Discovery of Orally Bioavailable Purine-Based Inhibitors of the Low-Molecular-Weight Protein Tyrosine Phosphatase.

Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.

Serine-Threonine Kinase TAO3-Mediated Trafficking of Endosomes Containing the Invadopodia Scaffold TKS5α Promotes Cancer Invasion and Tumor Growth.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

TGR5 contributes to hepatic cystogenesis in rodents with polycystic liver diseases through cyclic adenosine monophosphate/Gαs signaling.

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5-/- ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq ) proteins was increased 2-fold to 3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/- ;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI-115 alone and by ∼50% when SBI-115 was combined with pasireotide. CONCLUSION: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).

Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis.

Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.

Small-molecule IAP antagonists sensitize cancer cells to TRAIL-induced apoptosis: roles of XIAP and cIAPs.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-inducing activity in transformed, but not in normal, cells. As with most anticancer agents, however, its clinical use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that they do so in a caspase-8-dependent manner. We further show that the compounds have no cytotoxicity as single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the most efficacious in binding to X-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2 degradation. Our studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell lines.

Design, synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP.

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure-activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.

Expedient synthesis of highly potent antagonists of inhibitor of apoptosis proteins (IAPs) with unique selectivity for ML-IAP.

A series of novel, potent antagonists of the inhibitor of apoptosis proteins (IAPs) were synthesized in a highly convergent and rapid fashion (≤6 steps) using the Ugi four-component reaction as the key step, thus enabling rapid optimization of binding potency. These IAP antagonists compete with caspases 3, 7, and 9 for inhibition by X chromosome-linked IAP (XIAP) and bind strongly (nanomolar binding constants) to several crucial members of the IAP family of cancer pro-survival proteins to promote apoptosis, with a particularly unique selectivity for melanoma IAP (ML-IAP). Experiments in cell culture revealed powerful cancer cell growth inhibitory activity in multiple (breast, ovarian, and prostate) cell lines with single agent toxicity at low nanomolar levels against SKOV-3 human ovarian carcinoma cells. Administration of the compounds to human foreskin fibroblast cells revealed no general toxicity to normal cells. Furthermore, computational modeling was performed, revealing key contacts between the IAP proteins and antagonists, suggesting a structural basis for the observed potency.

Design, synthesis and evaluation of monovalent Smac mimetics that bind to the BIR2 domain of the anti-apoptotic protein XIAP.

We report the systematic rational design and synthesis of new monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Characterization of compounds in vitro (including 9i; ML101) led to the determination of key structural requirements for BIR2 binding affinity. Compounds 9h and 9j sensitized TRAIL-resistant breast cancer cells to apoptotic cell death, highlighting the value of these probe compounds as tools to investigate the biology of XIAP.

Agonist and antagonist of retinoic acid receptors cause similar changes in gene expression and induce senescence-like growth arrest in MCF-7 breast carcinoma cells.

Biological effects of retinoids are mediated via retinoic acid (RA) receptors (RAR) and retinoid X receptors (RXR). The best-characterized mechanism of retinoid action is stimulation of transcription from promoters containing RA response elements (RARE). Retinoids induce senescence-like growth arrest in MCF-7 breast carcinoma cells; this effect is associated with the induction of several growth-inhibitory genes. We have now found that these genes are induced by RAR-specific but not by RXR-specific ligands. Genome-scale microarray analysis of gene expression was used to compare the effects of two pan-RAR ligands, one of which is a strong agonist of RARE-dependent transcription, whereas the other induces such transcription only weakly and antagonizes the inducing effect of RAR agonists. Both RAR ligands, however, produced very similar effects on gene expression in MCF-7 cells, suggesting that RARE-dependent transcription is only a minor component of retinoid-induced changes in gene expression. The effects of RAR ligands on gene expression parallel changes associated with damage-induced senescence, and both ligands induced G(1) arrest and the senescent phenotype in MCF-7 cells. The RAR ligands up-regulated many tumor-suppressive genes and down-regulated multiple genes with oncogenic activities. Genes that are strongly induced by RAR ligands encode secreted bioactive proteins, including several tumor-suppressing factors. In agreement with these observations, retinoid-treated MCF-7 cells inhibited the growth of retinoid-insensitive MDA-MB-231 breast carcinoma cells in coculture. These results indicate that RARE-independent transcriptional effects of RAR ligands lead to senescence-like growth arrest and paracrine growth-inhibitory activity in MCF-7 breast carcinoma cells.