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

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 \u3e5,000 unique phosphopeptides and \u3e13,000 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\u3c0.05). In addition, a β-arrestin-biased small molecule potentiated the cutaneous inflammatory responses in wild-type mice (p\u3c0.05), 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 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. 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. 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. 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. 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. 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.https://digitalcommons.psjhealth.org/ppmc_internal/1003/thumbnail.jp

Antibody‐independent targeted quantification of TMPRSS2‐ERG fusion protein products in prostate cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135707/1/mol22014871169-sup-mmc1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135707/2/mol22014871169.pd

Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations

Context: Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitamin D binding protein (DBP) used to calculate f25OHD.  Objectives: Our objective was to assess racial-geographic differences in f25OHD and to understand inconsistencies in racial associations with DBP and calculated f25OHD.  Design: This study used a cross-sectional design.  Setting: The general community in the United States, United Kingdom, and The Gambia were included in this study.  Participants: Men in Osteoporotic Fractures in Men and Medical Research Council studies (N = 1057) were included.  Exposures: Total 25OHD concentration, race, and DBP (GC) genotype exposures were included.  Outcome Measures: Directly measured f25OHD, DBP assessed by proteomics, monoclonal and polyclonal immunoassays, and calculated f25OHD were the outcome measures.  Results: Total 25OHD correlated strongly with directly measured f25OHD (Spearman r = 0.84). Measured by monoclonal assay, mean DBP in African-ancestry subjects was approximately 50% lower than in whites, whereas DBP measured by polyclonal DBP antibodies or proteomic methods was not lower in African-ancestry. Calculated f25OHD (using polyclonal DBP assays) correlated strongly with directly measured f25OHD (r = 0.80–0.83). Free 25OHD, measured or calculated from polyclonal DBP assays, reflected total 25OHD concentration irrespective of race and was lower in African Americans than in US whites.  Conclusions: Previously reported racial differences in DBP concentration are likely from monoclonal assay bias, as there was no racial difference in DBP concentration by other methods. This confirms the poor vitamin D status of many African-Americans and the utility of total 25OHD in assessing vitamin D in the general population

Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrPC) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrPC interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrPC paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrPC and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrPC with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrPSc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrPC organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease