1. A Structure-Function Analysis of Water Soluble Inhibitors of the Catalytic Domain of Exotoxin A from Pseudomonas aeruginosa

2. Inhibition of ETA Previous work from our Research Group –Characterized a series of small, non-polar competitive inhibitors against the catalytic domain of ETA (PE24H) Most potent inhibitor was NAP (1,8-napthalamide)  IC 50 = 87 nM  Model of NAP bound to ETA proposed Lack of water-solubility limited the usefulness of these compounds as potential therapeutic drugs Purpose of this study –in vitro characterization of a series of water-soluble inhibitors of PE24H –Co-crystallization of an inhibitor (PJ34) bound to PE24H

3. Similar Enzymes Catalytic domain of ETA is functionally and structurally similar to both mono-ADPRTs and PARPs Diphtheria toxin (DT) –Mono-ADPRT that also catalyzes the ADP-ribosylation of eEF2 PARPs (Poly-(ADP-ribosyl) polymerases) –Eukaryotic nucleus –Catalyze the covalent attachment of ADP-ribose units from NAD + to itself and nuclear DNA-binding proteins –Responds to DNA strand breakage –Rapid activation of PARP depletes NAD + within the cell Disruption of energy production processes

4. The Inhibitors Common structural motif of inhibitors –Benzamido group fused into a heteroring to lock amide in s-trans conformation Mimics the nicotinamide moiety of NAD + –R-group substitutions that include the addition of hydrogen donors and/or acceptors to increase water solubility ( ) n=0 or 1 Nicotinamide moiety of NAD +

5. Tricyclic Lactams – [6,6,6] Ring System PJ34GP-L GP-G GP-N GP-M

6. Tricyclic Lactams – [5,6,7]-Ring Systems GP-D GP-FGP-H GP-I

7. Other Inhibitors Classes 5-AIQGP-P 2’-F-ribo-NAD + Bicyclic LactamTetracyclic Lactam NAD + Analog

8. IC 50 Values

9. Looking at the 3D Inhibitor Structures The Dundee PRODRG2 Server –http://davapc1.bioch.dundee.ac.uk/programs/prodrg /prodrg.html –Generates PDB file (with and without hydrogens) X-ray refinement topology and parameter files for use with CNS  CHECK THIS WEBSITE OUT! Let’s look at the inhibitor structures in 3D

10. Correlate IC 50 to Structure GP-D IC 50 = 165 nM Planar PJ34 IC 50 = 284 nM Planar GP-M IC 50 = 287 nM Planar GP-P IC 50 = 453 nM Non-Planar GP-F IC 50 = 478 nM Non-Planar GP-L IC 50 = 610 nM Non-Planar

11. Correlate IC 50 to Structure GP-GGP-N IC 50 = 688 nM Planar *Exception GP-H IC 50 = 964 nM Non-Planar IC 50 = 1.05 M Planar *Exception GP-I IC 50 = 4.46 M Non-Planar 5-AIQ IC 50 = 22.8 M Planar F-NAD + IC 50 = 82.4 M Planar

12. Summary of Inhibitors Importance of a locked benzamido group More potent inhibitors have a core ring structure that is planar –Exceptions are GP-G and GP-N Piperazine moieties as their R-group  Unfavourable Active site prefers compounds that are more rigid and compact –Non-flat ring systems may be too big to fit into the active site Positioning of the hydrogen bonding lactam is critical GP-D is the most potent inhibitor in this study –[5,6,7] tricyclic lactam containing a indole Potential H-bond to Glu-553 analogous to PARPs?

13. PJ34 – Its History Originally synthesized by Inotek Pharmaceuticals to target PARP –Now commercially available from Sigma Water-soluble phenanthridinone derivative Well-characterized compound –in vitro and in vivo studies in several PARP related systems Stroke, heart disease and transplantation, diabetes, cancer, exposure to cytotoxic oxidants etc….

14. Inhibition of PE24H by PJ34 Characterization of PJ34 –IC 50 = 284 ± 69 nM –K D = 820 ± 54 nM (A) 70x tighter binding to PE24H compared to NAD + –Competitive inhibitor (B) Preliminary work using wheat germ eEF2 suggest that the K i is ~300 nM AB

15. Let’s Crystallize PE24H with PJ34! Make Crystals Collect Diffraction Data Build Model with ‘O’ Refinement with CNS Check Quality of Model with PROCHECK Create Figures using PyMOL Molecular Replacement

16. The Program ‘O’ Macromolecular crystallographic modeling tool –used to look at macromolecular structures, analyze them, compare them, modify them and to build them from scratch Helpful website: –http://xray.bmc.uu.se/alwyn/A-Z_of_O/everything.html

17. More ‘O’

18. CNS Crystallography & NMR System (CNS) –international collaborative effort among several research groups –designed to provide a flexible multi-level hierarchical approach for the most commonly used algorithms in macromolecular structure determination include heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets Website: –http://cns.csb.yale.edu/v1.0/ –Copy scripts from the website and save them in XEmacs –Run CNS after scripts have been edited in XEmacs

19. Crystallization of PJ34 with PE24H

20. Ramachandran Plots

21. PE24H-PJ34 Structure Includes residues 399 to 602 –Poor density for C- terminal residues, 603 to end Monomers A and B superimpose with only minor alterations Monomer B –Residues 459 to 464 unresolved Monomer A Monomer B

22. PE24H-PJ34 Structure Monomer A Monomer B Glu-522 Lys-590 Crystal Packing

23. Interactions of PJ34 within the Active Site Hydrophobic Pocket –60% of the surface of PJ34 buried within the toxin –Trp-466, Tyr-470, Ile-471, Ala-472, Leu-477, Ala-478 and Tyr-481

24. Omit Map of PJ34 within the Active Site Gln-485 Glu-553 Tyr-470 Leu-477 Ala-478 Gly-441 His-440 Tyr-481 Ala-472 2F o -F c omit map of PJ34 bound within the active site of ETA contoured at 1 .

25. PJ34-PE24H Structure 1234 1 = 2.74 Å, 2= 2.45 Å, 3 = 2.53 Å, 4=3.08 Å Tyr-481 is 4 Å and Tyr-470 is 6-7 Å (and at 40°) away from PJ34 Glu-553 Tyr-470 Leu-477 Ala-472 Ala-478 His-440 Gly-441 Tyr-481 Gln-485 231 4

26. Superposition of Modeled Loop Toxin-PJ34 Toxin with hydrolyzed NAD + Li et al., 1995 Toxin--TAD Li et al., 1996

27. Comparisons with DT and PARP Tyr-470 Glu-553 Ala-472 His-440 Gly-441 Tyr-481 LOOP Ala-478 NU1025 Tyr-470 Glu-553 Ala-472 His-440 Gly-441 Tyr-481LOOP Ala-478 PJ34 PE24H-PJ34 vs DT PE24H-PJ34 vs PARP

28. Final Thoughts First report of a structure of a mono-ADPRT in complex with an inhibitor Confirmed the hydrogen bonding to the lactam moiety as seen in PARP and as predicted earlier with the NAP-ETA model Planar compounds may sandwich better into the nicotinamide binding pocket than more flexible compounds –Steric interactions? Similarities and differences between ETA/DT and PARP –Exploit the differences to preferentially target one enzyme over the other