The plasmids and strains used in this study.

a<p>MG 1655 was from the <i>E. coli</i> Genetic Stock Center (Yale University, New Haven), CGSC# 7740.</p>b<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048600#pone.0048600-Casadaban1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048600#pone.0048600-Ferenci1" target="_blank">[50]</a>.</p>c<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048600#pone.0048600-Kumar1" target="_blank">[13]</a>.</p

FCS autocorrelation curves.

<p>(A) A typical FCS autocorrelation curve <i>G</i>(τ) for TetA-YFP in the strain DGC103 (black dots), along with the fitted theoretical curve (red line). This curve yields <i>D</i> = 6.4 µm²/s. (B) The diffusion and fast dynamics terms in the fitting function for <i>G</i>(τ) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048600#s3" target="_blank">Methods</a>) plotted separately to illustrate how they contribute. The red curve is the entire model <i>G</i>(τ), identical to the curve in (A). The blue curve is <i>G_D</i>(τ) + <i>B</i>, the diffusion contribution. The green curve is <i>G_T</i>(τ)/N + <i>B</i>, the fast dynamics contribution. (B, inset) Logarithms of the contributions to <i>G</i>(τ) with the background <i>B</i> subtracted: ln(<i>G</i>(τ)-<i>B</i>) (red), ln(<i>G_D</i>(τ)) (blue), ln(<i>G_T</i>(τ)) (green). The three curves satisfy ln(<i>G</i>(τ)-<i>B</i>)  =  ln(<i>G_D</i>(τ)) + ln(<i>G_T</i>(τ)).</p

TetA-YFP fluorescence.

<p>Fluorescence images of cells expressing TetA-YFP. (A) A cell growing in minimal glucose medium. (B) A cell grown with cephalexin, causing filamentation, in order to perform FRAP measurements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048600#s3" target="_blank">Methods</a>). The scale bars indicate 1 µm.</p

Synthesis and Evaluation of the Metabolites of AMG 221, a Clinical Candidate for the Treatment of Type 2 Diabetes

All eight of the major active metabolites of (<i>S</i>)-2-((1<i>S</i>,2<i>S</i>,4<i>R</i>)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5<i>H</i>)-one (AMG 221, compound <b>1</b>), an inhibitor of 11β-hydroxysteroid dehydrogenase type 1 that has entered the clinic for the treatment of type 2 diabetes, were synthetically prepared and confirmed by comparison with samples generated in liver microsomes. After further profiling, we determined that metabolite <b>2</b> was equipotent to <b>1</b> on human 11β-HSD1 and had lower in vivo clearance and higher bioavailability in rat and mouse. Compound <b>2</b> was advanced into a pharmacodynamic model in mouse where it inhibited adipose 11β-HSD1 activity

5‑Alkyl-2-urea-Substituted Pyridines: Identification of Efficacious Glucokinase Activators with Improved Properties

Two 1-(4-aryl-5-alkyl-pyridin-2-yl)-3-methylurea glucokinase activators were identified with robust <i>in vivo</i> efficacy. These two compounds possessed higher solubilities than the previously identified triaryl compounds (i.e., <b>AM-2394</b>). Structure–activity relationship studies are presented along with relevant pharmacokinetic and <i>in vivo</i> data

Discovery of Potent and Simplified Piperidinone-Based Inhibitors of the MDM2–p53 Interaction

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone–pyridine inhibitors <b>6</b>, <b>7</b>, <b>14</b>, and <b>15</b> with improved pharmacokinetic properties in rats. Reducing structure complexity of the <i>N</i>-alkyl substituent led to the discovery of <b>23</b>, a potent and simplified inhibitor of MDM2. Compound <b>23</b> exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model

Discovery and Optimization of Potent GPR40 Full Agonists Containing Tricyclic Spirocycles

GPR40 (FFAR1 or FFA1) is a target of high interest being pursued to treat type II diabetes due to its unique mechanism leading to little risk of hypoglycemia. We recently reported the discovery of AM-1638 (<b>2</b>), a potent full agonist of GPR40. In this report, we present the discovery of GPR40 full agonists containing conformationally constrained tricyclic spirocycles and their structure–activity relationships leading to more potent agonists such as AM-5262 (<b>26</b>) with improved rat PK profile and general selectivity profile. AM-5262 enhanced glucose stimulated insulin secretion (mouse and human islets) and improved glucose homeostasis in vivo (OGTT in HF/STZ mice) when compared to AM-1638

C5-Alkyl-2-methylurea-Substituted Pyridines as a New Class of Glucokinase Activators

Glucokinase (GK) activators represent a class of type 2 diabetes therapeutics actively pursued due to the central role that GK plays in regulating glucose homeostasis. Herein we report a novel C5-alkyl-2-methylurea-substituted pyridine series of GK activators derived from our previously reported thiazolylamino pyridine series. Our efforts in optimizing potency, enzyme kinetic properties, and metabolic stability led to the identification of compound <b>26</b> (<b>AM-9514</b>). This analogue showed a favorable combination of <i>in vitro</i> potency, enzyme kinetic properties, acceptable pharmacokinetic profiles in preclinical species, and robust efficacy in a rodent PD model

Rational Design and Binding Mode Duality of MDM2–p53 Inhibitors

Structural analysis of both the MDM2–p53 protein–protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone <b>10</b>, an MDM2 inhibitor with a biochemical IC₅₀ of 1.0 μM. The cocrystal structure of <b>10</b> with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones <b>16</b> and <b>27</b> possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone <b>27</b> (IC₅₀ = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21^WAF1 mRNA induction (2.7-fold over vehicle) upon oral dosing of <b>27</b> at 300 mg/kg

Novel Inhibitors of the MDM2-p53 Interaction Featuring Hydrogen Bond Acceptors as Carboxylic Acid Isosteres

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (<b>4</b>) emerged as a potent inhibitor with remarkable biochemical (HTRF IC₅₀ = 0.1 nM) and cellular potency (SJSA-1 EdU IC₅₀ = 16 nM), as well as favorable pharmacokinetic properties. Compound <b>4</b> also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED₅₀ of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein