AIDS, Vienna 2010

MOAA01 Novel therapeutic strategies

The LEDGINs: rational design of first in

class LEDGF/p75-integrase inhibitorswith potent antiviral activity

Frauke Christ

Molecular Virology and Gene TherapyCatholic University Leuven

Belgium

LEDGF/p75 in HIV replication

Nucleus

Cytoplasm

Reverse transcription complex

Preintegration complex (PIC)

TRN-SR2

LEDGF/p75IN

LEDGF/p75IN

2003 LEDGF/p75 is a co-factor of HIV replication (Cherepanov et al., J. Biol. Chem.)

LEDGF/p75 tethers IN to the chromatin (Maertens et al., J. Biol. Chem. 2003, Ciuffi et al., Nature Medicine, 2005, Llano et al., Science 2006)

2006 Overexpression of the LEDGF/p75 integrase binding domain (IBD) inhibits HIV replication (De Rijck et al., J. Virol.)

Rational design of LEDGINs

CCD+IBD CCD+small molecule

CCD+CCD CCD+CCD

SuperpositionInput Consensus pharmacophore

Collection of features which are present in 3 or more structures resulted in 16 features constructing the initial pharmacophore

From in silico towards in cellulo

CX00287

N ON

OHHN

N

N

N

O

CX00287-6

NH

OO

OHCl

CX00482

Moderate in vitro activity

(36% inhibition at 100µM)

AlphaScreen IC50= 27.27 µM

AlphaScreen IC50= 12.2+/-3.4 µM

MTT/MT-4EC50=41.9+/-1.1µM

CC50>150µM

N ON

OHHN

N

N

N25 initial hit structures

from in silico screening

Molecular modeling

Optimization by medicinal chemistry

Optimization of 2-(quinolin-3-yl)acetic acids (LEDGINs)

AlphaScreenTM (IC50 in µM)ELISA (IC50 in

µM)MTT/MT-4 [µM]

LEDGF/p75-IN

LEDGF/p75-JPO2

LEDGF/p75-PogZ

IN-DNA IN-ININ-Strand transfer

EC50 CC50 SI

CX00287-6 27.27

CX00482 12.2+/-3.4 41.9+/-1.1 >150 >3

CX01978 9.2+/-0.8 >>100 >>100 >>100 >>100 >250 10.8+/-1.1 64.4+/-2.5 6

CX02260 13.2+/-2.8 >>100 >>100 >>100 >>100 >250 12.4+/-1.2 67.3+/-6.5 6

CX04328 1.4+/-0.4 >>100 >>100 >>100 >>100 54.9+/-24.4 2.4+/-0.3 59.8+/-0.5 25

N ON

OHHN

N

N

N

O

NH

OO

OHCl

N O

Cl OH

ONH

OO

OHCl

N

Cl

O

OH

CX00287-6 CX00482 CX01978 CX01978 CX04328

In addition LEDGINs are active in primary PBMCs and macrophages.

LEDGINs inhibit HIV replication at the integration step

0

50000

100000

150000

200000

250000

0 20 40 60 80hours post infection

p2

4 [

pg

/ml]

0

0,5

1

1,5

2

2,5

3

0 10 20 30

hours post infection

co

py

nu

mb

ers

vir

al

DN

A/R

NA

se

P

0

0,5

1

1,5

2

2,5

3

3,5

4

0 20 40 60

hours post infection

co

py

nu

mb

ers

2 L

TR

/RN

As

eP

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60

hours porst infection

cop

y n

um

ber

s in

teg

rate

d

DN

A/R

NA

seP

no inhibition CX04328 raltegravir AZT

Late RT

2LTRs proviral DNA

LEDGINs profile as integrase inhibitors during TOA*

0,00

1,00

2,00

3,00

4,00

5,00

6,00

1 2 3 4 5 6 7 8 9 10 11 12 26

time (hours)

log

( p

24

pg

/ml)

control 1

SIG/DS10000

CX06387(150)

MK-518

NIH/efavi

NIH/AZT

NIH/riton

* Time of addition

In combination experiments strand transfer inhibitors and LEDGINs behave neither synergistic nor antagonistic but additive. LEDGINs might be used in combination therapy.

Crossresistance profile of CX04328

Resistance Virus strainCX004328 raltegravir elvitegravir AZT

AMD3100

Efavirenz

IBD A128T/E170G >17* 1 1 2 1 1

raltegravir E92Q 1 5 6 3 1 1

Q148H 1 9 1 2 1 1

N155H 1 5 3 2 1 1

G140S/Q148H 1 486 154 2 1 1

L74M/F121Y/D232N 1 97 135 1 1 1

AZT RTMC 1 1 1 18 1 1

AMD3100 AMD3100 1 2 1 3 3260 1

Efavirenz A17RIIIB 4 1 1 1 1 23

* fold resistance with respect to the activity against the wt-strain

Resistance selection (CX04328)

125T V K 128A A C 131W

ACA GTT AAG GCC GCC TGT TGG

ACA GTT AAG ACC GCC TGT TGG

ACA GTT AAG ACC GCC TGT TGG

NL4.3 HIV-1 integrase

Passage #18 (13 x IC50)

Passage #29 (17.5 x IC50)

125T V K 128T A C 131W

GAA GTA AAG ATG GTA GCA TGG

125E V K 128M V A 131W

HIV-2 integrase

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40

N° of passages

% r

es

ista

nc

e (

A1

28

T)

(13%)

(63%)

(90%)(99%)

IN A128T

IN A128T (passage #40) is >10 fold resistant to CX04328

LEDGINs are allosteric integration inhibitors

CX04328 binds into a small molecule binding pocket in the dimer interface different from the strand transfer inhibitor binding pocket

1.84 Å co-crystal structure

CX04328 does not alter the overall shape of the IN structure and competes with LEDGF

(gray) for the binding to integrase.

Christ et al., Nat. Chem. Biol. May 2010

Lead optimizationcompound class LEDGF/p75-IN MTT/MT-4

IC50 [µM] EC50 [µM] CC50 [µM] SI [CC50/EC50]

CX06387 A 0,0554+/-0,00052 0,114+/-0,064 131,33+/-22,27 1152CX06458 A 1,57 0,33+/-0 71,66+/-14,3 217CX05272 A 0,75+/-0,44 0,44+/-0,21 80,6+/-30,14 183CX05193 A 0,78 0,53+/-0,07 54,29+/-8,28 102CX07522 D 1,08 0,53+/-0,28 82+/-0 155CX06077 A 1,37 0,78 38,5 49CX05221 A 2,85 0,79+/-0,17 82,94+/-34,16 105CX07707 B 0,99 1,17+/-0,16 43,5+/-9,5 37CX05192 A 0,62 1,21+/-0,37 14,73+/-1,42 12CX06442 B 1,01 1,61+/-0,12 40,33+/-16,92 25CX06579 B 41,8+/-14,2 1,89 76+/-25 40CX06706 B 6,3 2,33+/-1,04 38,33+/-15,63 16CX06645 C 17,85 2,91+/-0,46 38,66+/-15,01 13CX07523 D 4,6 4,5+/-0,3 114,5+/-12,5 25CX06491 C 19,73 5,3 124 23CX05271 A 3,2 5,3 79,8 15CX06116 A 13,46 5,48+/-1,35 58,5+/-25,5 11CX06704 B 7,85 5,51+/-3,98 200+/-48,56 36CX06542 C 10,88 5,97 144 24CX07486 C 5,92 7,93+/-2,6 72,5+/-9,5 9CX06544 C 13,38 13,42 143 11CX06581 B 62,67 16,41 56 3CX06703 B 17,68 21,16+/-1,8 127+/-5 6CX05792 B 2,85 23,23 235 10

Multiple small molecules demonstrate a clear SAR.

Conclusions

Rational design has led to the development of LEDGINs, a novel class of antivirals

LEDGINs are potent inhibitors of the LEDGF/p75-IN interaction in vitro and in vivo.

Their lack of cross-resistance to raltegravir and elvitegravir as well as their allosteric nature demonstrate their potential as second generation integrase inhibitors

Medicinal chemistry optimization has led to a clear SAR of several compound classes

Preliminary ADMEtox data, pharmacokinetics, dynamics and metabolism assays demonstrate their potential for further clinical development.

Nucleus

Cytoplasm

Reverse transcription complex

Preintegration complex (PIC)

TRN-SR2

LEDGF/p75IN

LEDGF/p75IN

Thank you….

Molecular Medicine: Belete A. Desimmie, Barbara Van Remoortel, Nam Joo Van der Veken, Zeger Debyser

Department of Pharmaceutical Sciences: Stefan Nicolet, Sergei Strelkov

Laboratory for Biomolecular Modeling: Arnout Voet, Abel Jonckheer, Marc De Maeyer

CD3 Leuven, CISTIM Leuven vzw: Damian Marchand, Arnaud Marchand, Dorothée Bardoit, Patrick Chaltin