Volume 24, Issue 7, Pages 858-869.e5 (July 2017) Mechanisms of Skin Toxicity Associated with Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators  Falgun Shah, Antonia F. Stepan, Alison O'Mahony, Sharlene Velichko, Alexandra E. Folias, Christopher Houle, Christopher L. Shaffer, John Marcek, Jessica Whritenour, Robert Stanton, Ellen L. Berg  Cell Chemical Biology  Volume 24, Issue 7, Pages 858-869.e5 (July 2017) DOI: 10.1016/j.chembiol.2017.06.003 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 858-869. e5DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Chemical Structures for mGluR5 NAMs (A) Chemical structures of clinical mGluR5 NAMs AFQ056 (1), ADX48621 (2), RG7090 (3), and AZD9272 (4); (B) Pfizer alkyne-lacking mGluR5 NAMs PF-06297470 (5), PF-06422913 (6), and PF-06462894 (7). Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 BioMAP Profiles for the Pfizer and Clinical mGluR5 NAMs BioMAP Diversity PLUS panel activity profiles for the Pfizer mGluR5 NAMs (A) 5, (B) 6, (C) 7, (D) 1, and (E) 3. These compounds were tested in 12 BioMAP systems (3C, 4H, LPS, SAg, BT, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, MyoF, /Mphg, as shown above) at 30 μM (red), 3.3 μM (orange), 1.1 μM (yellow), and 0.37 μM (green). The protein readouts measured are indicated along the x axis. See STAR Methods for details on readouts measured in each system. The y axis shows the log expression ratios (log10 [parameter value with drug/parameter value of control]) of the readout protein levels relative to the solvent or media controls. Values falling outside of the 95% confidence interval (shown as gray region) are significantly different from control. Data are shown in Table S2. Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Magnified View of Activities of mGluR5 NAMs in BT and LPS Systems A zoomed view of activity of Pfizer mGluR5 NAMs (A) 5, (B) 6, and (C) 7, and clinical mGluR5 NAM (D) 1 and (E) 3 in BioMAP LPS and BT systems. See Figure 2 for additional description. Data are in Table S2. Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 BioMAP Profiles of Select Reference Compounds in BT and LPS Systems (A) Activity profiles are shown for known skin irritants, 2-chloroethyl ethyl sulfide (green), calcitriol (pink), retinol (blue), and all-trans-retinoic acid (cyan), along with 5 (black). The concentrations at which these profiles were generated are shown in the legend; (B) BioMAP activity profiles of the AhR antagonist CH 223191 versus 5 in the BT system. Pearson's r correlation of the comparison profiles was 0.996. See Figure 2 for additional description. Data are in Table S2. Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 TF Profiles of the mGluR5 NAMs 5–7, 10 μM, 24 hr, Are Shown in a Spider Plot TF activities were determined as X-fold of induction values versus vehicle-treated (DMSO) control cells. The spider plot shows an average (n = 2) fold induction data at 24/48 hr plotted in a logarithmic scale. The circle marked with 1-fold change suggests that the modulation of a TF by a compound is no different than the control treatment, whereas circles labeled with 10- or 0.1-fold indicate a 10-fold induction or suppression, respectively. Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Proposed Toxicity Mechanism for mGluR5 NAMs 5 and 6 as an Adverse Outcome Pathway Construct This framework shows potential molecular initiating events identified for 5 and 6 in the present studies, connected through key events to the type IV hypersensitivity skin toxicity outcome observed in NHP. Cell Chemical Biology 2017 24, 858-869.e5DOI: (10.1016/j.chembiol.2017.06.003) Copyright © 2017 Elsevier Ltd Terms and Conditions