1. Sept 21, 2006 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. Sept 21, 2006 SPECIFIC AIMS 1.Optimize the in vitro LF inhibitory activity of the validated hit series a)Lead compound SAR b)Modeling/X-ray crystallography c)Biochemical activity against LF—Fluorescence-based/HPLC d)Continued screening of the MBX compound library against LF 2.Demonstrate potent selective inhibitory activity in cellular models of LF action a)Medicinal chemistry b)Cellular protection and toxicity assays 3.Identify LF inhibitors with in vitro ADME properties suitable for oral dosing 4.Identify lead LF inhibitors that are active in an in vivo model

3. Sept 21, 2006 LF Inhibitor NSC 12155 Compound NSC 12155 emerged as a lead compound with: –K i of 0.5  M in the LF assay –EC 50 < 25  M in a cell-based assay. NSC 12155 has also been used as a starting point for the SAR studies.

4. Sept 21, 2006 Quinoline Core

5. Sept 21, 2006 ANALYSIS OF LF INHIBITOR MEDICINAL CHEMISTRY

6. Sept 21, 2006 LF ASSAY REAGENT COMPOSITION AND GENERAL PROCEDURE 1) Combine the following reagents. Component[Stock][Final] 1 μL DMSO or Compound100 %1% 64 μL Water*55MN/A 10 μL HEPES pH 8.2*200mM20 mM 10 μL Tween 20*0.5%0.05% 5 μL diluted LF* ¥ 10 μg/mL0.5 μg/mL 10 μL Peptide Substrate200 μM20 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. Sept 21, 2006 12155 Activity & Specificity

8. Sept 21, 2006 SAR: Ureas

9. Sept 21, 2006 SAR: Heteroaryl Amides

10. Sept 21, 2006 SAR: Substituted Phenyl Amides

11. Sept 21, 2006 SAR: Extended Tether

12. Sept 21, 2006 SAR: Phenylpropionamides

13. Sept 21, 2006 SAR: Cinnanamides

14. Sept 21, 2006 SAR: Tether Length

15. Sept 21, 2006 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. Sept 21, 2006 SAR: What Next? Inhibitor-Bound X-Ray structure may provide more ideas and/or refinements

17. Sept 21, 2006 Computer Model

18. Sept 21, 2006 Proposed Compounds

19. Sept 21, 2006 Hybrid Compounds