Identification of “Hot Spots” in

Druggable Binding Pockets by

Computational Solvent Mapping of
Proteins
Melissa R. Landon1, Jessamin Yu1, Spencer C.
Thiel2, David R. Lancia2, Jr., Sandor Vajda1,3
1
Bioinformatics Graduate Program, Boston University, Boston MA
2
SolMap Pharmaceuticals, Cambridge MA
3
Department of Biomedical Engineering, Boston University, Boston MA
 Terms
• Druggability: the ability of a protein’s
binding pocket(s) to bind lead-like
molecules with high affinity

• Hot Spots: specific residues within a

binding pocket for which ligands display
high affinity
 Protein mapping and druggability

Observation based on SAR by NMR:

• Druggable sites bind a variety of small molecules

• Binding of probes is restricted to ligand binding sites
• “Hit rate” in mapping is a predictor of druggability

Hajduk PJ, Huth JR, Fesik SW: Druggability indices for protein targets derived from
NMR-based screening data. J Med Chem (2005) 48(7):2518-2525.

Hajduk PJ, Huth JR, Tse C: Predicting protein druggability. Drug Discov Today
(2005) 10(23-24):1675-1682.
CS-Map: Introduction

C. Mattos and D. Ringe.

Nature Biotech. 14: 595-599
(1996)

CS-Map is based on an experimental

method for ligand binding site identification
by the co-crystallization of a protein in
multiple organic solvents
 Step 1A: Probe Placement

222 initial probe positions

 Steps 1B-2: Rigid Body Search and
Minimization

•Simplex search
•Free energy-based score
•Second minimization in
CHARMM includes Van der
Waal term
 Step 3: Clustering of Bound Probes

• Interaction-based
clustering
Step 4: Creation of Consensus Sites

•5-10 lowest free energy clusters for each probe used

 Example 1: Mapping of lysozyme
Binding of solvents to lysozyme (Liepinsh & Otting, 1997)

NMR data on the binding of methanol, isopropanol, acetone, acetonitrile, t-

butanol, urea, DMSO, and methylene chloride

Based on observed NOEs:

 All ligands bind at site C

9 NOEs: N59 NH, W63 CH, W63 NH, I98 CH, I98 CH,
A107 CH, W108 CH, W108 CW108 NH

 In addition to site C, methanol and methylene chloride bind to an internal site

 A few week NOEs for isopropanol and acetone show binding at the rim of site C
Dennis, S., Kortvelyesi T., and Vajda. S.
Computational mapping identifies the binding sites of
organic solvents on proteins.
Proc. Natl. Acad. Sci. USA., 99: 4290-4295, 2002.

Kortvelyesi, T., Dennis, S., Silberstein, M.,

Brown III, L., and Vajda, S. Algorithms for
computational solvent mapping of proteins.
Proteins. 51: 340-351, 2003.
 Lowest free energy clusters for eight ligands

Methanol
Isopropanol
W108 A107 Acetone
Tert-butanol
Urea
I98 DMSO
Acetonitrile
N59 Methylene chloride

W63
 Subclusters of methanol and isopropanol

A107
W108
W108 isopropanol
A107

methanol

Q57 Q57
I98

I98
N59
W63 W63
N59
 Conclusions I: The nature of binding
• sites
Each ligand binds in several rotational states.

• The van der Waals energy is low in each rotational state: a

well defined pocket that can burry the ligands and exclude
water

• The site includes a hydrophobic patch created by hydrophobic

side chains

• The site also includes several hydrogen bond donor or

acceptor groups:
(for lysozyme N59 NH, W62 NH, W63 NH, A107 O, and
Q57 O)
Example 2: Thermolysin

Experimental mapping
English, et al. Proteins 37,
628-640 (1999) Protein Eng. 14,
47-59 (2001).

Probes:
Isopropanol (IPA)
Acetone (ACN)
Acetonitrile (CCN)
Phenol (IPH)
All in the S’1 pocket
Thermolysin – Computational Mapping

Consensus sites 1 and 2

Obtained by the CS-Map
algorithm

Dennis, S., Kortvelyesi T., and Vajda. S.

Computational mapping identifies the
binding sites of organic solvents on
proteins. Proc. Natl. Acad. Sci. USA.,
99: 4290-4295, 2002.

Kortvelyesi, T., Dennis, S., Silberstein, M.,

Brown III, L., and Vajda, S. Algorithms for
computational solvent mapping of proteins.
Proteins. 51: 340-351, 2003.
Comparison of mapping results to contacts in the PDB
Hydrogen bonds in thermolysin
Textbook-type representation of H-
Bonds
Why does CS-Map give better results than
earlier methods ?

1. Improved sampling of the regions of interest

2. A scoring potential that accounts for desolvation

3. Clusters are ranked, not individual conformations

4. Consensus site: The binding of different solvents reduces

the probability of finding false positives
 Detection of Hot Spots within Druggable
Binding Pockets by CS-Map

• Purpose of study: To determine the

predictive power of CS-Map toward the
identification of hot spots within a
binding pocket

• Comparisons are based on known

ligand interactions and NMR data
 Part 1: Identification of hot spots in peptide
binding pocket of Renin
• Major target for the
treatment of hypertension

• Over 25 years of research

into small molecule inhibitors

• Most inhibitors are

peptidomimetics

•Novartis in Phase III trials of

Aliskiren, a novel non-
peptidomimetic renin http://www.merck.com/mmhe/sec03/ch022/ch022a.html
inhibitor
 Part 1: Identification of hot spots in peptide
binding pocket of Renin

•First orally available inhibitor, Aliskiren, binds in a different

conformation than peptidomimetic inhibitors
-Wood, JM. et. al. Biochem. Biohphys. Res. Commun. 308(4): 698-705 (2003
•Used the GOLD algorithm for docking
-Verdonk, M.L, et. al. Proteins. 52:609-623 (2003)
 Identification of Peptide Binding Pocket by
CSMap

1RNE
1BIL
1BIM
1HRN
2REN

Top two consensus sites for each structure

are located in the binding pocket
 Identification of Preferred Binding
Modality of Aliskiren using CSMap
S2
S1’
S4

S3SP

S3 S1 S2’

PDB/Subsite S4 S3 S2 S1 S1’ S2’

1RNE NP 1(28) NP 1(28) 6(7) 2(15)
1HRN NP 1(24) 4(12) 1(24) 4(12) 2(15)
1BIL NP 1(19) NP 1(19) NP 2(15)
1BIM NP 1(24) NP 1(24) 2(14) 2(14)
2REN NP 1(11) NP 2(8) NP NP
 CS-Map Based Identification of Hot Spots
in Peptide Binding Pocket of Renin
18
S1 CS-Map Probes
Percent Atom Interactions (%)

Peptidomimetics
Aliskiren

S3SP S2
12
S1
S2’ S1
S2
6
S3 S4

S1’

0
W42

Q13
P113
R77

L215
D35

L116

Y157

T218

Y222

T296

T299
T15

Y17

T36

Y78

T80

A220

I292
G16

F34

S38

S79

F114

A117
F119
V122

D217

S221

A304
V22

G37

G81

G219

D291

G297
Residue

•Atom-Based Interactions calculated using HBPlus

I.K. McDonald and J.M.Thornton. J. Mol. Biol. 238:777-793 (1994)
•Pearson Correlation between Probes & Aliskiren = .73
•Pearson Correlation between Probes & Peptidomimetic = .17
Conclusions IV

Conclusions: Part 1
• Mapping results indicate the druggable pockets in the renin active site

• Pockets S2 and S4 are not “hot spots” and should not be targeted.

• The most important pockets are S1 and S3

• Pockets S1’ and S2’ are of intermediate importance, but contribute to the
binding.

• Some of these regions, primarily S2’, is not utilized by Aliskiren, suggesting

that a higher affinity drug may be developed.
 Ketopantoate Reductase

*Figures reproduced from Ciulli, et. al.

• NMR studies of E.coli Ketopantoate Reductase using NADPH

fragments and co-factor analogues revealed two hot spots located
on opposite ends of the NADPH binding region
-Ciulli, et. al. J.Med. Chem. 2006 Vol. 49

•Mutational analysis of residues on opposite ends of the binding

region, R31 and N98, confirmed these results.
 Mapping Results: Ketopantoate
Reductase
N98

R31

•Mapping analysis of three CS-Map Results

(% Interaction/Residue):
structures, PDB IDs 1YJQ, Red: 4.15/residue
1YON, 1KS9, yielded hot Green: 4.13/residue
spots on either end of the White: 2.52/residue
NADPH binding region, in Blue: 4.63/residue
agreement with the
experimental study
 Part 2: Hot Spot Identification for Proteins
used in NMR druggability study

Protein Druggable Pocket(s) PDB ID Predicted Hot Spots/Verified

FKBP FK-506 1FKJ 8/8
Akt-PH IP3 1H10 8/8
ErmAM SAH 1QAM 5/6
MDM2 P53 1YCR 7/7
MurA UDPNAG 1UAE 6/9
PTP1B Catalytic pTyr, non- 1PH0 8/9
catalytic pTyr
SCD Substrate 1G4K 7/8
UK Peptide 1FV9 7/7
LFA IDAS 1RD4 9/10
NMR study published by Hajduk, et. al. J. Med Chem. 2005

*Verified by structural and/or NMR data

Example from Study: FK-506 Binding Protein

Important Target for Immunosuppression

CS-Map Results Correspond to NMR data
Shuker, S.B., et. al. Science 274(5292): 1531-4 (1996)
Comparison of Residue Interactions between
CS-Map Probes and Bound Ligands

18
Percent Atom Interactions (%)

CS-Map Probes
Bound Ligands
15

12

0
E54

I56

H87
I90
I91
L97
Q53

W59
F46

R57
Y26

F36
D37
R42

F48

V55

A81
Y82

F99
G28

Residue
 Conclusions: Part 2

• CS-Map is capable of determining hot

spots within binding pockets of
druggable proteins, supported both by
NMR and structural data
 General Conclusions and Future Directions

• The computational prediction of residues important

for ligand binding is crucial to structure-based drug
design efforts, as well as providing further insight into
protein-ligand interactions.

• Future work will focus on the use of CS-Map derived

data to predict hot spots on proteins for which no
experimental binding data exists, namely to build
pharmacophore models of ligand interactions and to
predict hydrogen bonding patterns.
 Many Thanks
The Vajda Group: This work was funded by
Melissa Landon National Institutes of Health
Karl Clodfelter SolMap Pharmaceuticals
Jessamin Yu
Spencer Thiel
David Lancia, Jr.

SolMap Pharmaceuticals:
Frank Guarnieri
Patrick Devaney