Structure-based ligand discovery for the protein–protein interface of chemokine receptor CXCR4

Abstract

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, proteinrecognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC 50 values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.drug design | virtual screening | promiscuous aggregation G -protein-coupled receptors (GPCRs) play a central role in many normal physiological pathways and altered diseased states, and are the targets of approximately 30% of marketed drugs (1). Ligand discovery against small-molecule GPCRs such as the bioamine receptors has been particularly productive, as have structure-based screens against their crystal structures (2-5). Targeting larger-molecule-recognizing GPCRs has been more difficult. Although multiple reagents are available for lipid and peptidergic GPCRs, their molecular weights are substantially higher than those typical for bioamine receptors, and they are less ligand efficient. This reflects the challenges faced in ligand discovery against peptide-protein and lipid-protein interfaces. These difficulties are still more acute against chemokine GPCRs, which recognize folded proteins of ∼100 amino acids in length and are thus protein-protein interface (PPI) targets (6). Although there are several example drugs in this class, such as maraviroc, plerixafor, and vorapaxar, finding organic molecules with good affinity and the physical properties of oral drugs is notoriously difficult for PPI targets, as reflected in the high molecular weight and hydrophobicity of the few PPI drugs (7).A public competition to predict ligand complexes with the structure of C-X-C chemokine receptor 4 (CXCR4) inspired us to bring structure-based discovery to bear against a key member of the chemokine family (8). CXCR4 natively recognizes the CXCL12 chemokine, an 8-kDa protein. Like many other PPI targets, CXCR4 plays a key signaling role: it is constitutively expres...

Testing inhomogeneous solvation theory in structure-based ligand discovery

“…Following convention, negative GIST energies reflect favorable, costly-todisplace waters. We used adjusted logAUC to measure docking enrichment (39,(42)(43)(44)(45); this metric weights each factor of 10 in docking rank order equally, beginning from the top 0.1%, prioritizing the performance of the very top ranking ligands or decoys in the docking screen (44). Scaled enthalpy performed the best (adjusted logAUC of 57.46 ± 1.84), closely followed by unscaled free energy (56.08 ± 1.42).…”

Fragment-based lead discovery on G-protein-coupled receptors

“…The limited availability of experimental GPCR structures made homology models popular for virtual screening applications. On the other hand, however, recent developments in GPCR structural biology resulted in a high number of GPCR structures initiating a significant number of virtual screening studies (18)(19)(20)(21)(22). Fast and often parallelized docking algorithms allow the prediction of the binding mode for hundreds of thousands of potential ligands in reasonable time.…”

“…Mysinger and colleagues reported a virtual screening-based approach, followed by high concentration testing of hits in a functional assay (20). In this case leadlike (logP<3.5, molar weight<350 Da, number of rotatable bonds<8) compounds were virtually screened followed by testing hits at high concentration (100 µM) in a calcium mobilization and subsequently in a cell migration assay.…”

“…In this case leadlike (logP<3.5, molar weight<350 Da, number of rotatable bonds<8) compounds were virtually screened followed by testing hits at high concentration (100 µM) in a calcium mobilization and subsequently in a cell migration assay. Several hits displayed activity in both in vitro models with no sign of acute toxicity assessed via Trypan Blue exclusion (20). Interestingly, when assessed for binding activity, two of the five hits lacked the ability to displace labeled CXCL12, emphasizing at the complexity of the chemokine-receptor interaction.…”

Seeing CCR5

“…CXCR4 structure-based virtual ligand screening studies exhibited hit rates of 20%-70%. [3][4][5][6] Now, a member of Stevens' original team has used what she learned from the CXCR4 study to tackle CCR5. Beili Wu, a professor at the Shanghai Institute of Materia Medica (SIMM) of the Chinese Academy of Sciences, led her own team and determined the structure of CCR5.…”