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Session 78 Poster Abstracts
New Antiretroviral Agents: Entry Inhibitors
Wednesday, 1:30 - 3:30 pm
Poster Hall


540    
Determination of Binding Sites of a Unique CCR5 Inhibitor AK602 on Human CCR5
K Maeda*1, H Ogata1, S Harada1, Y Tojo1, T Miyakawa1, H Nakata1, Y Takaoka2, S Shibayama2, K Sagawa2, F Daikichi2, J Moravek3, E Arnold4, and H Mitsuya1,5
1Kumamoto Univ. Sch. of Med., Japan; 2ONO Pharm. Co. Ltd., Osaka, Japan; 3Moravek Biochemicals, Inc., Brea, CA; 4Rutgers Univ., Piscataway, NJ, USA; and 5NCI, NIH, DHHS, Bethesda, MD, USA

Background: A novel spirodiketopiperazine derivative, AK602/ONO4128/GW873140 exerts potent activity against R5 HIV-1 in vitro.  We characterized the CCR5 binding profile of AK602 and its interactions with HIV-1 gp120, CC-chemokines, and CCR5 in comparison with those of other previously published CCR5 inhibitors.

Methods: A variety of mutant CCR5 were expressed on various cell lines and assays for CCR5 binding of CC-chemokines, cytosolic Ca2+ mobilization, and susceptibility to HIV-1 were conducted. Effects of various CCR5 inhibitors on CCR5/gp120 interactions were also examined.  Using selected 3H-labeled CCR5 inhibitors and cells expressing a variety of mutated CCR5, the binding sites of CCR5 inhibitors were determined.

Results: Three selected CCR5 inhibitors (AK602, SCH-C, and TAK-779) exerted potent activity against R5 HIV (IC50; 0.2, 1, and 4 nM) and had high affinity to CCR5 with KD values of 3, 16, and 30 nM, respectively. AK602, unlike SCH-C and TAK-779, completely blocked the binding of an extracellular loop (ECL) 2-specific monoclonal antibodies (45531), suggesting AK602 directly interacts with ECL2.  When mutations were introduced in the interface of ECL2 (G163A/R and K191A), the affinity of AK602 to CCR5 was totally nullified.  Several mutations (e.g., Y108A and E283A) also decreased AK602's affinity to CCR5, a common feature to the binding profiles of TAK-779 and/or SCH-C.  We also determined the effects of CCR5 mutations on CC-chemokine binding/functions and gp120 binding to CCR5.  A variety of mutations in the transmembrane domain of CCR5 and the K191A mutation decreased the affinity of CC-chemokines and/or gp120 binding, indicating that these mutations induce structural/conformational changes in the helices and/or ECL domains.  Taken together, the data suggest that unlike SCH-C and TAK-779 whose binding sites are reportedly located in the transmembrane domains, the binding sites of AK602 are clustered around the interface of ECL2.

Conclusions: The present data suggest that the binding sites of AK602 on CCR5 are substantilly differennt from those of previously published CCR5 inhibitors. The data also might help to construct the optimal combination regimens of CCR5 inhibitors.

Keywords: CCR5 Inhibitor; CCR5; antiretroviral therapy