DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN - PowerPoint PPT Presentation

DRUG DISCOVERY AND DEVELOPMENT: THE BICYCLAM AMD3100, A CASE IN POINT Erik DE CLERCQ Rega Institute for Medical Research, K.U.Leuven B-3000 Leuven, Belgium

LAV Replication, tested by reverse transcriptase (RT) activity in supernatants of T cell cultures, during HPA 23 treatment of a patient with AIDS HPA-23: (NH 4 ) 18 (NaW 21 Sb 9 O 86 ) 17 3 0 HPA 23 HPA 23 2 0 (cp m ) y t vi i act 1 0 3 R T x I 1 0 NS NS NS 0 July Aug Dec Nov 84 May 83 Sep Oct Nov Jan 84 The two periods of treatment were from July 27 to August 20, 1983, and from October 17 to December 8, 1983. Cumulative doses were, respectively, 890 mg and 1980 mg. HPA 23 doses were slightly increased from 1 mg/kg to 3.3 mg/kg. Solid columns show RT activity (NS = no virus detectable). Rozenbaum et al ., Lancet i: 450-451 (1985)

JM1493 H 4 SiW 12 O 40 De Clercq, Clin. Microbiol. Rev. 8, 200-239 (1995)

JM1590 K 13 [Ce(SiW 11 O 39 ) 2 ].26H 2 0 De Clercq, Metal-Based Drugs 4, 173-192 (1997)

JM2820 De Clercq, Clin. Microbiol. Rev. 8, 200-239 (1995)

Anti-HIV activity of polyoxometalates Compound EC 50 CC 50 Selectivity (µg/ml) (µg/ml) index JM1583: K 5 [BW 12 O 40 ] 1.4 654 467 JM1590: K 13 [Ce(SiW 11 O 39 ) 2 ].26H 2 O 0.7 230 328 JM1591: K 12 H 2 P 2 W 12 O 48 .24H 2 O 0.3 339 1130 JM1596: K 10 [P 2 W 18 Zn 4 (H 2 O) 2 O 68 ].20H 2 O 0.7 466 666 JM1809: K 8 HP 2 W 15 V 3 O 62 .34H 2 O 1.1 293 266 JM2766: K 6 [BW 11 Ga(H 2 O)O 39 ] 2.8 > 500 > 178 JM2815: K 5 [SiW 11 (C 5 H 5 )TiO 39 ] 1.9 > 500 > 263 JM2820: [Me 3 NH] 8 [Si 2 W 18 Nb 6 O 77 ] 3.2 > 500 > 156 De Clercq, Metal-Based Drugs 4, 173-192 (1997)

Anti-HIV activity of metalloporphyrins R R N N M N N R R R M EC 50 CC 50 Selectivity (µg/ml) (µg/ml) index H 2 0.9 23 25 Fe 0.5 > 90 > 180 COOH Ni 1 > 90 > 90 Song et al ., Antiviral Chem. Chemother. 8, 85-97 (1997)

M NH HN N N M NH HN N N 4H Metal-cyclam complex Cyclam

Anti-HIV activity of JM1657 in MT-4 cells Compound Structure EC 50 (µM) CC 50 (µM) HIV-1(III B ) HIV-2(ROD) H H JM1657 0.144 1.01 319 NH HN NH HN NH HN NH HN NH HN JM1498 399 150 1248 NH HN De Clercq et al ., Proc. Natl. Acad. Sci. USA 89: 5286-5290 (1992)

Anti-HIV activity of JM2763 in MT-4 cells Structure EC 50 (µM) CC 50 (µM) HIV-1(III B ) HIV-2(ROD) 0.248 1.00 > 622 NH N N HN NH HN NH HN De Clercq et al ., Proc. Natl. Acad. Sci. USA 89: 5286-5290 (1992)

Anti-HIV activity of JM2987 in MT-4 cells Structure EC 50 CC 50 Selectivity (µg/ml) (µg/ml) index HIV-1(III B ) 0.005 > 500 > 100,000 NH N N HN NH HN NH HN 8 HBr 2 H 2 O De Clercq et al ., Antimicrob. Agents Chemother. 38: 668-674 (1994)

Anti-HIV activity of bicyclam JM2987 Virus Strain Cell line EC 50 CC 50 Selectivity (µg/ml) (µg/ml) index HIV-1 III B MT-4 0.005 > 500 > 100,000 RF MT-4 0.001 > 500 > 500,000 HE MT-4 0.003 > 500 > 167,000 HIV-2 ROD MT-4 0.007 > 500 > 71,400 EHO MT-4 0.004 > 500 > 125,000 SIV MAC-251 MT-4 > 100 > 100 AGM-3 MOLT-4 > 100 > 100 MND-GB1 MOLT-4 > 100 > 100 De Clercq et al ., Antimicrob. Agents Chemother. 38: 668-674 (1994)

NH N N HN NH HN NH HN 8 HCl 2 H 2 O JM3100 AMD3100 1,1’-[1,4-phenylene-bis(methylene)]-bis(1,4,8,11-tetra- azacyclotetradecane) octahydrochloride dihydrate

Mutations detected in gp120 of bicyclam-resistant NL4-3 viruses: N 269 Y R 272 T S 274 R Q 278 H I 288 V N 293 H A 297 T ∆ ∆ ∆ ∆ FNSTW P 385 L Q 410 E S 433 P V 457 I Passage Fold resistance to Mutations detected ∆ FNSTW no JM2763 JM3100 N 269 Y R 272 T S 274 R Q 278 H I 288 V N 293 H A 297 T P 385 L Q 410 E S 433 P V 457 I JM2763 resistant 0 0 + + 0 0 0 0 0 0 0 0 16 2 3 0 0 + + + 0 0 + 0 0 0 0 25 200 11 0 0 + + + 0 + + + 0 0 0 28 200 6 JM3100 resistant 0 0 + + + 0 + 0 + + 0 0 28 450 7 0 0 + + + 0 + + + + + 0 0 + + + + + + + + + + + 42 > 740 60 + + + + + + + + + + + + 60 > 740 200 De Vreese et al ., Antimicrob. Agents Chemother. 41, 2616-2620 (1997)

Mechanism of action of bicyclams: interaction with monoclonal antibody binding to CXCR4 . A M D3100 25 µ µ µ µ g/m l i sotype 1 2 G 5 R 0 .5 % 8 9 % 1 % E B M FI 23 M FI 3 M FI 4 M U N L L E AM D3100 1 µ µ g/m l 2 µ µ g/m l A M D3100 5 µ µ g/m l µ µ µ µ µ µ C A M D3100 0. E V I T 2 % 4 7 % A 1 % L E M FI 4 M FI 10 M FI 4 R R CXCR- 4 EXPRESSI O N Schols et al ., Antiviral Res. 35: 147-156 (1997)

Anti-HIV activity profile of AMD3100 correlated with coreceptor use Strain Coreceptor used EC 50 (ng/ml) SDF-1 α α α α AMD3100 RANTES T-tropic HIV-1 IIIB CXCR4 2 20 > 1,000 HIV-1 RF CXCR4 5 50 > 1,000 HIV-1 NL4-3 CXCR4 3 100 > 1,000 HIV-2 ROD CXCR4 7 55 > 1,000 M-tropic HIV-1 BaL CCR5 > 25,000 > 1,000 25 HIV-1 SF-162 CCR5 > 25,000 > 1,000 5 HIV-1 ADA CCR5 (CCR2b, CCR3) > 25,000 > 1,000 10 HIV-1 JR-FL CCR5 (CCR2b, CCR3) > 25,000 > 1,000 4 Schols et al ., J. Exp. Med. 186: 1383-1388 (1997)

Concentration-dependent inhibition of SDF-1-induced Ca 2+ flux in Sup-T1 cells by AMD3100 2000 Fluorescent change (counts) 1500 1000 500 0 -500 -1000 0 50 100 150 200 250 300 Time (seconds) Control AMD3100 200 ng/ml AMD3100 40 ng/ml AMD3100 8 ng/ml De Clercq, Int. J. Antimicrob. Agents 18: 309-328 (2001)

Correlation between inhibitory effects of AMD3100 on HIV-1 replication, CXCR4 mAb binding and SDF-1-mediated signal transduction 120 — ■ ■ — Binding of CXCR4 mAb ■ ■ — ■ ■ — SDF-1-induced Ca 2+ flux ■ ■ — ● ● ● — HIV-1 NL4.3 replication ● 100 80 % of inhibition 60 40 20 0 0 1000 100 10 1 0.1 Concentration of AMD3100 (ng/ml) De Clercq, Mol. Pharmacol. 57: 833-839 (2000)

Concentration-dependent inhibition of SDF-1/CXCR4-mediated intracellular calcium flux by AMD3100 in U87.CD4.CXCR4 cells U87.CD4.CXCR4 6000 Fluorescence change (counts) 5000 Control 5000 ng/ml 4000 1000 ng/ml 3000 200 ng/ml 40 ng/ml 2000 1000 0 -1000 0 50 100 150 200 250 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

Concentration-dependent inhibition of SDF-1/CXCR4-mediated intracellular calcium flux by AMD3100 in freshly isolated human PBMCs PBMCs 3500 Fluorescence change (counts) Control 2500 1000 ng/ml 100 ng/ml 10 ng/ml 1500 1 ng/ml 500 -500 0 50 100 150 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

Concentration-dependent inhibition of SDF-1/CXCR4-mediated intracellular calcium flux by AMD3100 in human T-lymphoid HSB-2 cells HSB-2 3500 3000 Fluorescence change (counts) Control 2500 1000 ng/ml 2000 200 ng/ml 1500 40 ng/ml 8 ng/ml 1000 500 0 -500 0 50 100 150 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

Concentration-dependent inhibition of SDF-1/CXCR4-mediated intracellular calcium flux by AMD3100 in murine B-lymphoblastic leukemia L1210 cells L1210 7000 6000 Fluorescence change (counts) Control 5000 1000 ng/ml 200 ng/ml 4000 40 ng/ml 3000 8 ng/ml 2000 1000 0 -1000 0 50 100 150 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

Lack of inhibitory effect of AMD3100 (25 µg/ml) on chemokine-induced signaling mediated by CXCR1, CXCR2, CXCR3, CCR1, CCR2 and CCR3 5500 3000 CXCR1 CXCR2 CXCR3 2500 2000 3500 1500 1000 Fluorescence change (counts) 1500 500 0 -500 -500 -1000 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 2000 4500 CCR1 CCR2 CCR3 2500 1500 3500 1000 1500 2500 500 1500 500 0 500 -500 -500 -500 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

Lack of inhibitory effect of AMD3100 (25 µg/ml) on chemokine-induced signaling mediated by CCR4, CCR5, CCR6, CCR7, CCR8 and CCR9 9500 3000 CCR4 CCR5 CCR6 5500 2500 7500 2000 5500 3500 1500 Fluorescence change (counts) 1000 3500 1500 500 1500 0 -500 -500 -500 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 1200 CCR7 CCR8 CCR9 1000 4000 4000 800 3000 3000 600 2000 2000 400 1000 1000 200 0 0 0 -1000 -200 -1000 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 Time (seconds) Hatse et al ., FEBS Lett. 527, 255-262 (2002)

AMD3100/CXCR4 Interactions Hatse et al ., Mol. Pharmacol. 60: 164-173 (2001)

Correlation of the anti-HIV-1 (NL4-3) activity and inhibitory effect of different ! : SDF-1 α α α α bicyclam analogues ( ) on binding of CXCR4 mAb (12G5) ! ! ! Esté et al ., Mol. Pharmacol. 60: 67-73 (1999)

Correlation of the anti-HIV-1 (NL4-3) activity and inhibitory effect of different bicyclam analogues ( ) on SDF-1 α α α -dependent intracellular Ca 2+ flux α Esté et al ., Mol. Pharmacol. 60: 67-73 (1999)