Post on 14-Jan-2016
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Conformational Flexibility and Ligand DesignConformational Flexibility and Ligand Design
Dr. Eamonn F. HealyDr. Eamonn F. HealyProfessor of ChemistryProfessor of Chemistry
St. Edward’s University, Austin, Tx. St. Edward’s University, Austin, Tx.
Our research focuses on the design of structure-activity probes to elucidate enzymatic activity. The interdisciplinary approach includes molecular modeling for the simulation of inhibitor binding , overexpression of wild-type and mutant target proteins and in vitro assays of enzymatic activity and inhibition. Our targets include HIV-1 integrase, the c-Kit and src-abl proteins , and the metalloproteinases associated with CXCL16 shedding. Large flexible ligands and conformationally mobile proteins present two distinct, but related challenges. Lessons learned from investigating these target systems will be presented.
A process for drug design which bases the design of the drug upon the structure of its protein target.
1. Structural mapping of the receptor (protein, P) active site
2. Identification of ligands (L) of complementary shape and appropriate functionality
3. Docking of the ligand to the receptor site - predicting a range of PL complexes with different GPL values
4. Scoring i.e. ranking GPL and correlating with experimentally determined properties such as IC50 values
PROTEIN
1. Structural mapping of the receptor active site
Crystal structure available for Integrase but :I. Limitations of crystal structure:
Often only one domain Membrane or DNA attachment sites
usually not shown Crystal structure vs. physiologically
active structureII. Position of hydrogens undetermined III. Residues missing or ill-definedIV. Protonation of His undeterminedV. SolvationVI.Conformational Dynamics
LIGAND
2. Identification of ligands (L) of complementary shape and appropriate functionality
Crystal structure often available for Inhibitor bound to catalytic core but :
I. Position of hydrogens undeterminedII. Tautomeric structures possibleIII. Influence of pHIV. Need to limit conformational flexibility based
on experimental and theoretical crteria
Fixed and planar
Based on HF/6-31G* calculationsLimited to +/- 45 degrees
DOCKING
3. Docking of the ligand to the receptor site - predicting a range of PL complexes with different GPL values
The prediction of the ligand conformation and orientation within a targeted binding site
involves:I. Positioning ligand and evaluating
quality of bindingII. Refining ligand positionIII. Energy minimization (electrostatic,
steric, strain and h-bond)
SCORING
4. Scoring i.e. ranking GPL and correlating with experimentally determined properties such as IC50 values
The prediction of the optimum ligand conformation
and orientation within a targeted
binding site involves:I. Posing : Determining the fit
of the ligandII. Conformational SearchingIII. Scoring and Ranking
Predicted Gbind
(kcal mol-1)
-12.7
Predicted Gbind
(kJ mol-1)
-53.0
Predicted Ki
(M)5.10-10
pKi 9.3
Expected IC50
< nanomolar
A B
DC
(A) 6.7 degree tilt on the N-lobe towards the C-lobe for the conversion of inactive c-Kit (yellow) to the active form (blue) ; (B) The induced fit of imanitib
mesylate to c-Kit, shown as a superimposition of the inactive form in the absence of inhibitor (yellow) and c-Kit+inhibitor ( gray); (C) , (D) Key
structural elements of inactive (C) and active (D) conformations
FEBS Lett. 2009, 583, 2899-2906.
Surface representation showing Ile653 side chain in red. Electrostatic map of bound inhibitor
Solvent accessible surfaces for Ile653 side chain + Aryl ring of inhibitor.
Trajectory analysis for solvent-exposed intra-helix backbone hydrogen bond distance between Leu644 and
Gly648 of the C-helix for a 1 ns molecular dynamics run for c-Kit with ( )nd without ( )bound ligand .
Final conformation for the 1ns MD run for c-Kit with bound ligand.
Final conformation for the 1ns MD run for c-Kit without bound ligand.
N
NO
H
H
HO
Thr670
O
OGlu640
HN
H
H
Lys623
1
34
56 7
89
10
2
A B
C D
(A) Inhibition of c-Kit through the binding of 9-hydroxy ellipticine. (B) Inhibition of c-Abl through the binding of 9-hydroxy ellipticine. (C) Docked structures for 9-hydroxy ellipticene in blue,9-methoxyellipticine in green and
ADP in gray with 1OPK. (D) Postion of the 9-methoxyellipticine ligand in relation to the backbone hydrogen bond between Phe401 and Leu403.
Alignments
Repositioning of DFG motif through movement of activation loop in going from inactive (yellow)
to active (blue) c-Kit
Dominant Patterns of Drug Resistance for certain Tyrosine Kinases
DCQ acids; DCT acids
DKAs
Quinolone derived
PDP SQL
HIV-1 Integrase Inhibitors
OO
OH
OH
HO
HO
COOHHOOC
OO
L-CA
Cl
NH
HO
NH
NH
NN
O
CITEP ( a DKA)
J Mol. Graph. Model. 2009, 27 , 14.
L-Chicoric Acid and HIV-1 Integrase
Isomer Erel (kcal mol-1) % Population (T=298 K)AM1 HF/6-31G** MP2/6-31G** AM1 HF MP2
cis-cis / syn-syn 0.0 0.0 0.0 75.6 77.3 74.9
cis-trans / syn-syn 1.2 1.2 1.1 24.2 21.6 23.9
trans-trans / syn-syn 3.7 2.6 2.5 0.2 1.1 1.2cis-cis / syn-anti 6.0 5.5 10.9 <1.10-4 <2.10-4 <2.10-6Ligand IN Protein Cluster
Occa.Gbind (kcal mol-1) Ki (M) IC50(relative)b h-bonded residues
L-CA (s-cis /s-cis)
1QS4 44 -8.1 1.1 1 D116,Q148, K156, K159
L-CA (s-cis /s-trans)
1QS4 29 -7.6 2.6 1 T66, H67,D92, Q148, K156, K159
L-CA (s-cis /s-cis)
Q148A 27 -6.2 31.7 20 D116, A148, K156
L-CA tetraacetylated (s-cis /s-cis)
1QS4 33 -6.6 14.0 9 T66, H67, Q148, K156, K159
Conformer energies, and predicted conformer populations, for the various L-CA conformer geometries. Results of 100 independent docking runs for the ligands L-CA and its tetraacetylated derivative with proteins 1QS4 and the mutant Q148A.
OO
OH
OH
HO
HO
COOHHOOC
OO
L-CA
Cl
NH
HO
NH
NH
NN
O
CITEP ( a DKA)
O
R
H
H
X
R
H
H
O
X
s-trans s-cis
RO
OR'
RO
OR'
syn anti
Top-ranked binding modes for s-cis /s-cis L-CA (top left), s-cis /s-trans L-CA (top right), tetracetylated derivative of L-CA (bottom
left) and a surface image of the inhibitor binding pocket with bounds-cis /s-cis L-CA overlaid with the experimentally observed
5CITEP inhibitor (bottom right).
Hydrogen bonded interactions and electrostatic map for the top-ranked s-cis /s-
cis L-CA solution
Druggability and Drug Resistance
ADAM 17 and ADAM 10
A zinc-binding group provides high affinity but low selectivity
Major determinant of potency and selectivity.
Long chains can provide selectivity.
Wide range of substituents tolerated
and are determinants of potency. Steric bulk beneficial for oral
availability.
Wide range of substituents
tolerated. Charged polar
groups influence bilary excretion.
ADAM Ligand Design
S1’-S3
’ Channel
S1'
S1
E
B
S2'
A TAPI-2
C R=CH3 (TMI-1)D R=CH2NH2
S1' S1'
S3'
S3'
S3'
S1'
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
S
1'
S1
E
B
S2'
A TAPI-2
C R=CH3 (TMI-1)D R=CH2NH2
S1' S1'
S3'
S3'
S3'
S1'
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
Acetylenic Sufonamide Inhibitor Design
B
S2'
A TAPI-2
S1' S1'
S3'S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
B
S2'
A TAPI-2
S1' S1'
S3'S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
TACE: Potency
S1'
C R=CH3 (TMI-1)D R=CH2NH2
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
S
1'
C R=CH3 (TMI-1)D R=CH2NH2
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
TACE: Potency
TACE: Potency
S2'
A TAPI-2
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
S2'
A TAPI-2
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
ADAM17 vs ADAM10: Selectivity
S1'
C R=CH3 (TMI-1)
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
S
1'
C R=CH3 (TMI-1)
S3'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
R
O
S
O
O
N
NH
OH
OO
SOO
O
SO
ADAM17 vs ADAM10: Selectivity
S1
S2
S1
E
S3'
S1'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
RO
S
O
O
N
NH
OH
OO
SOO
O
SO
S1
E
S3'
S1'
OHNH
O
O
NH O
NH
NH
NH2
O OHNH
O
N
S
O
O
O
OHNH
O
N S
RO
S
O
O
N
NH
OH
OO
SOO
O
SO
Future Ligand Design
S2
S1
S3'
S1'
O
O
OS
O
NO
NSOH
S
O
N
O
NH
OH
S
2
S1
S3'
S1'
O
O
OS
O
NO
NSOH
S
O
N
O
NH
OH
Future Ligand Design: Electrostatic map of S2 binding pocket
S1 S1’ S3
’ S2 S2’
loop
Protein Alignment for ADAM17 and ADAM10