Supplemental Material: Synthesis of ADX47273 (4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5yl)piperidin-1-yl)methanone, 2: The synthetic scheme for synthesis of ADX47273 is illustrated in Figure 1. The first step required synthesis of Ethyl 1-(4- fluorobenzoyl)piperidine-3-carboxylate (1). To a solution of (s)-nipecotic acid ethyl ester (2.0 g, 12.7 mmol) and DIPEA (3.7 mL, 20.4 mmol) in CH2Cl2 (20 mL) was added 4fluorobenzoyl chloride (2.61 g, 16.5 mmol). The reaction was stirred at room temperature for 18 h. The reaction was washed with 1 N HCl (15 mL) and sat. NaHCO 3 (15 mL). Dried over MgSO4, filtered, concentrated under vacuum and purified by column chromatography (silica gel) using 0 to 40% EtOAc/hexanes to afford 1 (3.35 g, 95%) as a yellow oil; 1H-nmr (400 mHz, CDCl3) δ 7.45-7.38 (m, 2H), 7.11 (t, J = 10.5 Hz, 2H), 4.81-4.32 (m, 1H), 4.28-4.09 (m, 3H), 3.98-3.52 (m, 1H), 3.38 (bd s, 1H), 3.09 (td, J = 13.5, 3.0 Hz, 1H), 2.56 (bd s, 1H), 2.21-2.11 (m, 1H), 1.92-1.72 (m, 2H), 1.281.19 (m, 3H). To a solution of 1 (2.32 g, 8.37 mmol) in THF (20 mL) was added MeOH (4 mL) and a solution of LiOH (1.34 g, 33.5 mmol) in water (4 mL). The reaction was stirred at room temperature for 30 mins. Quenched upon addition of 1 N HCl (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated under vacuum. The residue was dissolved in dioxane (20 mL), added 4-fluoro-N’-hydroxybenzimidamide (1.61 g, 10.0 mmol), EDC (2.33 g, 12.5 mmol) and HOBt (1.13 g, 8.37 mmol) and stirred at room temperature for 6h, then at 80 °C for 18 h. Cooled to room temperature, diluted with EtOAc (80 mL), washed with water (60 mL), 1 N NaOH (60 mL) and brine (80 mL). The organic extract was dried over MgSO4, filtered, concentrated under vacuum and purified by column chromatography (silica gel) using 0 to 45% EtOAc/hexanes to afford 2 (770 mg, 25%) as a clear colorless oil; 1H-nmr (400 mHz, CDCl3) δ 8.16-8.02 (m, 2H), 7.49-7.39 (m, 2H), 7.22-7.05 (m, 4H), 4.81-3.98 (bd m, 2H), 3.52 (bd s, 1H), 3.27 (td, J = 13.5, 3.0 Hz, 2H), 2.36 (dd, J = 9.5, 4.5 Hz, 1H), 2.12-1.82 (m, 2H), 1.72 (bd s, 1H). Figure S1. VU-29 (500 nM) has no effect on A. paired pulse facilitation, B. fiber volley amplitude or C. input-output curves when compared to vehicle treated slices at the Schaffer collateral – CA1 synapse of the rat hippocampus (n = 4; p>0.05). Error bars represent S.E.M. Figure S2. VU-29 has no effect on passive membrane properties recorded from CA1 pyramidal cells in whole-cell voltage clamp mode. Representative traces from cells treated with vehicle or VU-29 (1 µM). Input resistance (100.23±2.7% of predrug), membrane potential (-59.2±1.3mV in the absence of VU-29 vs -59.5±2.0mV in the presence of VU-29), and action potential firing are not affected by pre-treatment of the slice with VU-29 (n = 4). Figure S3. CDPPB enhances mGluR5-mediated ERK1/2 phosphorylation in cortical astrocytes. Secondary rat cortical astrocytes were cultured in 12-well plates in G-5 supplement-containing medium for 3 days and then starved in glutamine-free DMEM for 20 h. The day of assay, cells were incubated with CDPPB (1 µM) for 10 min and then stimulated with DHPG (1 µM) for 10 min. ERK1/2 phosphorylation was measured with Western blotting for dually phosphorylated ERK1/2 and normalized with Western blotting for total ERK1/2 and then expressed as percentage of maximal ERK1/2 phosphorylation. A. Representative Western blot analysis in duplicate and B. densitometry analysis of the modulation of DHPG-induced ERK1/2 phosphorylation by CDPPB from three independent experiments. Error bars represent S.E.M. Figure S4. Synthetic scheme for synthesis of ADX47273 (4-fluorophenyl)(3-(3-(4- fluorophenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone. Figure S5. ADX47273 is a novel mGluR5-selective PAM from a different structural family. Using secondary cultured rat cortical astrocytes, intracellular calcium mobilization responses were measured in response to an EC30 concentration of glutamate in the absence or presence of different concentrations of ADX47273. The EC30 concentration for glutamate was determined each day which was about 300 nM. A. Representative raw fluorescence traces from the calcium mobilization assay. B. Shown is the effect of increasing concentrations of ADX47273 on the calcium fluorescence response to an EC30 concentration of glutamate. Data were normalized to the maximum response of each reaction to 10 µM glutamate. The EC50 value for ADX47273 was determined to be 108 ± 41 nM. Concentration-response curves were generated from three independent experiments each performed in duplicate. Error bars represent S.E.M. Figure S6. ADX47273 does not potentiate mGluR1-, -2-, or -4-mediated responses. A concentration of ADX47273 that maximally potentiated mGluR5 responses (10 µM) neither potentiated nor antagonized responses to glutamate in HEK 293A cells transiently transfected with mGluR1 (A), mGluR2 (B), or mGluR4 (C) as measured using the calcium mobilization assay. Concentration-response curves were generated from three to four independent experiments performed in duplicate. ADX47273 had no significant effect on the glutamate EC50 value in cells expressing any of these receptor subtypes (p>0.05). Error bars represent S.E.M. Figure S7. ADX47273 facilitates threshold TBS induced LTP and DHPG-LTD. A. In control slices, TBS delivered in the presence of vehicle resulted in a slight potentiation of the fEPSP. The same threshold TBS delivered in the presence of 10 µM ADX47273, induced a robust potentiation of the fEPSPs that was significantly larger than that induced by TBS alone up to 45 minutes after stimulation (n = 9-12; p<0.005). B. In control slices, 25 μM DHPG induced modest depression of the fEPSP. The same concentration of DHPG in the presence of 10 µM ADX47273 induced a significantly larger depression of the fEPSPs (n = 5; p<0.01). Error bars represent S.E.M.