Biological characterization of ligands targeting the human CC chemokine receptor 8 (CCR8) reveals the biased signaling properties of small molecule agonists.

The human CC chemokine receptor 8 (CCR8) is a promising drug target for cancer immunotherapy and autoimmune disease. Besides human and viral chemokines, previous studies revealed diverse classes of CCR8-targeting small molecules. We characterized a selection of these CCR8 ligands (hCCL1, vCCL1, ZK756326, AZ6; CCR8 agonists and a naphthalene-sulfonamide-based CCR8 antagonist), in in vitro cell-based assays (hCCL1AF647 binding, calcium mobilization, cellular impedance, cell migration, ß-arrestin 1/2 recruitment), and used pharmacological tools to determine G protein-dependent and -independent signaling pathways elicited by these ligands. Our data reveal differences in CCR8-mediated signaling induced by chemokines versus small molecules, which was most pronounced in cell migration studies. Human CCL1 most efficiently induced cell migration whereby Gßγ signaling was indispensable. In contrast, Gßγ signaling did not contribute to cell migration induced by other CCR8 ligands (vCCL1, ZK756326, AZ6). Although all tested CCR8 agonists were full agonists for calcium mobilization, a significant contribution for Gßγ signaling herein was only apparent for human and viral CCL1. Despite both Gαi- and Gαq-signaling regulate intracellular Ca2+-release, cellular impedance experiments showed that CCR8 agonists predominantly induce Gαi-dependent signaling. Finally, small molecule agonists displayed higher efficacy in ß-arrestin 1 recruitment, which occurred independently of Gαi signaling. Also in this latter assay, only hCCL1-induced activity was dependent on Gßγ-signaling. Our study provides insight into CCR8 signaling and function and demonstrates differential CCR8 activation by different classes of ligands. This reflects the ability of CCR8 small molecules to evoke different subsets of the receptor's signaling repertoire, which categorizes them as biased agonists.

Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.

Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 µM) and CCR8 (EC50: 0.28 µM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.

Multistep continuous-flow synthesis in medicinal chemistry: discovery and preliminary structure-activity relationships of CCR8 ligands.

A three-step continuous-flow synthesis system and its application to the assembly of a new series of chemokine receptor ligands directly from commercial building blocks is reported. No scavenger columns or solvent switches are necessary to recover the desired test compounds, which were obtained in overall yields of 49-94%. The system is modular and flexible, and the individual steps of the sequence can be interchanged with similar outcome, extending the scope of the chemistry. Biological evaluation confirmed activity on the chemokine CCR8 receptor and provided initial structure-activity-relationship (SAR) information for this new ligand series, with the most potent member displaying full agonist activity with single-digit nanomolar potency. To the best of our knowledge, this represents the first published example of efficient use of multistep flow synthesis combined with biological testing and SAR studies in medicinal chemistry.

Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions.

BACKGROUND AND PURPOSE: Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. EXPERIMENTAL APPROACH: The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS: All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS: Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.

Reversed binding of a small molecule ligand in homologous chemokine receptors - differential role of extracellular loop 2.

BACKGROUND AND PURPOSE: The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine-based compound with structural similarity to previously described CCR8-specific agonists, but containing a unique phenyl-tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1. EXPERIMENTAL APPROACH: Single point mutations were introduced in CCR1 and CCR8, and their effect on small molecule ligand-induced receptor activation was examined through inositol trisphosphate (IP(3) ) accumulation. The molecular interaction profile of the agonist was verified by molecular modeling. KEY RESULTS: The chemokine receptor conserved glutamic acid in TM-VII served as a common anchor for the positively charged amine in the piperidine ring. However, whereas the phenyl-tetrazol group interacted with TyrIV:24 (Tyr(172) ) and TyrIII:09 (Tyr(114) ) in the major binding pocket (delimited by TM-III to VII) of CCR8, it also interacted with TrpII:20 (Trp(90) ) and LysII:24 (Lys(94) ) in the minor counterpart (delimited TM-I to III, plus TM-VII) in CCR1. A straightening of TM-II by Ala-substitution of ProII:18 confirmed its unique role in CCR1. The extracellular loop 2 (ECL-2) contributed directly to the small molecule binding site in CCR1, whereas it contributed to efficacy, but not potency in CCR8. CONCLUSION AND IMPLICATIONS: Despite high ligand potency and efficacy and receptor similarity, this dual-active and bitopic compound binds oppositely in CCR1 and CCR8 with different roles of ECL-2, thereby expanding and diversifying the influence of extracellular receptor regions in drug action.

A multistep continuous-flow system for rapid on-demand synthesis of receptor ligands.

A multistep continuous-flow system for synthesis of receptor ligands by assembly of three variable building blocks in a single unbroken flow is described. The sequence consists of three reactions and two scavenger steps, where a Cbz-protected diamine is reacted with an isocyanate, deprotected, and reacted further with an alkylating agent.

Chapter 8. Activation mechanisms of chemokine receptors.

Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand binding. Attempts to unravel the activation mechanism of 7TM receptors have led to the conclusion that activation involves movements of the transmembrane segments VI and VII in particular, as recently gathered in the Global Toggle Switch Model. However, to understand the activation mechanism completely, more research has to be done in this field. Chemokine receptors are interesting tools in this matter. First, the chemokine system has a high degree of promiscuity that allows several chemokines to target one receptor in different ways, as well as a single chemokine ligand to target several receptors in different ways. Second, the endogenous ligands are large proteins that mainly activate their cognate receptors by interacting with various extracellular-located receptor regions. It is, however, also possible to introduce agonism of simple ligands like metal ions. Thus, the chemokine system offers the possibility to test and compare the activation profiles of several chemically diverse ligands. This also brings up the interesting discussion of allosterism, because small molecules in the chemokine field often interact with allosteric receptor sites.