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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
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
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
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