1. AIDS, Vienna 2010 MOAA01 Novel therapeutic strategies The LEDGINs: rational design of first in class LEDGF/p75-integrase inhibitors with potent antiviral activity Frauke Christ Molecular Virology and Gene Therapy Catholic University Leuven Belgium

2. LEDGF/p75 in HIV replication Nucleus Cytoplasm Reverse transcription complex Preintegration complex (PIC) TRN-SR2 LEDGF/p75 IN LEDGF/p75 IN 2003LEDGF/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) 2006Overexpression of the LEDGF/p75 integrase binding domain (IBD) inhibits HIV replication (De Rijck et al., J. Virol.)

3. Rational design of LEDGINs CCD+IBD CCD+small molecule CCD+CCD SuperpositionInputConsensus pharmacophore Collection of features which are present in 3 or more structures resulted in 16 features constructing the initial pharmacophore

4. From in silico towards in cellulo CX00287 CX00287-6 CX00482 Moderate in vitro activity (36% inhibition at 100µM) AlphaScreen IC 50 = 27.27 µM AlphaScreen IC 50 = 12.2+/-3.4 µM MTT/MT-4 EC 50 =41.9+/-1.1µM CC 50 >150µM 25 initial hit structures from in silico screening Molecular modeling Optimization by medicinal chemistry

5. Optimization of 2-(quinolin-3-yl)acetic acids (LEDGINs) AlphaScreen TM (IC 50 in µM) ELISA (IC 50 in µM) MTT/MT-4 [µM] LEDGF/p75- IN LEDGF/p75- JPO2 LEDGF/p75- PogZ IN-DNAIN-IN IN-Strand transfer EC 50 CC 50 SI CX00287-627.27 CX0048212.2+/-3.441.9+/-1.1>150>3 CX019789.2+/-0.8>>100 >25010.8+/-1.164.4+/-2.56 CX0226013.2+/-2.8>>100 >25012.4+/-1.267.3+/-6.56 CX043281.4+/-0.4>>100 54.9+/-24.42.4+/-0.359.8+/-0.525 CX00287-6 CX00482 CX01978 CX04328 In addition LEDGINs are active in primary PBMCs and macrophages.

6. LEDGINs inhibit HIV replication at the integration step no inhibition CX04328 raltegravir AZT Late RT 2LTRsproviral DNA

7. LEDGINs profile as integrase inhibitors during TOA* * 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.

8. Crossresistance profile of CX04328 ResistanceVirus strain CX004328raltegravirelvitegravirAZT AMD 3100 Efavirenz IBDA128T/E170G >17*11211 raltegravirE92Q 156311 Q148H 191211 N155H 153211 G140S/Q148H 1486154211 L74M/F121Y/D 232N 197135111 AZTRTMC 1111811 AMD3100 121332601 EfavirenzA17RIIIB 4111123 * fold resistance with respect to the activity against the wt-strain

9. Resistance selection (CX04328) 125 T V K 128 A A C 131 W ACA GTT AAG GCC GCC TGT TGG ACA GTT AAG ACC GCC TGT TGG NL4.3 HIV-1 integrase Passage #18 (13 x IC 50 ) Passage #29 (17.5 x IC 50 ) 125 T V K 128 T A C 131 W GAA GTA AAG ATG GTA GCA TGG 125 E V K 128 M V A 131 W HIV-2 integrase (13%) (63%) (90%) (99%) IN A128T IN A128T (passage #40) is >10 fold resistant to CX04328

10. 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

11. Lead optimization compound classLEDGF/p75-INMTT/MT-4 IC 50 [µM] EC 50 [µM]CC 50 [µM]SI [CC 50 /EC 50 ] CX06387A0,0554+/-0,000520,114+/-0,064131,33+/-22,271152 CX06458A1,570,33+/-071,66+/-14,3217 CX05272A0,75+/-0,440,44+/-0,2180,6+/-30,14183 CX05193A0,780,53+/-0,0754,29+/-8,28102 CX07522D1,080,53+/-0,2882+/-0155 CX06077A1,370,7838,549 CX05221A2,850,79+/-0,1782,94+/-34,16105 CX07707B0,991,17+/-0,1643,5+/-9,537 CX05192A0,621,21+/-0,3714,73+/-1,4212 CX06442B1,011,61+/-0,1240,33+/-16,9225 CX06579B41,8+/-14,21,8976+/-2540 CX06706B6,32,33+/-1,0438,33+/-15,6316 CX06645C17,852,91+/-0,4638,66+/-15,0113 CX07523D4,64,5+/-0,3114,5+/-12,525 CX06491C19,735,312423 CX05271A3,25,379,815 CX06116A13,465,48+/-1,3558,5+/-25,511 CX06704B7,855,51+/-3,98200+/-48,5636 CX06542C10,885,9714424 CX07486C5,927,93+/-2,672,5+/-9,59 CX06544C13,3813,4214311 CX06581B62,6716,41563 CX06703B17,6821,16+/-1,8127+/-56 CX05792B2,8523,2323510 Multiple small molecules demonstrate a clear SAR.

12. 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/p75 IN LEDGF/p75 IN

13. 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