Antagonists for Human and Mouse Structure Function Differences in Nonpeptide

A useful strategy for identifying ligand binding domains of G protein-coupled receptors has been the exploitation of species differences in antagonist potencies. We have used this approach for the CCR1 chemokine receptor with a novel series of antagonists, the 4-hydroxypiperidines, which were discovered by high throughput screening of human CCR1 and subsequently opti-mized. The structure-activity relationships for a number of different 4-hydroxypiperidine antagonists for human and mouse CCR1 were examined by receptor binding and functional assays. These compounds exhibit major differences in their rank order of potency for the human and mouse chemokine receptor CCR1. For example, the initial lead template, BX 510, which was a highly potent functional antagonist for human CCR1 ( K i (cid:1) 21 nM) was > 400-fold less active on mouse CCR1 ( K i (cid:1) 9150 nM). However, increasing the length of the linker between the piperidine and dibenzothiepine groups by one methylene group generated a compound, BX 511, which was equipotent for both human and mouse CCR1. These and other analogs of the lead template BX 510, which have major differences in potency for human and mouse CCR1, are described, and a model for their interaction with human CCR1 is presented. The Journal of Immunology, 2003, 170: 1910–1916. environment surrounding CCR1. A distance-dependent dielectric of 2r was used. The receptor-antagonist complex was minimized and subjected to 2 ns of MD using the AMBER 6.0 program package. Harmonic restraints were applied to keep the structure close to the original structure. Harmonic position restraints of 2.0 kcal/mol (cid:1) Å 2 were applied to backbone atoms of the intracellular side of the receptor away from the binding site: residues 53–75, 125–154, 215–245, and 298–345. Restraints of 0.2 kcal/mol (cid:1) Å 2 were applied to the rest of the receptor backbone. After 2 ns of dynamics at 298 K, the structure was minimized for 3000 steps with the same restraints.Thestructures from 1 and 2 ns were compared and were found to be very similar except in the tilt angle of the piperidine ring. After dynamics, the root-mean-squared deviation of the (cid:1) carbons from the initial model was 1.0 Å after 1 or 2 ns. This represents a fairly small deviation in structure from the homology model. A small portion of extracellular loop 2 (ECL2), residues 177–180, deviated more signiﬁcantly (2.9 Å (cid:1) carbon root-mean-squared deviation) during dynamics. This larger deviation may be due to interactions with the antagonist or sequence differences with respect to the rhodopsin structure that was the source of the homology model. The portion of ECL2 that is close to BX 510, residues 182–185, did not deviate signiﬁcantly from its initial position during 2 ns of restrained MD. The only residues showing signiﬁcant shifts in their side chain positions during the MD were Tyr 113 and Trp 252 , which opened up a binding pocket to accommodate the phenylpiperidine moiety. The binding pocket of the dibenzothiepine ring remained very similar to its initial coordinates. The fused ring system itself ﬂexed into several different conformations during the dynamics, but the cyano group remained in a fairly constant position.