Controlling Ibrutinib's Conformations about Its Heterobiaryl Axis to Increase BTK Selectivity.

Ibrutinib is a covalent BTK inhibitor that is approved for several indications in oncology. Ibrutinib possesses significant off-target activities toward many kinases, often leading to adverse events in patients. While there have been robust medicinal chemistry efforts leading to more selective second-generation BTK inhibitors, there remains a need for new strategies to rapidly improve the selectivity of kinase inhibitors. An analysis of PDB data revealed that ibrutinib binds BTK in dihedral conformations that are orthogonal of ibrutinib's predicted low energy conformational range. Synthesis of a series of analogues with ground state conformations shifted toward orthogonality led to the discovery of an analogue with two incorporated ortho-methyl groups that possessed markedly increased BTK selectivity. This work suggests that conformational control about a prospective atropisomeric axis represents a strategy to rapidly program a compound's selectivity toward a given target.

1,5-Disubstituted Acylated 2-Amino-4,5-dihydroimidazoles as a New Class of Retinoic Acid Receptor-Related Orphan Receptor (ROR) Inhibitors.

A growing body of evidence suggests a pathogenic role for pro-inflammatory T helper 17 cells (Th17) in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis-diseases for which no curative treatment is currently available. The nuclear retinoic acid receptor-related orphan receptors alpha and gamma (RORα/γ), in particular the truncated isoform RORγt that is specifically expressed in the thymus, play a critical role in the activation of a pro-inflammatory Th17 response, and RORγ inverse agonists have shown promise as negative regulators of Th17 for the treatment of autoimmune diseases. Our study underscores the screening of a large combinatorial library of 1,5-disubstituted acylated 2-amino-4,5-dihydroimidazoles using a demonstrated synthetic and screening approach and the utility of the positional scanning libraries strategy for the rapid identification of a novel class of ROR inhibitors. We identified compound 1295-273 with the highest activity against RORγ (3.3 µM IC50) in this series, and almost a two-fold selectivity towards this receptor isoform, with 5.3 and 5.8 µM IC50 against RORα and RORß cells, respectively.

Discovery of cyclic guanidine-linked sulfonamides as inhibitors of LMTK3 kinase.

Lemur tyrosine kinase 3 (LMTK3) is oncogenic in various cancers. In breast cancer, LMTK3 phosphorylates and modulates the activity of estrogen receptor-α (ERα) and is essential for the growth of ER-positive cells. LMTK3 is highly expressed in ER-negative breast cancer cells, where it promotes invasion via integrin ß1. LMTK3 abundance and/or high nuclear expression have been linked to shorter disease free and overall survival time in a variety of cancers, supporting LMTK3 as a potential target for anticancer drug development. We sought to identify small molecule inhibitors of LMTK3 with the ultimate goal to pharmacologically validate this kinase as a novel target in cancer. We used a homogeneous time resolve fluorescence (HTRF) assay to screen a collection of mixture-based combinatorial chemical libraries containing over 18 million compounds. We identified several cyclic guanidine-linked sulfonamides with sub-micromolar activity and evaluated their binding mode using a 3D homology model of the LMTK3 KD.

Leveraging Atropisomerism to Obtain a Selective Inhibitor of RET Kinase with Secondary Activities toward EGFR Mutants.

Unstable atropisomerism is innate in many common scaffolds in drug discovery, commonly existing as freely rotating aryl-aryl bonds. Such compounds can access the majority of dihedral conformations around the bond axis; however, most small molecules bind their target within a narrow range of these available conformations. The remaining accessible conformations can interact with other proteins leading to compound promiscuity. Herein, we leverage atropisomerism to restrict the accessible low-energy dihedral conformations available to a promiscuous kinase inhibitor and achieve highly selective and potent inhibitors of the oncogenic target rearranged during transfection (RET) kinase. We then evaluate our lead inhibitor against kinases that were predicted to bind compounds in a similar conformational window to RET, discovering a potent inhibitor of drug-resistant epidermal growth factor receptor (EGFR) mutants including L858R/T790M/C797S EGFR. Leveraging atropisomerism to restrict accessible conformational space should be a generally applicable strategy due to the prevalence of unstable atropisomerism in drug discovery.

Tetrahydrobenzothiophene carboxamides: Beyond the kinase domain and into the fatty acid realm.

A series of tetrahydrobenzothiophene carboxamides, inspired by structural features present in kinase and SCD1 inhibitors, are presented here. Prototype compound 8 (MMDD13) modulates fatty acid elongase and desaturase indexes, lipid accumulation, while preserving kinase inhibitory activity. This chemotype represents a stepping stone towards chemical probes to study the consequences of lipid metabolism modulation through non-redundant pathways.

Synthesis of dihydroimidazole tethered imidazolinethiones and their activity as novel antagonists of the nuclear retinoic acid receptor-related orphan receptors (RORs).

Targeting the transcriptional activity of nuclear hormone receptors has proven an effective strategy to treat certain human diseases, and they have become a major focus point to develop novel therapies for the treatment of cancer, inflammation, autoimmune diseases, metabolic disorders, and others. One family of nuclear receptors that has attracted most interest in recent years is the retinoic acid receptor-related orphan receptors (RORs), in particular RORγ. RORγ is a critical regulator of the immune system and RORγ antagonists have shown activity in animal models of inflammatory autoimmune diseases. Here we present the synthesis and biological evaluation of dihydroimidazole tethered imidazolinethiones. We have identified several dual RORγ/α and pan-ROR antagonists with significant activity in cellular assays that could serve as starting points for future optimization efforts to generate potent and selective RORγ modulators.

Diversity oriented synthesis and IKK inhibition of aminobenzimidazole tethered quinazoline-2,4-diones, thioxoquinazolin-4-ones, benzodiazepine-2,3,5-triones, isoxazoles and isoxazolines.

The derivatization of resin-bound aminobenzimidazole toward the parallel solid-phase synthesis of aminobenzimidazole tethered pharmacologically important heterocycles such as quinazoline-2,4-diones, thioxoquinazolin-4-ones, benzodiazepine-2,3,5-triones, isoxazoles and isoxazolines is reported. All the compounds were tested for IKK inhibition. Only one compound elicited significant inhibition of IKKε, TBK-1 and IKK2.

Inhibition of IκB kinase-ß and IκB kinase-α by heterocyclic adamantyl arotinoids.

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKß, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKß inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKß, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.

Adamantyl arotinoids that inhibit IκB kinase α and IκB kinase ß.

A series of analogues of the adamantyl arotinoid (AdAr) chalcone MX781 with halogenated benzyloxy substituents at C2' and heterocyclic derivatives replacing the chalcone group were found to inhibit IκBα kinase α (IKKα) and IκBα kinaseâ€…ß (IKKß) activities. The growth inhibitory capacity of some analogues against Jurkat T cells as well as prostate carcinoma (PC-3) and chronic myelogenous leukemia (K562) cells, which contain elevated basal IKK activity, correlates with the induction of apoptosis and increased inhibition of recombinant IKKα and IKKß in vitro, pointing toward inhibition of IKK/NFκB signaling as the most likely target of the anticancer activities of these AdArs. While the chalcone functional group present in many dietary compounds has been shown to mediate interactions with IKKß via Michael addition with cysteine residues, AdArs containing a five-membered heterocyclic ring (isoxazoles and pyrazoles) in place of the chalcone of the parent system are potent inhibitors of IKKs as well, which suggests that other mechanisms for inhibition exist that do not depend on the presence of a reactive α,ß-unsaturated ketone.

Synthesis and pharmacological evaluations of novel 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as a new class of anti-cancer agents.

The synthesis of novel 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives has been carried out using trifluoroacetic anhydride/phosphoric acid mediated C-C bond forming reaction as a key step. This method does not require the use of environmentally harmful AlCl(3) or moisture sensitive acid chloride. A number of compounds containing the benzooxazinone moiety attached to a five-membered central heterocyclic ring was synthesized and tested for their anti-cancer properties in vitro against three cell lines e.g. A549 (lung), DLD-1 (colorectal adenocarcinoma) and MV4-11 (acute myeloid leukemia). Some of them showed anti-cancer activities along with a number of reference compounds tested. Few of them showed promising anti-leukemic properties. A brief Structure-Activity-Relationship study within the series is presented. An imidazole derivative 9c containing benzene ring with a para-CF(3) group at C-2 position was identified as a potent anti-leukemic agent.

Necrotic concentrations of cisplatin activate the apoptotic machinery but inhibit effector caspases and interfere with the execution of apoptosis.

Cisplatin is a chemotherapeutic drug whose cytotoxicity is key to its therapeutic and side effects. Nephrotoxicity, mainly due to renal tubular injury, poses its most important therapeutic limitation. Tubular necrosis is derived from epithelial cell death by apoptosis and necrosis in the proximal and distal tubuli. The mode of cell death has been related to drug concentration, with necrosis occurring with high concentrations and apoptosis with lower concentrations. To fully understand the toxic effects of cisplatin to potentially improve its pharmaco-toxicological profile, it is necessary to unravel the cellular events and signaling pathways implicated in the appearance of both modes of cell death. We used cultured human lymphoma and renal tubule cells to investigate the biochemical and phenotypic characteristics of the death mode induced by increasing concentrations of cisplatin. Our results indicate that pronecrotic concentrations of cisplatin early activate the apoptotic machinery, which is in turn directly blocked by cisplatin at the level of effector caspases. Aborted apoptosis induces a death phenotype lacking some typical characteristics of this process, which more closely resembles necrosis. Furthermore, unidentified Bcl-2- and mitochondria-independent pathways are induced by pronecrotic and not by proapoptotic concentrations of cisplatin. Cisplatin-induced cell necrosis is the result of an aborted apoptosis at the level of effector caspases. Yet, Bcl-2-independent effects lead to cell death, which may pose potential targets for pharmacological intervention aimed at reducing cisplatin nephrotoxicity.

Highly twisted adamantyl arotinoids: synthesis, antiproliferative effects and RXR transactivation profiles.

Retinoid-related molecules with an adamantyl group (adamantyl arotinoids) have been described with selective activities towards the retinoid receptors as agonists for NR1B2 and NR1B3 (RARbeta,gamma) (CD437, MX3350-1) or RAR antagonists (MX781) that induce growth arrest and apoptosis in cancer cells. Since these molecules induce apoptosis independently of RAR transactivation, we set up to synthesize novel analogs with impaired RAR binding. Here we describe adamantyl arotinoids with 2,2'-disubstituted biaryl rings prepared using the Suzuki coupling of the corresponding fragments. Those with cinnamic and naphthoic acid end groups showed significant antiproliferative activity in several cancer cell lines, and this effect correlated with the induction of apoptosis as measured by caspase activity. Strikingly, some of these compounds, whereas devoid of RAR binding capacity, were able to activate RXR.

Inhibition of IkappaB kinase-beta and anticancer activities of novel chalcone adamantyl arotinoids.

On the basis of the observations that chalcone 7 (MX781) and some related adamantyl arotinoids (AdArs) inhibit IkappaB alpha kinase beta (IKKbeta) activity, inhibit cell growth, and induce apoptosis in cancer cells, a new series of AdArs structurally related to 7 have been designed and synthesized. Modifications were intended to reduce or eliminate RAR activity, and we evaluated the effect of the novel analogues of 7 on IKKbeta activity and proliferation of a variety of cancer cell lines (leukemia, Jurkat; prostate, PC-3; breast carcinomas, T47D, MDA-MB-468). Consistent with the design principles, the biological activities of these AdArs do not appear to be RAR-mediated, since most analogues are unable to activate RAR-mediated transactivation and exhibit significantly diminished antagonist activity. All compounds are capable of inducing apoptosis in Jurkat cells, as demonstrated by elevated DEVDase activity and externalization of phosphatidylserine. Several of the analogues elicit stronger growth inhibitory activity against prostate (PC-3) and breast (MDA-MB-468) carcinoma cells, which contain elevated basal IKK activity; this antiproliferative activity correlates with increased inhibition of recombinant IKKbeta in vitro, suggesting that the anticancer activities of these AdArs might be related to the inhibition of IKK/NFkappaB signaling.

The human myeloperoxidase gene is regulated by LXR and PPARalpha ligands.

Myeloperoxidase (MPO) is an oxidant-generating enzyme expressed in macrophages and implicated in atherosclerosis and cholesterol homeostasis. LXRalpha and PPARalpha regulate genes involved in cholesterol metabolism and the inflammatory response in macrophages. Here, we examine the effect of LXR and PPARalpha ligands on MPO expression. LXR and PPARalpha, as heterodimers with RXR, are shown to bind overlapping sites in an Alu receptor response element (AluRRE) in the MPO promoter. The LXR ligand T0901317 suppresses MPO mRNA expression in primary human macrophages, and in bone marrow cells and macrophages from huMPO transgenic mice. The PPARalpha ligand GW9578 downregulates MPO expression in GMCSF-macrophages, while upregulating in MCSF-macrophages. In contrast, the mouse MPO gene, which lacks the primate-specific AluRRE, is not regulated by LXR or PPARalpha ligands. These findings identify human MPO as a novel LXR and PPARalpha target gene, consistent with the role of these receptors in regulation of proinflammatory genes in macrophages.

The extrinsic and intrinsic apoptotic pathways are differentially affected by temperature upstream of mitochondrial damage.

It is well known that mild hypothermia prevents neuronal cell death following cerebral ischemia, although it can also cause apoptosis in other cell types. Thus, incubation at room temperature (RT) has been shown to induce apoptosis in hematopoietic cells, including Jurkat T leukemia cells. To further understand the apoptotic events that can be activated at RT, we compared the induction of apoptosis by several apoptotic insults in Jurkat cells stimulated at 37 degrees C or RT. Retinoid-related molecules, which induce apoptosis via the intrinsic pathway, failed to induce apoptosis when cells were treated at RT, as determined by various apoptotic parameters including cytochrome c release and activation of caspase 3. In contrast, most apoptotic events were enhanced by lower temperatures when cells were stimulated with anti-Fas antibody via the extrinsic pathway. Ultraviolet radiation produced partial effects at RT, correlating with its capacity to activate both pathways. Our results indicate that the core caspase machinery is operational under mild hypothermia conditions. Experiments using purified recombinant caspases and cell-free assays confirmed that caspases are fully functional at RT. Other hallmark events of apoptosis, such as phosphatidylserine externalization and formation of apoptotic bodies were variably affected by RT in a stimulus-dependent manner, suggesting the existence of critical steps that are sensitive to temperature. Thus, analysis of apoptosis at RT might be useful to (i) discriminate between the extrinsic and intrinsic pathways in Jurkat cells treated with prospective stimuli, and (ii) to unravel temperature-sensitive steps of apoptotic signaling cascades.

In silico identification of novel EGFR inhibitors with antiproliferative activity against cancer cells.

Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase activity by small molecules has proved effective for the treatment of cancer. To the best of our knowledge, the crystal structure of EGFR has been used for the first time to identify novel inhibitor chemotypes by docking-based in silico screening of a large virtual chemical library followed up by experimental validation. We identified several compounds with antiproliferative effects on cancer cells. Amongst them, a C(4)-N(1)-substituted pyrazolo[3,4-d]pyrimidine MSK-039 (39) was discovered as a low-micromolar inhibitor of EGFR tyrosine kinase activity. The predicted binding mode of 39 opens a new avenue toward the optimization of novel chemical entities to develop potent and selective inhibitors of EGFR signaling.

The retinoid antagonist MX781 induces clusterin expression in prostate cancer cells via heat shock factor-1 and activator protein-1 transcription factors.

Retinoids mediate numerous biological responses through the transcriptional activation of nuclear retinoid receptors. Due to their antiproliferative activity, retinoids have shown promise as anticancer agents. Synthetic analogs have been described that selectively activate one subset of the retinoid receptors or inhibit their transcriptional activity. Some of these compounds exhibit strong anticancer activity, which is associated with their ability to induce apoptosis. Here we describe that the retinoid antagonist MX781 causes a substantial increase of clusterin mRNA and protein levels in prostate carcinoma cells. In contrast, retinoic acid and other synthetic agonists and antagonists show no effect on clusterin mRNA/protein levels. Induction of clusterin mRNA is associated with transcriptional activation of the clusterin promoter, which requires the proximal -218-bp region containing binding sites for heat shock factor (HSF)-1, activator protein (AP)-2, and AP-1 transcription factors. MX781 slightly induces AP-1 DNA binding activity, and mutation of the AP-1 site differentially affects the activation of the clusterin promoter in a cell type-specific manner. In contrast, a robust increase of HSF-1 DNA binding activity is observed in all cancer cell lines examined, and mutation of the heat shock element site in the clusterin promoter completely abolishes MX781-induced transcriptional activation in PC3 and DU145 cells. Other agonist retinoid-related molecules also induce AP-1 activity, but not HSF-1, and elicit no effect on clusterin expression levels. These data point to HSF-1 as an important factor regulating clusterin expression in response to MX781, although AP-1 activity may also participate in a cell type-specific manner.

Peroxisome proliferator-activated receptor gamma ligands regulate myeloperoxidase expression in macrophages by an estrogen-dependent mechanism involving the -463GA promoter polymorphism.

A functional myeloperoxidase (MPO) promoter polymorphism, -463GA, has been associated with incidence or severity of inflammatory diseases, including atherosclerosis and Alzheimer's disease, and some cancers. The polymorphism is within an Alu element encoding four hexamer repeats recognized by nuclear receptors (AluRRE). Here we show that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists strongly regulate MPO gene expression through the AluRRE. Opposite effects were observed in granulocyte/macrophage colony-stimulating factor (GMCSF)- versus macrophage colony-stimulating factor (MCSF)-derived macrophages (Mphi): Expression was markedly up-regulated (mean 26-fold) in MCSF-Mphi and down-regulated (34-fold) in GMCSF-Mphi. This was observed with rosiglitazone and three other PPARgamma ligands of the thiazolidinedione class, as well as the natural prostaglandin metabolite 15-deoxy-Delta(12,14) prostaglandin J(2). The selective PPARgamma antagonist, GW9662, blocked both the positive and negative effects on MPO expression. Gel retardation assays showed PPARgamma bound hexamers 3/4, and estrogen receptor-alpha bound hexamers 1/2, with -463A in hexamer 1 enhancing binding. Estrogen blocked PPARgamma effects on MPO expression, especially for the A allele. Charcoal filtration of fetal calf serum eliminated the block of PPARgamma, whereas replenishing the medium with 17beta-estradiol reinstated the block. These findings suggest a model in which estrogen receptor binds the AluRRE, preventing PPARgamma binding to the adjacent site. The positive and negative regulation by PPARgamma ligands, and the block by estrogen, was also observed in transgenic mice expressing the G and A alleles. The mouse MPO gene, which lacks the primate-specific AluRRE, was unresponsive to PPARgamma ligands, suggesting the human MPO transgenes will enhance the utility of mouse models for diseases involving MPO, such as atherosclerosis and Alzheimer's.

Reduced concentrations of serum enhance the antiproliferative activity of retinoid-related molecules and accelerate the onset of apoptosis.

Retinoid-related molecules (RRMs) that are selective agonists for the retinoic acid receptor-gamma and one retinoid antagonist are potent inducers of apoptosis in various cancer cell lines. This cell-killing activity makes them promising candidates for their use as anticancer drugs. We have observed that reducing the amount of serum in the cell culture medium significantly increased the antiproliferative activity of these RRMs in a serum concentration dependent manner. The induction of caspase activity, DNA fragmentation, and externalization of phosphatidylserine by the RRMs was markedly reduced when cells were treated in medium containing 10% serum, as compared to cells treated in low serum. High concentrations of serum also inhibited the activation of stress kinases by RRMs and higher amounts of the retinoid derivatives were necessary to cause quantitatively similar effects as compared to treatments in medium containing low serum. We have demonstrated that high concentrations of serum in the culture medium prevented the intracellular accumulation of MX3350-1 (agonist). Moreover, pre-incubation of cells in low serum-containing medium accelerated the onset of apoptosis as evidenced by the rapid activation of caspases and formation of apoptotic bodies. The release of cytochrome c and Smac induced by RRMs occurred earlier in cells that had been pre-incubated in 0.5% serum, while the activation of JNK and p38 stress kinases was unaffected.

Z-FA-fmk inhibits effector caspases but not initiator caspases 8 and 10, and demonstrates that novel anticancer retinoid-related molecules induce apoptosis via the intrinsic pathway.

Synthetic retinoid-related molecules (RRMs) have been described that show strong antiproliferative activity and induce apoptosis in cancer cells. These RRMs induce caspase activity independently of the retinoid receptors in Jurkat T cells. We observed that the inhibitor of cathepsins B and L Z-FA-fmk blocks the induction of DEVDase activity, DNA fragmentation, and externalization of phosphatidylserine by selective RRMs. Z-FA-fmk can inhibit caspase activity in vitro and selectively inhibits recombinant effector caspases 2, -3, -6, and -7. In contrast, purified initiator caspases 8 and 10 are not affected, whereas the apoptosome-associated caspase 9 is only partially inhibited by Z-FA-fmk in vitro. These data correlate with the covalent binding of biotinylated Z-FA-fmk to the active large subunit of effector caspases. This selective targeting of effector caspases is also observed in Jurkat cells and has been used to demonstrate that RRMs induce apoptosis through the mitochondrial pathway and activate caspase 8 in a Z-FA-fmk-sensitive manner. Thus, Z-FA-fmk fails to inhibit Fas-mediated activation of caspase 8, but completely inhibits RRM-induced processing of caspase 8. Z-FA-fmk does not prevent the autoproteolytic cleavage of caspase 9 in Jurkat cells and partially inhibits the processing and full maturation of effector caspases induced by the RRMs. Moreover, Z-VAD-fmk and Z-FA-fmk have no effect on the release of cytochrome c induced by the RRMs. Other cathepsin inhibitors elicit no effect on RRM-induced apoptosis in Jurkat cells, suggesting that caspases are the major effectors of RRM action.