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The insulin-like growth factor-1 receptor (IGF1R) is a drug target for oncology, and many studies are ongoing to identify compounds that inhibit its tyrosine kinase activity. IGF1R is highly homologous to the insulin receptor (IR) and IGF1R inhibition might be beneficial for patients, while IR inhibition may lead to limiting toxicity. Therefore selectivity for IGF1R over IR is the aim for drug design in this context. A few compounds that selectively inhibit IGF1R over IR in cells have been identified, but none of them show the same levels of selectivity in enzymatic assays. To deter- mine whether this discrepancy is linked to the conditions used in the enzymatic assays, we have studied the interaction be- tween known IGF1R inhibitors (NVP-AEW541, OSI906, AG538, NVP-TAE226) and phosphorylated/unphosphorylated IGF1R/IR proteins with both biophysical (isothermal calorimetry and surface plasmon resonance) and enzymatic methods. In this report, we describe the results of this study and comment on the different degrees of selectivity IGF1R versus IR measured in biochemical and cellular assays. Finally, our study provides new information on the biochemical and mechanism of action of these small molecular weight IGF1R inhibitors.

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53–MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein–protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line.

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.

Two splice variants of the human uncoupling protein-3 (UCP3L and UCP3S) are highly expressed in skeletal muscle. The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter. Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis. The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability. Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3. A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein–protein interaction, we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53–MDM2 interaction.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhibitory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK properties in rats. Synthesis and structure–activity relationship data are also provided.

Epidermal growth factor (EGF) has been reported to stimulate carbohydrate, amino acid, and electrolyte transport in the small intestine, but its effects on lipid transport are poorly documented. This study aimed to investigate EGF effects on fatty acid uptake and esterification in a human enterocyte cell line (Caco-2). EGF inhibited cell uptake of [14C]palmitate and markedly reduced its incorporation into triglycerides. In contrast, the incorporation in phospholipids was enhanced. To elucidate the mechanisms involved, key steps of lipid synthesis were investigated. The amount of intestinal fatty acid-binding protein (I-FABP), which is thought to be important for fatty acid absorption, and the activity of diacylglycerol acyltransferase (DGAT), an enzyme at the branch point of diacylglycerol utilization, were reduced. EGF effects on DGAT and on palmitate esterification occurred at 2–10 ng/ml, whereas effects on I-FABP and palmitate uptake occurred only at 10 ng/ml. This suggests that EGF inhibited palmitate uptake by reducing the I-FABP level and shifted its utilization from triglycerides to phospholipids by inhibiting DGAT. This increase in phospholipid synthesis might play a role in the restoration of enterocyte absorption function after intestinal mucosa injury.

Abstract 62 Mutation within the kinase domain of tyrosine kinases is a common mechanisms of resistance to enzymatic inhibitors. Inhibitors of janus kinase 2 (JAK2) are under evaluation in patients with myeloproliferative neoplasms (MPNs), B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, and other tumors with constitutive JAK2 signaling. To identify resistance mutations in JAK2, we randomly mutagenized human JAK2 R683G, which is observed in approximately half of CRLF2-rearranged B-ALL. We transduced the mutagenized JAK2 cDNA library into murine Ba/F3 cells that express CRLF2. Expression of CRLF2 and JAK2 R683G confers IL3 independent growth in Ba/F3 cells. The transduced population was selected in the JAK2-selective inhibitor NVP-BVB808 in the absence of IL3. Multiple BVB808-resistant clones were recovered that harbored either E864K, Y931C or G935R mutations in JAK2. Alignment of homologous regions of the JAK2 kinase domain (JH1) with ABL1 demonstrated that the three mutations are located in regions homologous to imatinib resistance hotspots in ABL1. Codons Y931 and G935 are within the hinge region of the kinase domain. Based on structural modeling, Y931C is likely to inhibit substrate binding. E864K is located in the middle of b3 following the P-loop in the N-lobe and may modify the structure and flexibility of the preceding P-loop, thus destabilizing the conformation required for inhibitor binding. We expressed JAK2 V617F alleles harboring Y931C, G935R or E864K in Ba/F3-EPOR cells and exposed the cells to the JAK2 enzymatic inhibitors JAK inhibitor-1, NVP-BSK805, TG101348, tofacitinib (formerly tasocitnib), ruxolitinib (formerly INCB18424) and BVB808. All three mutations conferred 2- to >10-fold resistance against BVB808, NVP-BSK805, TG101348, ruxolitinib and JAK inhibitor-1. Y931C and E864K but not G935R conferred resistance to tofactinib. Modeling of G935R indicated that a 935R side-chain would occlude the hydrophobic channel of the ATP-binding pocket. As a consequence, this mutation would decrease the binding affinity of compounds occupying the hydrophobic channel like JAK inhibitor-1 or BSK805, but not affect the potency of tofactinib, which does not bind in this region. Mutation of G935 to arginine, histidine or glutamine reduced the inhibitory effects of JAK inhibitor-1, but not tofacitinib, on JAK2 kinase domain activity. None of the codon 935 mutations had significant effects on Km or Vmaxin vitro. BVB808 treatment partially reduced activation state-specific phosphorylation of STAT5 in Ba/F3-EPOR/JAK2 V617F cells but not in Ba/F3-EPOR/JAK2 V617F/G935R or G935H cells. JAK2 is a known client of HSP90, and HSP90 inhibitors promote the degradation of both wild-type and mutant JAK2. We hypothesized that resistance mutations within the JAK2 kinase domain would not affect JAK2 degradation induced by HSP90 inhibitors. We assayed the cytotoxicity of the resorcinylic isoxazole amide NVP-AUY922 and the benzoquinone ansamycin 17-AAG in Ba/F3 cells that express the erythropoietin receptor (EPOR) and JAK2 V617F, which is observed in more than half of MPNs. Mutation of JAK2 V617F to include E864K, Y931C or G935R did not affect sensitivity to either AUY922 or 17-AAG. In fact, AUY922 was more active against cells harboring G935R (GI50, 3.87 nM) or E864K (GI50, 6.14 nM) compared to cells with no resistance mutation (GI50, 14.7 nM; p Disclosures: Gaul: Novartis: Employment. Vangrevelinghe:Novartis: Employment. De Pover:Novartis: Employment. Regnier:Novartis: Employment. Erdmann:Novartis: Employment. Hofmann:Novartis: Employment. Eck:Novartis: Consultancy, Research Funding. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Abstract 3777 Poster Board III-713 The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and from primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative disorders and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild type enzymes by novel small molecule inhibitors, which act in an ATP-competitive manner. The profile of a lead compound from this class will be presented that displays more than twenty-fold selectivity towards JAK2 within the JAK family, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, the inhibitor exhibited good oral bioavailability and is efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model as well as polycythemia and extramedullary erythropoiesis in mouse and rat models. Disclosures: Radimerski: Novartis: Employment, Equity Ownership. Baffert:Novartis: Employment. Regnier:Novartis: Employment. De Pover:Novartis: Employment. Pissot:Novartis: Employment. Gerspacher:Novartis: Employment. Brueggen:Novartis: Employment. Tavares:Novartis: Employment. Blasco:Novartis: Employment. Ledieu:Novartis: Employment. Nolan:Novartis: Employment. Ruetz:Novartis: Employment. Chene:Novartis: Employment. Erdmann:Novartis: Employment. Drueckes:Novartis: Employment. Furet:Novartis: Employment. Lang:Novartis: Employment. Trappe:Novartis: Employment. Vangrevelinghe:Novartis: Employment. Wartmann:Novartis: Employment. Hofmann:Novartis: Employment.

(Na+ + K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3.) has been prepared from human heart and guinea-pig heart, with respective specific activities of 10–15 μmol Pi · mg−1 · h−1 and of 25–30 μmol Pi · mg−1 · h−1. Residual Mg2+—ATPase activities were about 5 per cent. The parameters of (Na+ + K+)ATPase activity and of ouabain-interaction have been compared: (1) Half-maximal activity concentrations and Hill coefficients of Na+, K+, Mg2+ and ATP were similar for the two species. The apparent activation energies calculated from Arrhenius plots were also similar. A transition was observed at about 23°C. (2) Human heart (Na+ + K+)ATPase was 10 times more sensitive to ouabain-inhibition than that of guinea-pig. Hunter-Downs plots showed a competitive inhibition for K+ at low K+ concentration and noncompetitive inhibition at high concentration. (3) The Scatchard plot for [3H]ouabain binding was upward-concave with human heart and linear with guinea-pig heart. (4) The dissociation kinetics of [3H]ouabain from human preparations studied by an isotopic dilution technique indicated two classes of binding sites with kd of 0.058 min−1 and 0.0092 min−1. The dissociation kinetics with guinea-pig heart indicated one single class of binding sites with a kd of 0.43 min−1. (5) The time-course of 0.2 μM [3H]ouabain binding showed pseudo-first order association kinetics in man and in guinea-pig. ka for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 106 min−1 · M−1 and to 3.7 × 106 min−1 · M−1 · ka for guinea-pig heart was equal to 2.3.106 min−1 · M−1. (6) In guinea-pig heart, KD c from Scatchard plot and from kd/ka ratio were equal to the inhibition constant Ki calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na+ + K+)ATPase inhibition. (7) In human heart, KD of the low affinity binding sites was close to Ki, whereas KD of the high affinity binding sites was several times lower. This suggests that only low affinity binding sites might be involved in (Na+ + K+)ATPase inhibition by ouabain.