Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2

Abstract

The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have provided structural evidence for an allosteric, intracellular binding site. The high conservation of residues involved in this site suggests its presence in most chemokine receptors, including the close homologue CCR1. By using [3H]CCR2-RA-[R], a high-affinity, CCR2 intracellular ligand, we report an intracellular binding site in CCR1, where this radioligand also binds with high affinity. In addition, we report the synthesis and biological characterization of a series of pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several high-affinity intracellular ligands, including selective and potential multitarget antagonists. Evaluation of selected compounds in a functional [35S]GTPγS assay revealed that they act as inverse agonists in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular binding site enables the design of selective and multitarget inhibitors as a novel therapeutic approach.

Synthesis and Pharmacological Evaluation of Triazolopyrimidinone Derivatives as Noncompetitive, Intracellular Antagonists for CC Chemokine Receptors 2 and 5

“…23,24 To test whether they also bind to the intracellular site of the receptor, we synthesized “example 1” from the patent by Bengtsson et al, 23 corresponding to the triazolopyrimidinone derivative 8 in our study (Figure 1). Using a [ 3 H]-CCR2-RA-[ R ] binding assay as previously described, 19 we found that compound 8 fully displaced [ 3 H]-CCR2-RA-[ R ] binding from U2OS cells stably expressing hCCR2b (U2OS-CCR2) with high affinity and a pseudo-Hill slope ( n H ) close to unity, indicating a competitive interaction with [ 3 H]-CCR2-RA-[ R ] for the intracellular binding site. 8 displaced [ 3 H]-CCR2-RA-[ R ] with a p K i of 8.90 ± 0.04 ( K i = 1.3 nM, Figure 2a and Table 1), consistent with its previously reported activity in a CCR2 calcium flux assay (IC 50 = 16 nM).…”

“…Next, inspired by our work on CCR1/CCR2 selectivity of pyrrolone derivatives, 19 we investigated whether aromatic substituents are tolerated in this position. As expected from previous studies, 19,30 the introduction of aromatic groups decreased 20-fold ( 34 , 27 nM), 40-fold ( 36 , 52 nM), and 122-fold ( 35 , 159 nM) the affinity for CCR2 compared to 8 . When tested in CCR5, all derivatives showed a complete loss of activity at 1 μM, indicating that aromatic groups are not favorable for selectivity or dual activity.…”

“…For radioligand binding assays, K i values were determined using the Cheng–Prussoff equation using a K D of 6.3 nM for the radioligand. 19…”

“…Docking was performed in the Schrodinger suite, 54 as previously described for the docking of CCR2-RA-[ R ]. 19 Before docking, the CCR2b crystal structure was prepared by replacing the residues between L226 5×62 and R240 6×32 , which correspond to the M2 muscarinic acetylcholine receptor, with the CCR2b sequence using prime 55−57 and CCR5 as template (PDB code 4MBS). 35 Induced fit docking, with a substructure restraint on the right-hand phenyl (R 1 , SMARTS: “c1ccccc1”) was used to dock compound 43 in the hCCR2b model.…”

“…17,18 In addition, the high conservation of this intracellular site allows for the design of multitarget antagonists. 18,19 Several high-affinity intracellular ligands have been already identified for CCR2 20,21 but not for CCR5, although intracellular compounds developed for CCR2 or CCR4 have been reported to bind CCR5 with much lower potency. 21,22…”