • Complex, slow-growing organisms • Complex chemistry • Vast knowledge • Eventual Outcome: • Therapeutic access • Less expensive drugs • Widespread use • Challenges: • Uncompetitive production titers

  7. Approach II –Metabolic and process optimization

  8. Examples – Erythromycin and Taxol Saccharopolysporaerythraea Polyketide Propionyl-CoA 6 × (2S)-Methymalonyl-CoA 6-Deoxyerythronolide B Erythromycin CarbonSource(s) Isoprenoid 3 × IPP Taxusbrevifolia DMAPP Taxadiene Taxol

  9. Erythromycin – Complete heterologous biosynthesis Zhang H, et al. Chem Biol. 2010 Nov;17(11):1232-40. Pfeifer BA, et al. Science. 2001 Mar;291(5509):1790-2.

  10. Pyruvate 2C-methyl-D-erythritol-4-phosphate (MEP) 1-deoxy-D-xylulose-5-phosphate (DXP) DXR IspE DXS IspD CDP-MEP CDP-ME Glyceraldehyde 3-phosphate IspF IPP IspH IspG MEC HMBPP GGPPS 5α TXS IDI DMAPP Taxol – Isoprenoid pathway optimization Plasmid, promoter, strain combinations Ajikumar PK, et al. Science. 2010 Oct 1;330(6000):70-4.

  11. Opportunities I – Access Nature’s potential • 25 predicted polyketide, nonribosomal peptide, and terpene gene clusters • No products derived from these clusters (besides erythromycin) were indentified on 50 different types of solid and liquid media Boakes S, et al. J Mol Microbiol Biotechnol. 2004;8(2):73-80. Oliynyk M, et al. Nat Biotechnol. 2007 Apr;25(4):447-53.

  12. Opportunities II – Manipulate biosynthesis DEBS1 DEBS2 Tailoring Enzymes DEBS3 Propionate Sfp PrpE PCC DEBS1 Propionate DEBS2 Tailoring Enzymes DEBS3 Sfp PrpE PCC Benzoate Zhang H, et al. 2011. In preparation.

  13. Opportunities III – Production speed and economy ~Days ~Days Process Time: ~100 years ~Weeks ~Weeks ~Weeks 100 year old tree → 3 kg bark →300 mg Taxol → 1 dose! A. thalianaHeterologous E. coli Heterologous T. canadensisCell-Culture Tomato Heterologous S. cerevisiae Heterologous T. brevifoliaExtraction (Horwitz 1994 Nature) (Ketchum, et al.1998 B&B) (Besumbes, et al.2004 B&B) (Kovacs, et al. 2007 Transgenic Res) (Engels, et al. 2008Metab Eng) (Ajikumar, et al. 2010 Science)

  14. 6dEB (mg/L) Tryptone Trace Metals Opportunities IV – High(er) throughput process optimization -Media optimization through 96-well format Defined Medium: -Potassium Phosphates -Ammonium Sulfate -Glycerol -Magnesium Sulfate -Vitamins -Trace Metals LB Medium: -Yeast Extract -NaCl -Tryptone HPLC-ELSD Analysis 96-well format Plackett-Burman Screening: LB Medium: -Yeast Extract -NaCl -Tryptone Enhanced Medium: -Yeast Extract -Tryptone -Glycerol -NaCl -Vitamins -Trace Metals Defined Medium: -Potassium Phosphates -Ammonium Sulfate -Glycerol -Magnesium Sulfate -Vitamins -Trace Metals Pistorino M, Pfeifer BA. BiotechnolProg. 2009 Sep-Oct;25(5):1364-71.

  15. Acknowledgements • Students: • Haoran Zhang • Brett Boghigian • Jiequn Wu • Karin Skalina • Yong Wang • Funding/Collaborators: • NIH (AI074224; GM085323) • NSF (0712019; 0924699) • Milheim Foundation • Greg Stephanopoulos (MIT)

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