Volume 21, Issue 11, Pages 1486-1496 (November 2014) Inhibition of AMP Deaminase Activity Does Not Improve Glucose Control in Rodent Models of Insulin Resistance or Diabetes  Therese Admyre, Lena Amrot-Fors, Maria Andersson, Martin Bauer, Mikael Bjursell, Tomas Drmota, Stefan Hallén, Judith Hartleib- Geschwindner, Bo Lindmark, Jianming Liu, Lars Löfgren, Mattias Rohman, Nidhal Selmi, Kristina Wallenius  Chemistry & Biology  Volume 21, Issue 11, Pages 1486-1496 (November 2014) DOI: 10.1016/j.chembiol.2014.09.011 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 High-Throughput Screen and Hit Confirmation for the Identification of AMPD1 Inhibitors (A) Schematic showing the AMPD assay. α-KG, α-ketoglutarate. (B) Z’ factors of each run through the HTS. (C) The correlation between the negative logarithm of the IC50 (pIC50) from the HTS assay and the counterscreen. The x axis is the counterscreen, and the y axis is the HTS assay. (D) Correlation between three-point and seven-point concentration responses. The x axis shows seven-point data, and the y axis shows three-point data. Chemistry & Biology 2014 21, 1486-1496DOI: (10.1016/j.chembiol.2014.09.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Properties and Optimization of AMPD Inhibitors (A) The structure-activity relationship for series 106. (B) Structures for compounds 1, 2, 3, and 4. (C) Direct comparison of the inhibitory action of compound 2 (squares) and compound 3 (circles) in ob/ob muscle homogenates (MH, open symbols, n = 3) with recombinant murine AMPD1 (filled symbols, n = 2, upper panel) as well as rat liver extracts (LH, open symbols, n = 3) and recombinant AMPD2 (filled symbols, n = 6, lower panel). The values are mean ± SD. Chemistry & Biology 2014 21, 1486-1496DOI: (10.1016/j.chembiol.2014.09.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 Pharmacological Inhibition of AMPD in Primary Hepatocytes (A) Fructose induced ATP depletion in primary rat hepatocytes. Freshly prepared rat hepatocytes kept in suspension were ATP-depleted by a 5 min incubation with fructose (2.8 mM) that induced IMP formation. (B) IMP levels in ATP-depleted hepatocytes treated with AMPD inhibitors. cpd, compound. (C) Effects on AMP levels in hepatocytes treated with AMPD inhibitors. The values are mean ± SD. #, below the detection limit of the system; ∗p < 0.03 compared with the DMSO controls in a two-tailed Student’s t test. Chemistry & Biology 2014 21, 1486-1496DOI: (10.1016/j.chembiol.2014.09.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 Metabolic effects because of deletion of the Ampd1 gene in mice. (A) Body weight gain from 4–14 weeks of age in standard chow-fed (SCD) wild-type mice (n = 8, filled circles), Ampd1 KO mice (n = 8, filled circles), HFD-fed wild-type mice (n = 8, filled squares), and Ampd1 KO mice (n = 8, open squares). Statistical analysis was performed using repeated Student’s t test. (B and C) Oral glucose tolerance test in chow-fed wild-type mice (n = 7, filled circles), Ampd1 KO mice (n = 7, open circles), HFD-fed wild-type mice (n = 7, filled squares), and Ampd1 KO mice (n = 7, open squares). The results are presented as mean ± SEM. Statistical analysis of AUC values was performed using Student’s t test. Chemistry & Biology 2014 21, 1486-1496DOI: (10.1016/j.chembiol.2014.09.011) Copyright © 2014 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2014 21, 1486-1496DOI: (10. 1016/j. chembiol. 2014 Copyright © 2014 Elsevier Ltd Terms and Conditions