Structural Basis for Chemokine Function

Medical College Of Wisconsin, Milwaukee WI

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Abstract

PROJECT SUMMARY/ABSTRACT OF THE RESEARCH PLAN The goal of this project is to understand how chemokines bind and activate their cognate G protein-coupled receptors (GPCRs) to aid in development of new molecules that alter chemokine signaling for therapeutic benefit. With 46 chemokine ligands and 23 receptors, the human chemokine network represents the largest GPCR family with respect to the number of endogenous ligands and receptors. Chemokine-receptor specificity varies from strictly monogamous to highly promiscuous, as illustrated in the accompanying illustration. This "selectively promiscuous" signaling network orchestrates the trafficking of leukocytes and other cells in early development, homeostasis and immune responses. GPCRs are well represented in the pharmacopeia and many chemokines have been validated as therapeutic targets, yet only three drugs in this category are approved for clinical use. Biopharma companies and investors suspect that functional redundancy makes the chemokine family effectively undruggable. I posit that promiscuous chemokine-GPCR interactions have non-redundant functions in the mammalian immune system, and that a full understanding of selective promiscuity is essential for successful therapeutic development. As a first aim for the continuation period of this MERIT award, I propose to create The Chemokine Atlas as a comprehensive online resource linking all details of sequence, 3D structure, and quantitative molecular pharmacology for the entire human chemokine network. Using ortholog/paralog conservation and contact network analysis of available structures, we classified each chemokine and GPCR sequence position according to its contribution to ligand-receptor selectivity. We will populate the Atlas with high-quality homology models for every chemokine-GPCR pair and leverage productive collaborations with Andy Chevigne (Luxembourg Institute of Health) and industry partner Protein Foundry to carry out systematic all-by-all functional profiling of the chemokine network. Guided by standardized potency and efficacy values for G protein and B-arrestin signaling, we will work with Aashish Manglik (UCSF) to develop a streamlined process for purification and cryoEM structure determination of all functionally relevant chemokine-GPCR pairs. Aim 2 will test the hypothesis that 2D NMR is a sensitive tool for GPCR pharmacotyping. Ongoing NMR studies of the conformational dynamics of atypical chemokine receptor 3 (ACKR3) activation and inhibition will be expanded to include several types of non-chemokine ligands. Engineered nanobodies, synthetic and endogenous opioid peptides (recently discovered to be ACKR3 ligands by the Chevigne group), and photoswitchable small molecules from the lab of Rob Leurs (VU Amsterdam) will be compared to NMR spectral signatures of chemokine agonist, nanobody antagonist and unliganded receptor. We will identify NMR signatures that discriminate between the binding of ACKR3 superagonists, full agonists, partial agonists, antagonists and inverse agonists. To delineate G protein- and B-arrestin-specific conformational dynamics, the NMR activation and inhibition profiles of chemokine receptors CXCR3 and CXCR4 will be compared with B-arrestin-biased ACKR3. Our third aim will test the hypothesis that an ACKR3 nano body antagonist can be converted to an agonist using a structure-guided design process. Our solved NMR structure, mutational analysis, and homology modeling suggest that the extended CDR3 loop of VUN701 (from Martine Smit, VU Amsterdam) is a stable Bhairpin that occupies the chemokine binding pocket and makes specific contacts that prevent ACKR3 activation. We will solve cryoEM structures of ACKR3-nanobody complexes to validate our mechanistic models. Functional redesign of VUN701 will establish this nanobody as a development scaffold for GPCR research tools or drug candidates with tunable pharmacology and programmable target selectivity.