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.

Scheme 1. Synthesis Route of Pyrrolones 6–48, with Different R¹, R², and R³ Substituents

Reagents and conditions: (a) acetic acid, reflux for 2–4 h or THF, rt, overnight; (b) Na, EtOH, 0–20 °C, overnight; (c) p-toluenesulfonic acid, 2-propanol, reflux, 48 h.

Figure 1

(a) Homologous displacement curves of 3, 6, and 12 nM [³H]-CCR2-RA-[R] specific binding by increasing concentrations of CCR2-RA-[R] in U2OS-CCR1 at 25 °C. (b) Displacement curves of 6 nM [³H]-CCR2-RA-[R] specific binding by increasing concentrations of SD-24, JNJ-27141491, and BX471 in U2OS-CCR1 at 25 °C. BX471 significantly enhanced the binding of [³H]-CCR2-RA-[R] up to 120%. Statistical significance between binding in absence (100%) and presence of 10 μM BX471 (116 ± 2%) was determined using an unpaired, two-tailed Student’s t-test with Welch’s correction. (c,d) Displacement curves of 6 nM [³H]-CCR2-RA-[R] specific binding by compounds 39, 41, 43, and 45 (b) in U2OS-CCR1 or (c) in U2OS-CCR2 at 25 °C. In the case of U2OS-CCR2, compound 45 did not displace more than 50% of [³H]-CCR2-RA-[R], thus only single-point data at 1 μM is shown. The dashed blue line corresponds to the nonlinear regression fit for compound 45 by GraphPad Prism 7.0. Data shown are mean ± SEM of at least three experiments performed in duplicate.

Figure 2

Proposed binding mode of compound CCR2-RA-[R] in the homology models of CCR1 and CCR2, based on the crystal structure of CCR2 (PDB 5T1A). For CCR1, representative residues are shown as green “sticks” and for CCR2 as orange “sticks”. In all cases, oxygen and nitrogen atoms are represented in red and blue, respectively, and hydrogen bonds with dashed yellow lines. Residues are numbered based on the corresponding residue numbers and with structure-based Ballesteros–Weinstein numbers in superscript.

Figure 3

(a) [³⁵S]GTPγS binding upon stimulation of U2OS-CCR1 and U2OS-CCR2 by increasing concentrations of CCL3 and CCL2, respectively. In both cases, the response was corrected by subtracting the basal activity (approximately 8000 dpm for both CCR1 and CCR2). (b) Inhibition of CCL3-induced [³⁵S]GTPγS binding by compounds 39, 41, 43, and 45 in U2OS-CCR1. (c) Inhibition of CCL2-induced [³⁵S]GTPγS binding by compounds 39, 41, 43, and 45 in U2OS-CCR2. The level of basal activity in U2OS-CCR1 and U2OS-CCR2 is indicated by a dashed line. In all cases, data shown are mean ± SEM of at least three experiments performed in duplicate.