German Edition: DOI: 10.1002/ange.201607831 Membrane Proteins International Edition: DOI: 10.1002/anie.201607831 Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3 Lampros Milanos, Noureldin Saleh, RalfC. Kling, Jonas Kaindl, Nuska Tschammer, and Timothy Clark* Abstract: The chemokine receptor CXCR3 is a G protein- coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8- azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a “shallow” and a second “deeper” pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively. Chemokine receptors, class A G-protein coupled receptors (GPCRs), orchestrate functions of various immune cells in health and disease. [1] Their endogenous ligands chemokines, soluble proteins with sizes up to 10 kDa, guide leukocytes to inflammation sites in a physiological process known as chemotaxis. The human chemokine receptor CXCR3 directs activated T cells to the site of inflammation when bound to its chemokines CXCL9, CXCL10 or CXCL11. [2] CXCR3 is a promising therapeutic target because of its involvement in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis and also cancer. [3] A general “two-step” model has been proposed to describe the chemokine–receptor interactions and activation. Chemokines have been shown to occupy a relatively shallow binding pocket in their receptors. [4] This, however, is not the case for several small molecules that bind to distinct allosteric binding sites in the chemokine receptors, including CXCR3. [5] We recently reported the first b-arrestin biased CXCR3- agonist FAUC1036. [6] Although this biased agonist showed nanomolar potency (EC 50 = 224 nm), it only displaced the radiolabeled negative allosteric modulator RAMX3 [5d] at nearly 19-fold higher concentration (K i = 4.2 mm) (see Scheme 1 for structures of the ligands). [6] We proposed the existence of an alternative binding pocket for FAUC1036, which would explain strong negative cooperativity between the two ligands. [6] Multiple allosteric binding sites have been reported for e.g., free fatty acid 1 receptor FFA1. [7] Muta- genesis experiments indicated a deep binding pocket between the transmembrane helices of CXCR3 for the 8-azaquinazo- linone-based negative allosteric modulators NBI-74 330 [5a] and VUF10085 [8] (analogues of RAMX3). We performed extensive molecular-dynamics (MD) simulations combined with a novel enhanced sampling metadynamics scheme in order to identify the location of the FAUC1036 binding site. We now report the discovery of a surprisingly “shallow” binding pocket for the biased agonist FAUC1036 and an Scheme 1. Small molecules of CXCR3, RAMX3, [5d] NBI-74330 [5a] and VUF10085 [6] are antagonists that share a common core structure, whereas FAUC1036 [2] is a recently discovered b-arrestin-biased agonist. Atoms used to describe the reaction coordinates for RAMX3 and FAUC1036 are highlighted in blue. [*] L. Milanos, N. Saleh, Dr. R. C. Kling, J. Kaindl, Prof. Dr. T. Clark Computer-Chemie-Centrum Friedrich-Alexander-Universität Erlangen-Nürnberg Nägelsbachstr. 25, 91052 Erlangen (Germany) E-mail: Tim.Clark@fau.de L. Milanos, Dr. N. Tschammer Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander-Universität Erlangen-Nürnberg Schuhstraße 19, 91052 Erlangen (Germany) Dr. R. C. Kling Institute of Physiology, Paracelsus Medical University Nürnberg Prof.-Ernst-Nathan-Str. 1, 90419 Nürnberg (Germany) Dr. N. Tschammer Research and Development, NanoTemper Technologies GmbH Floessergasse 4, 81369 München (Germany) Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10. 1002/anie.201607831. A ngewandte Chemi e Communications 1 Angew. Chem. Int. Ed. 2016, 55,1–6  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü