We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

FFA2 (GPR43) has been identified as a receptor for short-chain fatty acids (SCFAs) that include acetate and propionate. FFA2 is highly expressed in islets, a subset of immune cells, and adipocytes. Although the potential roles of FFA2 activation in these tissues have previously been described, the physiological functions are still unclear. The potency for SCFAs on FFA2 is low, in the high micromolar to millimolar concentrations. To identify better pharmacological tools to study receptor function, we used high-throughput screening (HTS) to discover a series of small molecule phenylacetamides as novel and more potent FFA2 agonists. This series is specific for FFA2 over FFA1 (GPR40) and FFA3 (GPR41), and it is able to activate both the G␣ q and G␣ i pathways in vitro on Chinese hamster ovary cells stably expressing FFA2. Treatment of adipocytes with these compounds also resulted in G␣ i -dependent inhibition of lipolysis similar to that of endogenous ligands (SCFAs). It is noteworthy that these compounds not only acted as FFA2 agonists but also exhibited positive cooperativity with acetate or propionate. The observed allosteric modulation was consistent in all the functional assays that we have explored, including cAMP, calcium mobilization, guanosine 5Ј-[␥-thio]triphosphate binding, and lipolysis. Molecular modeling analysis of FFA2 based on human ␤ 2 -adrenergic receptor structure revealed potential nonoverlapping binding sites for the endogenous and synthetic ligands, further providing insight into the binding pocket for the allosteric interactions. This is the first report describing the identification of novel allosteric modulators with agonist activity for FFA2, and these compounds may serve as tools for further unraveling the physiological functions of the receptor and its involvement in various diseases.

Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium-and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits.

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Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3). We used gene targeting to introduce the human achondroplasia mutation into the murine FGFR3 gene. Heterozygotes for this point mutation that carried the neo cassette were normal whereas neo ؉ homozygotes had a phenotype similar to FGFR3-deficient mice, exhibiting bone overgrowth. This was because of interference with mRNA processing in the presence of the neo cassette. Removal of the neo selection marker by Cre͞loxP recombination yielded a dominant dwarf phenotype. These mice are distinguished by their small size, shortened craniofacial area, hypoplasia of the midface with protruding incisors, distorted brain case with anteriorly shifted foramen magnum, kyphosis, and narrowed and distorted growth plates in the long bones, vertebrae, and ribs. These experiments demonstrate that achondroplasia results from a gain-of-FGFR3-function leading to inhibition of chondrocyte proliferation. These achondroplastic dwarf mice represent a reliable and useful model for developing drugs for potential treatment of the human disease.Four fibroblast growth factor receptors (FGFRs) are known (1), and Ͼ50 mutations in three of them (FGFR1, 2, and 3) recently have been implicated in congenital skeletal and cranial disorders (reviewed in refs. 2-4). Achondroplasia, the most common form of dwarfism, was shown to be linked to a single point mutation, G380R, in the transmembrane region of FGFR3 (5, 6). FGFR3 is expressed mainly by developing bones, brain, lung, and spinal cord (7,8), and FGFR3-deficient mice show enhanced endochondral bone growth, expansion of their growth plate, and increased chondrocyte proliferation (9, 10). Thus, FGFR3 is a negative regulator of bone growth. Several experiments at the cellular level indicated that the Ach mutation (G380R) results in a constitutive activation of the receptor in a ligand-independent manner (11-13). It was suggested that this is because of stabilization of receptor dimers, a prerequisite for signal transduction in these receptors (14). This is also consistent with the constitutive activation by dimer formation described for an erbB2 (neu) receptor mutant, which carries a Val-to-Glu mutation in an analogous position to that of the FGFR3 variant in its transmembrane region (15). It is likely that, in many of the other mutations in FGFR1-3, the underlying mechanism of receptor activation is also through stabilization of receptor dimers because many of these mutations result in unpaired cysteines that may enhance inter-receptor disulfide bonds (2-4).To generate an animal model for this type of mutation and to study the role of the mutated FGFR3 in vivo, we used gene targeting to introduce the achondroplasia mutation (G380R) into murine FGFR3. This resulted in a dominant dwarf phenotype that exhibits many of the features of human achondroplasia. This is an indication from in vivo data for ...