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SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR

SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR. Phase I SBIR Project Period 7/01/05-6/30/07 P.I. Norton Peet John D. Williams. SPECIFIC AIMS. Optimize the in vitro LF inhibitory activity of the validated hit series Lead compound SAR Modeling/X-ray crystallography

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SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR

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  1. SMALL MOLECULE INHIBITORS OF ANTHRAX LETHAL FACTOR Phase I SBIR Project Period 7/01/05-6/30/07 P.I. Norton Peet John D. Williams

  2. SPECIFIC AIMS • Optimize the in vitro LF inhibitory activity of the validated hit series • Lead compound SAR • Modeling/X-ray crystallography • Biochemical activity against LF—Fluorescence-based/HPLC • Continued screening of the MBX compound library against LF • Demonstrate potent selective inhibitory activity in cellular models of LF action • Medicinal chemistry • Cellular protection and toxicity assays • Identify LF inhibitors with in vitro ADME properties suitable for oral dosing • Identify lead LF inhibitors that are active in an in vivo model

  3. LF Inhibitor NSC 12155 • Compound NSC 12155 emerged as a lead compound with: • Ki of 0.5 mM in the LF assay • EC50 < 25 mM in a cell-based assay. • NSC 12155 has also been used as a starting point for the SAR studies.

  4. Quinoline Core

  5. ANALYSIS OF LF INHIBITOR MEDICINAL CHEMISTRY

  6. LF ASSAY REAGENT COMPOSITION AND GENERAL PROCEDURE 1) Combine the following reagents.  Component [Stock] [Final] 1 μL DMSO or Compound 100 % 1% 64 μL Water* 55M N/A 10 μL HEPES pH 8.2* 200mM 20 mM 10 μL Tween 20* 0.5% 0.05% 5 μL diluted LF* ¥ 10 μg/mL 0.5 μg/mL 10 μL Peptide Substrate 200 μM 20 μM 2) Incubate Above Mix For 15 Minutes at 30 °C 3) Add stop solution [Stock] [Final] 10 μL Acetic Acid 5% 0.45% 3) Read excitation 324 nm and emission 395 nm *These reagents are combined proportionately into an assay mix, with 90 µL of mix added per microplate well. 10 µL of peptide substrate is added to initiate the reaction. ¥Enzyme dilution buffer contains 5 mM HEPES, pH 7.4, 50 mM NaCl, 100 mM Trehalose

  7. 12155 Activity & Specificity

  8. SAR: Ureas

  9. SAR: Heteroaryl Amides

  10. SAR: Substituted Phenyl Amides

  11. SAR: Extended Tether

  12. SAR: Phenylpropionamides

  13. SAR: Cinnanamides

  14. SAR: Tether Length

  15. SAR: General Trends • Simple aromatic compounds inactive • Polyaromatics generally more active • Tether of ~4 atoms seems to have highest activity • Phenylpropionamides need 2-substituent • Cinnanamides have lower requirements, lower activity

  16. SAR: What Next? Inhibitor-Bound X-Ray structure may provide more ideas and/or refinements

  17. Computer Model

  18. Proposed Compounds

  19. Hybrid Compounds

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