Design Of New Rac1 Inhibitors Through Computational

Approaches

Alessandro Contini, Nicola Ferri, Stefano Stragliotto – CDDD – L’Aquila 2011

What is Rac1?

-signal transducer

- GTPase

-activated by GEFs

-acts through effectors

Rac1 regulates:[1]

a) cellular mobility

b) cellular proliferation

c) superoxide production

[1] Bosco, E.E.; Mulloy, J.C.; Zheng, Y. Cell. Mol. Life Sci. 2009, 66, 370.

[2] Parri, M.; Chiarugi, P. Cell Communication and Signaling 2010, 8, 23.

[3] Sawada N.; Li Y.; Liao, J.K. Curr Opin Pharmacol. 2010, 10, 116.

Implication of Rac1 in cancer and cardiovascular disease![2,3]

Rac1 is activated by GEF

[4] Gao, Y.; Dickerson, J. B.; Guo, F.; Zheng, J.; Zheng, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7618.

GEF selectivity:

Tiam is the only Rac1 specific GEF (PAK → cytoskeleton reorganization)

NCI

DB

N

NH2 HN

N

NHN

N

NSC23766 [4]

Identification of Rac1 inhibitors: the library[5]

[5] Ferri, N.; Corsini, A.; Bottino, P.; Clerici, F.; Contini, A. J. Med. Chem. 2009, 52, 4087

The "mp3" compression…

1024 molecules with RMSD 0.1-1 Å

FP:BIT_MACCS

tanimoto index=0.8

overlap= 50%

lowest MW

Ph4 filtration

1.5 ≤logP(o/w) ≤4

643 molecules for docking

x: molar refractivity y: HB acceptor

z: logP(o/w)

Identification of Rac1 inhibitors: consensus docking

The receptor:

Rac1-NSC23766

complex

X-ray

coordinates "transcription"

Ph4 Hits

(643)

The docking:

MOE: TM (engine)

Affinity + LdG (scoring)

AD4: 80x80x80, spc 0.175 Ǻ

pop_size 150

ga_num_evals 500000

GA-LS runs 20

106 compounds

affinity < -4.2,

London dG < -7.5,

AD4 < -6.4 kcal/mol

(thresholds from

NSC23766)

Visual inspection Purchased

Hits

(50)

The results:

33 more active than

NSC23766 (11.1%)

5 with > 45% inhibitory

effect

G-LISA bioassay

selectivity specificity

N SO2

NHO

O

NHN

H2N SO2

NH

O

O

ON

4(45.8% inhibition)

5(65.6% inhibition)

MD

Amber ff03

TIP3P box

Current development: hit-to-lead optimization

bioassays

NH

O

YX

R2

R1

scale-up In vivo

Ph4, QSAR synthesis

Ar3

Ar4

docking NH

O

YX

R2

R1

similarity search G-LISA

Ar1

Ar2

Focussed library and method optimization

N SO2

NHO

O

NHN

H2N SO2

NH

O

O

ON

4

5

ZINC

DB

similarity search

170 cmpds

similarity=85%

NH

O

YX

R2

R1

consensus docking

56 cmpds purchased

NH

O

YX

R2

R1

G-LISA bioassay

active

(31)

inactive

(25)

Rate of acceptance:

active compounds (78) : 99%

decoys (374): 16%

New Ph4 model

Placement: Alpha Triangle

Scoring: Affinity dG

Refinement: MMFF94x

(5000 iteration, rmsd 0.001)

Rescoring: Affinity dG

Ph4 filtration

QSAR filter

binary model, r2=0,68

Improved docking protocol

Virtual library generation: COMBIGEN

SCAFFOLD-An + A0-R SCAFFOLD-R

Two different database of –R groups:

1. –R groups taken from molecules with

known activity

2. –R groups taken from most common

functional groups (i.e. hydroxil, alkyl,

halogen…)

An ATTACHMENT POINT

301 MOLECULES

2234 MOLECULES

Virtual library generation: BREED

“crossover” scheme for the generation of new ligands

1. Molecules with known activity are aligned

2. Substituents on superposed bonds are exchanged

Bond 3D superposition

exchange

1153

NEW

MOLECULES

molecules aligned in groups of 5

process reiterated until no new

molecules were generated

Virtual library screening

301

–R groups from

known molecules 2234

–R groups from

common funct. groups

1153 BREED

Eg(MOE)<-6.0 kcal/mol Eg(ADT)<-9.0 kcal/mol

Filtration by property

(Oprea, nonreactive, 2.1<logP<4.2)

5 29

17

COMBIGEN

Filtration by docking + Ph4

Filtration by QSAR

($PRED ≥0.5)

Synthesis of new compounds

+

TEA

CH2Cl2

Pharmacology, preliminary results, SMCs

Pharmacology, preliminary results, cancer lines[6]

[6] Prof. David Williams, Children’s Hospital, Boston MA

Acknoledgements

The modelers, actual The modelers, former

Degree thesis on Rac1:

Paolo Bottino

Stefano Assolari

Chiara Gregorio

Giada Maggioni Synthesis:

Dr. Emanuela Erba

Prof. Francesca Clerici

Fundings:

MIUR: FIRB – progetto giovani 2009

Regione Lombardia: Progetto Astil

Pharmacology:

Prof. Alberto Corsini

Prof. David Williams