A Structure-Function Analysis A Structure-Function Analysis of Water Soluble Inhibitors of Water Soluble Inhibitors of the Catalytic Domain of of the Catalytic Domain of

Exotoxin A from Exotoxin A from Pseudomonas aeruginosaPseudomonas aeruginosa

Inhibition of ETAInhibition 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)

IC50 = 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

Similar EnzymesSimilar 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

The InhibitorsThe 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+

NH2

O

R

N+

O

NH2

R

NH

O

( ) n=0 or 1

Nicotinamide moiety

of NAD+

Tricyclic Lactams – [6,6,6] Ring SystemTricyclic Lactams – [6,6,6] Ring System

NH

O

NH

NCH3 CH3

O

NH

N

NHN

O

ON

NH

N

N

O

O

F

NH

N

N

NCH3

O

NH

N

N

O

N

CH3CH3

PJ34 GP-L

GP-G GP-N

GP-M

Tricyclic Lactams – [5,6,7]-Ring SystemsTricyclic Lactams – [5,6,7]-Ring Systems

NH

NH

N

O

N O

N

N

NH

O

O

N

CH3

CH3

N

N

NH

N

CH3

CH3

O

N

N

NH

O

N

GP-D GP-F GP-H GP-I

Other Inhibitors ClassesOther Inhibitors Classes

NH

O

SO3H

O

NH

O

NH3+

Cl-

O

OHOP

F

N+

O

PO

O

OH

OH

O

O-O

O-

NH2

O

N

N

N

N

NH2

5-AIQ GP-P

2’-F-ribo-NAD+

Bicyclic Lactam Tetracyclic Lactam

NAD+ Analog

ICIC5050 Values Values

Code Compound Chemical Name IC50 (M)

GP-D 3-(morpholin-4-ylmethyl)-1,5-dihydro-6H-[1,2]diazepino[4,5,6-

cd]indol-6-one 0.165 0.026

PJ34 N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide 0.284 0.069

GP-M

N-(6-oxo-5,6-dihydrobenzo[c][1,5]naphthyridin-2-yl)-2-(4-pyrrolidin-1-ylpiperidin-1-yl)acetamide·HCl 0.287 0.043

GP-P 1,11b-dihydro-[1]benzopyrano[4,3,2-de]isoquinolin-3(2H)-one-10-

sulfonic acid 0.453 0.017

GP-F

1-[4-(3-dimethylamino-propoxy)-phenyl]-8,9-dihydro-7H-2,7,9a-triaza-benzo[cd]azulen-6-one HCl 0.478 0.037

GP-L

8-fluoro-2-(3-piperidin-1-ylpropanoyl)-1,3,4,5-2H-tetrahydrobenzo[c]-1,6-naphthyridin-6-one MsOH 0.610 0.144

GP-G

2-(4-methylpiperazin-1-yl)-5H-benzo[c][1,5]naphthyridin-6-one MsOH 0.688 0.126

GP-H

1-dimethylaminomethyl-8,9-dihydro-7H-2,7,9a-triaza-benzo[cd]azulen-6-one HCl 0.964 0.050

GP-N

2-(4-Isopropylpiperazin-1-yl)-5H-benzo[c][1,5]naphthyridin-6-one MsOH 1.05 0.04

GP-I

1-[2-(4-pyrrolidin-1-ylmethyl-phenyl)-ethyl]-8,9-dihydro-7H-2,7,9a-triaza-benzo[cd]azulen-6-one hydrochloride HCl 4.46 0.95

5-AIQ 5-amino-isoquinoline-HCl 22.8 2.7

F-NAD+ 2’-F-ribo-NAD+ 82.4 7.4

Looking at the 3D Inhibitor StructuresLooking 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

Correlate ICCorrelate IC5050 to Structure to Structure

NH

NH

N

O

N OGP-D

IC50 = 165 nMPlanar

NH

O

NH

NCH3 CH3

O

PJ34

IC50 = 284 nMPlanar

NH

N

NHN

O

ON

GP-M

IC50 = 287 nMPlanar

NH

O

SO3H

O

GP-P

IC50 = 453 nMNon-Planar

N

N

NH

O

O

N

CH3

CH3

GP-F

IC50 = 478 nMNon-Planar

NH

N

N

O

O

F GP-L

IC50 = 610 nMNon-Planar

Correlate ICCorrelate IC5050 to Structure to Structure

NH

N

N

NCH3

O

NH

N

N

O

N

CH3CH3

GP-G GP-NIC50 = 688 nM

Planar*Exception

N

N

NH

N

CH3

CH3

O

GP-HIC50 = 964 nM

Non-PlanarIC50 = 1.05 M

Planar*Exception

N

N

NH

O

N

GP-I

IC50 = 4.46 MNon-Planar

NH

O

NH3+

Cl-

5-AIQ

IC50 = 22.8 MPlanar

O

OHOP

F

N+

O

PO

O

OH

OH

O

O-O

O-

NH2

O

N

N

N

N

NH2

F-NAD+

IC50 = 82.4 MPlanar

Summary of InhibitorsSummary 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?

PJ34 – Its HistoryPJ34 – 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….

NH

O

NH

NCH3 CH3

O

Inhibition of PE24H by PJ34Inhibition of PE24H by PJ34

• Characterization of PJ34– IC50 = 284 ± 69 nM

– KD = 820 ± 54 nM (A)• 70x tighter binding to PE24H compared to NAD+

– Competitive inhibitor (B)• Preliminary work using wheat germ eEF2 suggest that

the Ki is ~300 nM

0.000 0.005 0.010 0.015 0.020 0.0250.00

0.05

0.10

0.15

0.20

0.25

0.30

1/v

(s/p

mo

l)

1/[NAD+] (M-1)

0 1000 2000 3000 4000 50000.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e F

luo

resc

ence

[PJ34], nM

A B

Let’s Crystallize PE24H with PJ34!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

The Program ‘O’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

 

More ‘O’More ‘O’

CNSCNS

• 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

Crystallization of PJ34 with Crystallization of PJ34 with PE24HPE24H

Data Space group P212121 Unit-cell parameters a (Å) 56.035 b (Å) 78.680 c (Å) 91.694 Wavelength (Å) 1.046 Resolution (Å)1 35-2.1 (2.3-2.1) Completeness1 (%) 99.8 (100) Mean I/(I)1 13.35 (7.65) Rmerge (%)1,2 10.8 (23.6) Redundancy1 6.8 (6.8) Refinement Reflections Used/Free 24275/1236 Atoms Protein/Inhibitor/Water 3090/44/391 R-factor (%)1,3 21.33 (24.44) Rfree-factor (%)1,4 23.46 (26.02) Resolution for outer shell (Å)

2.13-2.10

R.m.s. deviation Bonds (Å) 0.008 Angles ( ) 1.449 Ramachandran (%)5 Monomer A 98.8/1.2/0 Monomer B 98.7/0.6/0.66

Ramachandran PlotsRamachandran Plots

PE24H-PJ34 StructurePE24H-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

PE24H-PJ34 StructurePE24H-PJ34 Structure

Monomer A

Monomer B

Glu-522Lys-590

Crystal Packing

Interactions of PJ34 within the Active SiteInteractions 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

Omit Map of PJ34 within the Active SiteOmit 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

2Fo-Fc omit map of PJ34 bound within the active site of ETA contoured at 1 .

PJ34-PE24H StructurePJ34-PE24H Structure

11 = 2.74 Å, 22= 2.45 Å, 33 = 2.53 Å, 44=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-440Gly-441

Tyr-481

Gln-48522 3311

44

Superposition of Modeled LoopSuperposition of Modeled Loop

Toxin-PJ34

Toxin with hydrolyzed NAD+

Li et al., 1995

Toxin--TADLi et al., 1996

Comparisons with DT and PARPComparisons 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-481 LOOP

Ala-478

PJ34

PE24H-PJ34PE24H-PJ34 vsvs DT DT

PE24H-PJ34PE24H-PJ34 vsvs PARP PARP

Final ThoughtsFinal 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