1. XVIII International AIDS Conference Novel HIV-1 Protease Inhibitors Containing bis-Tetrahydrofuran or a Novel Polycyclic Ligand, Pyranofuran 1 Depts of Hematology and Infectious Diseases, Kumamoto Univ; 2 Kumamoto Health Science Univ; 3 Depts. of Chem & Med Chem, Purdue Univ; 4 Experimental Retrovirology Section, NCI, NIH. Kazuhiko Ide 1, M. Aoki 1,2, Y. Koh 1, M. Amano 1, S. Kulkarni 3, D.D. Anderson 3, B. Chapsal 3, R. Yedidi 4, D. Das 4, A.K. Ghosh 3, and H. Mitsuya 1,4

2. Background HAART made a major impact on the morbidity of HIV-1-infected individuals and significantly extended their survival. The emergence of multi-drug-resistant viruses (HIV-1 MDR ) is still one of the major concerns. Continuous efforts are required to develop more potent and safer therapeutics with high genetic barrier.

3. Darunavir DRV possesses a dual antiviral activity, i.e., protease inhibition and protease dimerization inhibition. Koh & Mitsuya. J Biol Chem. 282:28709-20, 2007 The presence of bis-THF is important for the antiviral activity of DRV. bis-THF bis-tetrahydrofuran Koh, Ghosh & Mitsuya. Antimicrob Agents Chemother 47:3123-3129, 2003

4. GRL-0878 GRL-0888 Major Isomer Minor Isomer bis-THF 5 5 6 6 ( Cis ) ( Trans ) GRL-1388GRL-1398 polycyclic ligand (pyranofuran)

5. DrugEC 50 (nM)CC 50 (μM)* Selectivity Index SQV6.219.73,100 APV19.6>100>5,100 ATV5.127.65,400 DRV3.3>100>30,300 08782.334.615,000 08885.133.66,200 13883.6>100>27,800 13980.237.8186,800 *Selectivity index = CC 50 / EC 50 Four New Compounds Are Comparably or More Potent against HIV Compared to DRV

6. Virus EC 50 (nM) ATVAPVDRV0878088813881398 HIV-1 WT (X4)1.9 (1)35.1 (1)3.5 (1)6.0 (1)6.8 (1)3.2 (1)0.3 (1) HIV-1 MDR/B (X4)228 (120)328.8 (9)19.4 (6)59.1 (10)82.3 (12)4.7 (2)4.8 (16) HIV-1 MDR/C (X4)25.4 (13)265.2 (8)5.2 (1)34.5 (6)58.5 (9)4.0 (1)1.1 (4) HIV-1 MDR/MM (R5)68.9 (36)402.2 (11)21.4 (6)301.0 (50)312.8 (46)21.3 (7)2.6 (9) HIV-1 MDR/EV (R5)39.0 (21)>1,000 (28)214.6 (61)>1,000 (167)>1,000 (147)530.3 (166)23.5 (78) (fold change), blue ： 5-10 times, red ： >10 times GRL-1398 Is More Potent against a Wide Spectrum of HIV MDR Isolates than DRV

7. Virus EC 50 (nM) DRV GRL-1388 GRL-1398 HIV-1 WT 3.3 (1)3.2 (1)0.3 (1) HIV-1 DRV R P10 29.1 (9)24.6 (8)3.3 (11) HIV-1 DRV R P20 214 (65)151 (47)21.9 (73) GRL-1398 Is Active against DRV-resistant HIV Variants (Fold-change)

8. CFP Monomer-monomer Interactions Protease or Polyprotein Dimerization FRET YFP RT Yellow Fluorescent Protein PR F/P Cyan Fluorescent Protein PR AAAAA Spacer pHIV-1 NL43sma 238 AA RT 99 AA Apa FRET-HIV-1 Expression System Protease or polyprotein Monomer Intermolecular FRET (Fluorescence Resonance Energy Transfer)-based If CFP A/B ratios are < 1.0, there is no FRET or dimerization Protease or polyprotein Monomer 1 - 10 nm

9. 1.0 0.9 0.8 0.7 0.6 0.5 1.1 1.2 1.3 1.4 1.5 0.86 CFP A/B ratio NoneGRL-1398GRL-1388DRV 10201 (μM) 1.12 1.02 0.83 1.10 0.96 0.88 0.92 0.85 0.81 GRL-1388 and -1398 have PDI activity Dimerization － Dimerization ＋ 1020110201 p = 0.07 p <0.0001 p = 0.15 p = 0.0005 p = 0.0001

10. Cells used : MT4 A28S/M46I Confer Resistance to GRL-1398 on HIV NL4-3 GRL-1388 GRL-1398 DRV L10F A28S M46I L10F A28S M46I V82I L10F V32I M46L APV L10F V32I L33F M46L I54M A71V Weeks

11. GRL-1398 Resists against Emergence of HIV DRV R with a Mixture of 8 HIV-1 MDR Isolates GRL-1388 GRL-1398 DRV Cells used : MT4 Weeks L10I L33I M36I M46I L63P A71V V82A L90M

12. VAL82 VAL32 ILE84 ASP30 ASP29 ALA28 ASP29’ ASP30’ GLY49 GLY48 ILE47 ASP25 PRO81’ VAL82’ ILE84’ ILE50’ H2OH2O GLY27’ ALA28’ GRL-1398 Has Tight Interactions with PR

13. Summary GRL-1388 and -1398, containing a novel structure-based designed ligand, were comparably or more potent compared to DRV. Although GRL-1398 had moderate PDI activity as compared to DRV, GRL-1398 exhibited more potent anti-HIV activity against DRV- resistant HIV variants.

14. Conclusions GRL-1398 has activity against protease’s enzymatic activity and dimerization activity with high genetic barrier. It is warranted that the compounds, especially GRL-1398, be further studied as potential therapeutics for HIV wt and HIV-1 MDR infection.

15. Thank you for your attention