The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 Å and 2.3 Å, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of ␤-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone ␣-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed. Keywords: Neurophysin; crystal structures; unliganded state; vasopressin complexThe protein neurophysin (NP) plays a central role in the targeting of the hormones oxytocin and vasopressin to regulated neurosecretory vesicles and to the storage of the hormones within these vesicles (for review, see Breslow and Burman 1990). Biologically, each hormone is compartmentalized with a separate neurophysin, but the oxytocin-and vasopressin-related neurophysins are very closely related structurally and each reacts similarly with either hormone in vitro. We previously have reported the structure of bovine vasopressin-related neurophysin (BNP-II) bound to oxytocin and to the dipeptide p-iodo-L-phenylalanyl-L-tyrosine amide, which also binds to the hormonebinding site (Chen et al. 1991). However, questions about the relationship between structure and function in this system, particularly as to mechanisms underlying allosteric behavior (e.g., Nicolas et al. 1980;Breslow et al. 1991) and the thermodynamics of ligand binding (e.g., Breslow and Burman 1990) are not answered by the structure of the bound state alone. Neurophysin was probably the first protein for which binding was found to be thermodynamically

Starting with our previously described(20) class of CC chemokine receptor-3 (CCR3) antagonist, we improved the potency by replacing the phenyl linker of 1 with a cyclohexyl linker and by replacing the 4-benzylpiperidine with a 3-benzylpiperidine. The resulting compound, 32, is a potent and selective antagonist of CCR3. SAR studies showed that the 3-acetylphenyl urea of 32 could be replaced with heterocyclic ureas or heterocyclic-substituted phenyl ureas and still maintain the potency (inhibition of eotaxin-induced chemotaxis) of this class of compounds in the low-picomolar range (IC(50) = 10-60 pM), representing some of the most potent CCR3 antagonists reported to date. The potency of 32 for mouse CCR3 (chemotaxis IC(50) = 41 nM) and its oral bioavailability in mice (20% F ) were adequate to assess the efficacy in animal models of allergic airway inflammation. Oral administration of 32 reduced eosinophil recruitment into the lungs in a dose-dependent manner in these animal models. On the basis of its overall potency, selectivity, efficacy, and safety profile, the benzenesulfonate salt of 32, designated DPC168, entered phase I clinical trials.

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b S Supporting Information M acrophages, which are derived from monocytes, a subset of leukocytes, are well-characterized mediators of tissue destruction. These cells can be activated to secrete proinflammatory cytokines such as TNFR and IL-1β, tissue-degrading enzymes such as MMPs, and other chemokines that mediate the influx of other inflammatory cells. 1 Excessive recruitment of these cells to sites of inflammation leads to significant tissue destruction and contributes to the morbidity of chronic inflammatory and autoimmune diseases. The trafficking of monocytes/ macrophages to sites of inflammation is believed to be predominantly mediated by monocyte chemoattractant protein-1 (MCP-1, CCL2) through interaction with its specific receptor, CCR2, which is a member of the super family of seven-transmembrane G-protein-coupled receptors (GPCRs) and is predominantly expressed on monocytes. Binding of MCP-1 to CCR2 induces chemotaxis, resulting in directed migration of monocytes/macrophages to disease sites where MCP-1 expression is elevated. 2 Studies in rodent models have demonstrated the critical role of MCP-1/CCR2 in inflammatory and autoimmune diseases and strongly suggest that CCR2 is an attractive therapeutic target. 3 As a result, inhibition of CCR2 has emerged as a novel therapeutic approach for pharmaceutical research, and a number of potent small molecule CCR2 antagonists have been identified. 4À10 We have reported the discovery of a novel series of CCR2 antagonists through rational design. 10 Our structureÀactivity relationship (SAR) studies on that series of compounds led to the identification of INCB3344 (Figure 1), a potent, selective, and orally bioavailable antagonist of human and murine CCR2 (hCCR2 and mCCR2). INCB3344 has been used as a tool compound for target validation in rodent models because of its potent inhibitory activity toward murine CCR2, its selectivity over other homologous chemokine receptors, and its good pharmacokinetics profile but was not suitable as a clinical candidate due to its moderate hERG activity (IC 50 = 13 μM) as assessed using a dofetilide binding assay, which did not meet our criteria in hERG binding activity. In addition, INCB3344 was an inhibitor of CYP3A4.In the course of SAR studies on the INCB3344 series in an attempt to identify a clinical candidate, we discovered that removal of the ethoxy at the 3-position on the pyrrolidine in the INCB3344 series as in 1 (Figure 2) resulted in a significant loss in mCCR2 activity but retained the hCCR2 activity as in 2 (Figure 2). An R configuration at the 3-position on the pyrrolidine as shown in 2 is required for activity as the S enantiomer of 2 displayed an IC 50 of >1 μM in hCCR2 MCP-1 assay. Although this des-ethoxy series is not superior to the INCB3344 series by comparison of 2 with 1 in hCCR2 activity and hERG binding activity, the 1,3-disubstituted pyrrolidine core in this series offers an advantage over the 1,3,4-trisubstituted pyrrolidine core in the INCB3344 series from a synthetic point of view as the former ...