Download

Download Full Text (2.3 MB)

Publication Date

4-29-2020

Disciplines

Internal Medicine

Abstract

Drugging Chemokine Receptors: Biased CXCR3 Agonists Differentially Regulate Chemotaxis And Inflammation

Jeffrey Smith, MD, PhD

Providence Portland Medical Center – Portland, OR

Additional Authors: Dylan Eiger, BS; Chia-Feng Tsai, PhD; Lowell Nicholson,MD; Rachel Glenn, BS; Priya Alagesan, BS; Amanda MacLeod, MD; John Jacobs, PhD; Tujin Shi, PhD; Sudarshan Rajagopal, MD,PhD

Introduction: G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and the target of ~30% of FDA approved drugs. It is now well established that GPCRs can signal through multiple transducers, including classical heterotrimeric G proteins but also GPCR kinases and β-arrestins (1). While these signaling pathways can be activated or blocked by ‘balanced’ agonists or antagonists, they can also be selectively activated in ‘biased’ responses. This new GPCR signaling paradigm of ‘biased signaling’ heralds drugs with increasing efficacy and fewer side effects (2). With over 50 ligands and 20 receptors, biased agonism is prominent within the chemokine system. Here, ligands and receptors bind one another with significant redundancy. For example, the GPCR CXCR3 is expressed on activated T cells and has three established endogenous ligands: CXCL9, CXCL10, and CXCL11 (3,4). Despite its role in inflammation, infectious disease, and cancer (5), no approved drugs target CXCR3. The purpose of my research is to measure biased signaling at CXCR3 and assess the therapeutic potential of selectively targeting certain CXCR3 signaling pathways with biased agonists.

Methods: Utilizing state-of-the-art transcriptomic and phosphoproteomic analyses (6), we show vast differences in CXCR3-regulated intracellular signaling. Responses were compared between vehicle, CXCL9, CXCL10, or CXCL11 treatment assessing >5,000 unique phosphopeptides and >13,000 genes. Biased responses were assessed in both immortalized cell lines and primary human T cells. Utilizing various second messenger reporter systems and bioluminescence resonance energy transfer assays (2,3), we identify an important proximal GPCR signaling pathway, β-arrestin, and demonstrate that CXCL11 acts as a β-arrestin-biased agonist at CXCR3. Furthermore, we screened small molecules to identify a G protein-biased and a β-arrestin-biased small molecule agonist of CXCR3. We then utilized these small molecules to measure physiological readouts of inflammation and chemotaxis in both mice and patients.

Results: Endogenous chemokines of CXCR3 activate divergent intracellular signaling pathways. Using both chemokines and small molecules, we show that β-arrestin pathway signaling through CXCR3 is necessary for full efficacy chemotaxis of activated T cells in both mice and patients (p<0.05). In addition, a β-arrestin-biased small molecule potentiated the cutaneous inflammatory responses in wild-type mice (p<0.05), but not in either β-arrestin KO (p=0.77) or CXCR3 KO (p=0.72) mice, indicating both CXCR3 and β-arrestin dependence in T cell mediated inflammatory responses.

Conclusions: Here we show that CXCL9, CXCL10, and CXCL11 activate distinct CXCR3 intracellular signaling pathways with divergent physiological effects. We clearly demonstrate that the multiple CXCR3 chemokines, CXCL9, CXCL10, and CXCL11, are not redundant in their CXCR3 signaling properties. We show that non-canonical β-arrestin signaling is necessary for certain CXCR3-regulated inflammatory responses and for chemotaxis in both mice and patients. These data strongly suggest that CXCR3 biased agonists have therapeutic promise to treat inflammatory conditions.

References

  1. Smith, J.S. and Rajagopal, S., 2016. The β-arrestins: multifunctional regulators of G protein-coupled receptors. Journal of Biological Chemistry, 291(17), pp.8969-8977.
  1. Smith, J.S., Lefkowitz, R.J. and Rajagopal, S., 2018. Biased signaling: from simple switches to allosteric microprocessors. Nature Reviews Drug Discovery, 17(4), p.243.
  1. Smith, J.S., Alagesan, P., Desai, N.K., Pack, T.F., Wu, J.H., Inoue, A., Freedman, N.J. and Rajagopal, S., 2017. CXC motif chemokine receptor 3 splice variants differentially activate beta-arrestins to regulate downstream signaling pathways. Molecular pharmacology, 92(2), pp.136-150.
  1. Smith, J.S., Nicholson, L.T., Suwanpradid, J., Glenn, R.A., Knape, N.M., Alagesan, P., Gundry, J.N., Wehrman, T.S., Atwater, A.R., Gunn, M.D. MacLeod, A.S., and Rajagopal, S., 2018. Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation. Sci. Signal., 11(555), p.eaaq1075.
  1. Chow, M.T., Ozga, A.J., Servis, R.L., Frederick, D.T., Lo, J.A., Fisher, D.E., Freeman, G.J., Boland, G.M. and Luster, A.D., 2019. Intratumoral activity of the CXCR3 chemokine system is required for the efficacy of anti-PD-1 therapy. Immunity.
  1. Tsai, C.F.*, Smith, J.S.*, Krajewski, K., Zhao, R., Moghieb, A.M., Nicora, C.D., Xiong, X., Moore, R.J., Liu, T., Smith, R.D. and Jacobs, J.M., 2019. TMT labeling facilitates RPLC-MS analysis of hydrophilic phosphopeptides. Analytical chemistry.

Specialty/Research Institute

Internal Medicine

Specialty/Research Institute

Graduate Medical Education

Conference / Event Name

Academic Achievement Day, 2020

Location

Providence Portland Medical Center, Internal Medicine Residency, Portland, Oregon

Drugging Chemokine Receptors: Biased CXCR3 Agonists Differentially Regulate Chemotaxis And Inflammation

Share

COinS