Transcript Altman et al. JACS 2008, 130 6099-6113 Presented By Swati Jain.

Altman et al. JACS 2008, 130 6099-6113
Presented By Swati Jain
Drug Resistance
 Mutations in drug target – selective lower
inhibitor affinity – maintenance of normal
function.
 Approach – drugs for known resistant mutants.
 Problems – potential to introduce new drug
resistant mutations.
 New techniques – not induce viable mutations,
work with unknown modes of resistance.
Substrate envelope Hypothesis
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Inverse Inhibitor Design Algorithm
 Generate substrate envelope.
 Select scaffolds. Choose functional groups.
 Generate conformational ensembles.
 Place scaffold in the substrate envelope – single
and pair-wise energies - DEE/A* - energy
ranked compounds.
 Refine the list - more accurate energy
functions.
HIV-1 Protease as target model
 Homodimer – each
subunit made up of
99 amino acids.
 Well studied protein
 Aspartic protease:
Asp-Thr-Gly active
site.
Figure taken from Wikipedia.
Known HIV-1 Substrates and
Inhibitor
Figure taken from King et al. Chem bio 11 1333-1338.
Substrate and Maximal Envelope
Substrate and Maximal Envelope
Scaffold and functional groups
Functional Groups
Amprenavir scaffold
 Carboxylic acids – R1.
Primary amines - R2.
Sulfonyl chlorides –
R3
 Criterion: < four
rotatable bonds.
(ignoring the bond to
the active group).
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Conformational Ensembles
 Hydrogen atoms placed at attachment sites for
both scaffold and functional groups.
 Geometry Optimization.
 Scaffold and Functional Groups: Sampling
dihedral angles about each rotatable bond.
(every 30 degrees for sp3-sp3, sp2-sp3 and every
45 degrees for sp2-sp2 bond).
Energy calculations
 Substrate bound protease structure
 Inactivating mutation reversed.
 Assigned force field parameters.
 Substrate envelope placed inside the active site.
 Three components: Van der Waal’s packing
term, screened electrostatic interaction term,
Desolvation penalties for both ligand and
receptor.
Grid based energy calculations
 Receptor shape and charges fixed.
 Basis points within the ligand – points of cubic
grid inside substrate envelope.
 Van der Waal’s energies – each atom type at
each grid point.
 Electrostatic – 1 electron charge at each grid
point.
 Desolvation – change in solvation potential for
all grid points when one grid point is charged.
Energy calculations contd …
 Van der Waal’s energy –
interpolating energies
from grid points.
 Electrostatic and
desolvation – projecting
partial charges to grid
points.
Figure taken from Wikipedia.
Scoring function
 Constant term – Binding energy of blunt
scaffold + receptor desolvation term.
 Self energy of functional group – Binding
energy with receptor + desolvation between
functional group and scaffold.
 Pair wise energies – desolvation penalties
between two functional groups.
 Clashes – energy infinite.
Scaffold into the Envelope
 Placed the scaffold in the envelope.
 Scaffold position accepted – all atoms within
the envelope + required hydrogen bonding +
no clashes.
 For each scaffold placement – discrete
ensembles of every functional group attached –
self energies.
 Pairs of functional groups attached – pair wise
energies.
DEE/A*
 Self and pair wise energies sum to the total energy
calculated.
 For each scaffold (backbone) conformation –
ensemble (rotamers) of functional groups (side-
chains) and the self and pair wise energy
contribution to the total energy.
 Used DEE/A* to generate the list of energy ranked
conformations.
 A common list for all scaffold positions.
Hierarchical energy functions
 Assumption – energies calculated using substrate
envelope.
 Generated list re-evaluated.
 More sophisticated energy function – true
molecular surface.
 Higher Grid resolution.
First Round Design
 Design repeated eight times
 Tight and loose substrate envelope
 Doubly deprotonated and deprotonated
protease structure.
 Rigid and flexible scaffold placement.
 20 compounds selected based on robustness to
parameters.
 15 synthesized and tested.
First round Inhibitor Affinities
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Second round design
 Selection of functional groups – based on
successful compounds from the first round.
 Inhibitor bound protease structure used for the
design.
 Only doubly-deprotonated protease structure.
 Tighter definition of substrate envelope.
 36 compounds synthesized and tested.
Second round design results
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Binding Affinities – Drug resistant
protease
inhibitor
WT
M1
M2
M3
M4
worst fold loss
ritonavir
0.055
3.0
0.46
2.8
ND
55
saquinavir
0.065
90
1.0
78
ND
1385
indinavir
0.18
34
0.73
21
ND
189
nelfinavir
0.28
15
3.5
19
ND
68
lopinavir
0.005
6.1
0.040
0.90
ND
1220
amprenavir
0.10
0.15
0.21
1.4
0.34
14
atazanavir
0.046
0.33
0.009
0.49
ND
11
tipranavir
0.088
0.014
0.001
0.032
ND
0.36
darunavir
0.008
0.005
0.041
0.025
0.33
41
11b
42
260
85
79
ND
6
11c
50
380
66
140
ND
8
12h
33
270
29
95
ND
8
12j
53
140
130
670
ND
13
Binding Affinities – Drug resistant
protease
inhibitor
WT
M1
M2
M3
M4
worst fold
loss
27a
0.14
1.5
0.020
2.0
0.84
14
27b
0.24
3.0
0.79
9.7
ND
40
28a
0.027
5.9
0.12
1.8
1.2
219
28b
0.12
7.6
0.45
2.6
ND
63
29a
0.12
0.99
0.064
1.6
4.1
34
29b
0.062
3.5
0.84
7.0
5.3
113
30a
0.036
0.44
0.31
0.57
0.10
16
30b
0.063
0.93
0.49
6.5
5.4
103
30d
0.063
1.1
0.88
5.0
1.3
79
32c
0.014
0.41
0.094
2.4
0.24
171
Correlation between calculated
and observed binding free energies
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Crystal structures of the inhibitors
 Structures done – four first round, five second
round.
 Scaffold preserved hydrogen bonding network.
 First round inhibitors – mostly inside substrate
envelope except one functional group.
 Second round inhibitors – Mostly inside
substrate envelope with one exception.
Predicted and Determined
structures
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Substrate envelope
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Crystal structures – Relation to
Resistance profile.
Figure taken from : Altman et al. JACS 2008 130 (19) 6099-6113
Testing the algorithm for
separating binders and non-binders
Figure taken from: Huggins et al. Proteins 75: 168-186.
Differences from earlier algorithm
 Geometry Optimization of the Protein structure.
 Scaffold and side groups - the set of known
binders and non binders.
 Maximal envelope
 Torsion angle of the bond attaching functional
group to scaffold – 10 degrees.
 Minimization.
Enrichment for binders
Figure taken from: Huggins et al. Proteins 75: 168-186.
Contribution of electrostatic
energy
Figure taken from: Huggins et al. Proteins 75: 168-186.
Explicit water model
Figure taken from: Huggins et al. Proteins 75: 168-186.
Issues and Improvement
 Inhibitors have lower binding energies outside the
substrate envelope – factors beyond substrate
envelope important.
 Finer Sampling - better results – generates too
many placements.
 Scoring functions – minimization gives better
results – MinDEE??.
 Flexible receptor.
 Certain functional groups and solubility
prediction.