Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective, and Orally Bioavailable Porcupine Inhibitors

Blockade of aberrant Wnt signaling is an attractive therapeutic approach in multiple cancers. We developed and performed a cellular high-throughput screen for inhibitors of Wnt secretion and pathway activation. A lead structure (GNF-1331) was identified from the screen. Further studies identified the molecular target of GNF-1331 as Porcupine, a membrane bound O-acyl transferase. Structure–activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound <b>19</b>, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1 xenograft tumor model

Itpkb is required for T cell development.

<p><i>Itpkb</i>^<i>+/+</i> and <i>Itpkb</i>^<i>fl/fl</i> mice were treated with tamoxifen for 5 days followed by 2 days of rest. Thymocytes and splenocytes from tamoxifen-treated mice were compared to WT and <i>Itpkb</i>^<i>-/-</i> mice via flow cytometry. (A) Gating schemes used for thymocyte analysis with antibodies to CD4, CD8, CD3, and TCRb; numbers in the plots indicate the percentages of each gated population. (B) Total numbers of each thymocyte subset. (C) Gating scheme for analysis of the splenocytes stained with antibodies to CD4, CD8, CD3, and B220; numbers in the plots indicate the percentages of each gated population. (D) Total numbers of the indicated splenocyte populations. DP: double positive. Data from one representative experiment is shown. *, P < 0.05; **, P < 0.01.</p

Itpkb inhibitors block rat antigen-induced arthritis.

<p>(A) Lewis rats were immunized intra-dermally on Day -21 and -14 with methylated BSA (mBSA), followed by daily oral dosing of GNF362, or dexamethasone (Dex) as a positive control. On Day 0, the rats received an intra-articular injection of mBSA into the right knee joint. (B) Serum was sampled on Day +7, and IgG antibody titers to mBSA were determined by ELISA. The antibody titers were calculated by determining the average dilution at which half-maximal absorbance is detected after subtraction of background. Fold reduction in antibody titer over vehicle is shown in the table. Data shown is one representative experiment. (C) The diameters of the right and left knees were measured on Days 0, 2, 4, and 7, and the ratio of right over left knee diameters (R/L) is shown. (D) Histological analysis of the knee joint was performed blindly and scored on a 5-point scale at the termination of the study. Data shown is one representative experiment. *, P < 0.05; **, P < 0.01.</p

Ins(1,3,4,5)P4 inhibits Orai1-mediated current.

<p>HEK293-Orai1-Stim1 cells in serum-free media were placed on the QPatch HT recording system. Following cell break-in (1), little current was measured. Orai1 currents were then activated in a time-dependent manner following passive store depletion with an EGTA/BAPTA solution to chelate intracellular Ca^<i>2+</i> (2), and then stabilized in an open state (3). Next, (4), Ins(1,3,4,5)P4 (A), a control Ins(1,4,5,6)P4 (B), or 0.1% DMSO (C) was applied for 15 minutes. At the end of each experiment, 2-APB (5) was applied to block any remaining Orai1 current. In (C), percent inhibition values are expressed as the difference in current amplitude before compound application and following 2-APB application (% inhibition = ((base pA–compound pA) / (base pA– 2-APB pA)) X 100. The numbers in parentheses indicate the number of cells tested for each condition on 2 separate experimental days. Data shown is representative of three independent experiments. *, P < 0.05; **, P < 0.01</p

Itpkb is required for T cell function by negatively regulating SOC channels.

<p>WT and <i>Itpkb</i>^<i>fl/fl</i> mice were immunized with either the T-independent antigen, TNP-Ficoll, or the T-dependent antigen DNP-KLH. ELISA of TNP-specific IgG3 (A) or DNP-specific IgG1 (B) antibody responses on day 12 following immunization. Data from one representative experiment is shown (**, P < 0.01). <i>In vitro</i>, Itpkb-deficient mature lymphocytes are diminished in their proliferative capacity as measured by thymidine incorporation of purified CD4^<i>+</i> cells following stimulation with various concentrations of anti-CD3 plus anti-CD28 (C). The data from one representative experiment are shown with values representing the mean counts per minute (cpm) ± SEM. *P < 0.05. Analysis of Ca^<i>2+</i> responses using the Ca^<i>2+</i> sensitive dyes Fluo-4 and Fura Red were evaluated after cross-linking the antigen receptor either in the presence or absence of exogenous Ca^<i>2+</i>. Splenocytes gated on CD4 were treated with anti-CD3-biotin, followed by cross-linking with streptavidin (S.A.) in the presence of exogenous calcium (top panel), or in the absence of exogenous calcium, followed by calcium re-addition to examine SOC channel function (bottom panel). Ionomycin (Iono.) stimulation was used at the end of each run to control for equivalent dye loading. Data is shown as the mean fluorescent ratio of Fluo-3 and Fura-Red. Representative data of three independent experiments are shown.</p

Discovery of GNF-5837, a Selective TRK Inhibitor with Efficacy in Rodent Cancer Tumor Models

Neurotrophins and their receptors (TRKs) play key roles in the development of the nervous system and the maintenance of the neural network. Accumulating evidence points to their role in malignant transformations, chemotaxis, metastasis, and survival signaling and may contribute to the pathogenesis of a variety of tumors of both neural and non-neural origin. By screening the GNF kinase collection, a series of novel oxindole inhibitors of TRKs were identified. Optimization led to the identification of GNF-5837 (<b>22</b>), a potent, selective, and orally bioavailable pan-TRK inhibitor that inhibited tumor growth in a mouse xenograft model derived from RIE cells expressing both TRKA and NGF. The properties of <b>22</b> make it a good tool for the elucidation of TRK biology in cancer and other nononcology indications

Itpkb negatively regulates pro-apoptotic gene expression and AICD in CD4^<i>+</i> T cells.

<p>Either purified (primary) CD4^<i>+</i> T cells (A) or in vitro-expanded (secondary) CD4^<i>+</i> T cells (B) were stimulated with anti-CD3/28 beads for the indicated time points. Following RNA isolation, cDNA was generated, and subjected to real-time quantitative PCR analysis for Bim, Bcl2, Fas, and FasL. The Y-axis represents the respective transcript normalized to either B2-microglobulin or GAPDH values determined for each sample. One representative experiment is shown with the mean values of each genotype ± SE. *, P < 0.05; **, P < 0.01. (C) Purified CD4^<i>+</i> cells were labeled with CFSE and stimulated with anti-CD3/28 beads in the presence of anti-FasL or an isotype control Ig. 72 hours following stimulation, cells were stained with Annexin V and DAPI. Numbers in the lower left quadrant indicate the percentage of live cells at the time of analysis. (D) Proliferation measured by CFSE dilution was followed after 72 hours in culture. Blue and red lines indicate stimulated and unstimulated cells, respectively. Numbers above bracketed lines indicate the percentage of divided cells. Data shown is representative of three independent experiments.</p

Identification and characterization of Itpkb inhibitors.

<p>(A) GNF362 was identified following a 2 million compound biochemical screen, co-crystallography with Itkpb, and additional medicinal chemistry optimization. (B) The biochemical activity of GNF362 was determined in Kinase Glo assays using purified Itpka, Itpkb, and Itpkc proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131071#pone.0131071.ref014" target="_blank">14</a>]. Data shown is one representative experiment. (C) The cellular activity of GNF362 was profiled on wild type B cells loaded with the calcium-sensitive dye, Fluo-4. Splenocytes were pre-incubated with varying concentrations of GNF362, and SOC-mediated Ca^<i>2+</i> entry, depicted as the mean value of Fluo-4 over time, was measured on the FLIPR following stimulation with anti-IgM and calcium add-back. (D) The peak calcium response, after calcium re-addition is shown as a function of the concentration of GNF362 in the assay, with an EC50 of 12nM. Data shown is one representative experiment. (E) Purified CD4^<i>+</i> T cells were stimulated in the presence of GNF362, along with the addition of either anti-FasL or a control Ig, to determine the effect on proliferation and FasL-mediated activation-induced cell death. Data shown is representative of three independent experiments. (F) Wild type mice were dosed orally with GNF362 at 3, 10, or 25mg/kg twice daily for 9 days. The percentage of CD4^<i>+</i> T cells in the thymus was determined by FACS analysis. Data shown is representative of three independent experiments. **, P < 0.01.</p

Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH)

The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of <b>1</b> (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC

(<i>R</i>)‑2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class of Potent and Selective Pan-TRK Inhibitors

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (<i>R</i>)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (<i>R</i>)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs