Identification of potent, selective non-peptide CC chemokine receptor-3 antagonist that inhibits eotaxin-, eotaxin-2-, and monocyte chemotactic protein-4-induced eosinophil migration.

Eosinophils have been implicated in the pathogenesis of asthma and other allergic diseases. Several CC chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES (CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating peptides acting through CC chemokine receptor-3 (CCR3). Thus, antagonism of CCR3 could have a therapeutic role in asthma and other eosinophil-mediated diseases. A high throughput, cellular functional screen was configured using RBL-2H3 cells stably expressing CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A small molecule CCR3 antagonist was identified, SK&F 45523, and chemical optimization led to the generation of a number of highly potent, selective CCR3 antagonists including SB-297006 and SB-328437. These compounds were further characterized in vitro and demonstrated high affinity, competitive inhibition of (125)I-eotaxin and (125)I-MCP-4 binding to human eosinophils. The compounds were potent inhibitors of eotaxin- and MCP-4-induced Ca(2+) mobilization in RBL-2H3-CCR3 cells and eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis induced by three ligands that activate CCR3 with similar potencies. Selectivity was affirmed using a panel of 10 seven-transmembrane receptors. This is the first description of a non-peptide CCR3 antagonist, which should be useful in further elucidating the pathophysiological role of CCR3 in allergic inflammatory diseases.

Identification of a truncated form of the CC chemokine CK beta-8 demonstrating greatly enhanced biological activity.

A new CC chemokine, designated CKbeta-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKbeta-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKbeta-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1-99, 24-99, and 25-99 of the secreted chemokine) showed a large variation in potency. CKbeta-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKbeta-8 (25-99) was >2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine's physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKbeta-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKbeta-8 was a very poor chemotactic factor for eosinophils.