Structure-Based Identification of Novel Ligands Targeting Multiple Sites within a Chemokine–G-Protein-Coupled-Receptor Interface

Abstract

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…The constraints imposed by the TM domain of the receptor as well as disulfide crosslinks between ACKR3 and CXCL12, suggest that the interaction results in the formation of an anti-parallel β-sheet between receptor and chemokine (Fig. 2d, e) in contrast to the parallel β-sheet suggested by NMR studies (119). The compatibility of this antiparallel orientation with earlier models of CXCR4:CXCL12 (70, 103) also prompted the proposal of a similar extended model for CXCR4:CXCL12 (not shown).…”

“…Mutation or deletion of amino acids within the first 10 residues of the receptor have been shown to affect chemokine binding and/or receptor activation in the case of CXCR4:CXCL12 (12, 141) and CXCR2 with CXCL1, CXCL7 and CXCL8 (62). An NMR structure of CXCL12 in complex with an N-terminal peptide (residues 1–38) from CXCR4 suggests that the distal end pairs with the β 1 -strand of CXCL12 (119), a region also implicated by cross-saturation NMR experiments (67). Mutagenesis and chemical shift perturbation studies of CXCR1:CXCL8 also support a role for the CXCL8 β 1 -strand (59, 78), as well as a potential direct interaction of the chemokine β 1 -strand with the receptor N-terminus (59).…”

Chemokines and their receptors: insights from molecular modeling and crystallography

“…Methyl transferred cross-saturation NMR experiments suggested the involvement of CXCL12 β 1 -strand in its interactions with CXCR4 in 2009 [72]. More recently, the interaction was explicitly observed in the NMR structure of monomeric CXCL12 with an N-terminal peptide of CXCR4 [43], although in this case, the observed β-sheet is parallel (Figure 2D). This discrepancy is likely due to the absence of the context normally provided by the receptor TM domain.…”

“…Unfortunately, these surfaces lack non-polar, enclosed pockets that are generally required for high-affinity binding of small molecules. The scarcity and low affinity of known small molecule binders to chemokines [39, 42, 43] reflects this conceptual limitation.…”

“…Attempts to “divide-and-conquer” the system and to structurally characterize CRS1 interaction by NMR outside of the context of the full-length receptor, using N-terminal receptor peptides [43, 49–51], provided ambiguous results, even in determining the orientation of the bound CRS1 with respect to the chemokine (Figure 2B–D). When analyzed retrospectively, this is because the TM domain of the receptor provides some critical anchoring contacts: without these contacts, the receptor N-terminus is free to bind chemokine in multiple energetically equivalent conformations.…”