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