Integrated Stress Response Potentiates Ponatinib-Induced Cardiotoxicity.

BACKGROUND: Mitochondrial dysfunction is a primary driver of cardiac contractile failure; yet, the cross talk between mitochondrial energetics and signaling regulation remains obscure. Ponatinib, a tyrosine kinase inhibitor used to treat chronic myeloid leukemia, is among the most cardiotoxic tyrosine kinase inhibitors and causes mitochondrial dysfunction. Whether ponatinib-induced mitochondrial dysfunction triggers the integrated stress response (ISR) to induce ponatinib-induced cardiotoxicity remains to be determined. METHODS: Using human induced pluripotent stem cells-derived cardiomyocytes and a recently developed mouse model of ponatinib-induced cardiotoxicity, we performed proteomic analysis, molecular and biochemical assays to investigate the relationship between ponatinib-induced mitochondrial stress and ISR and their role in promoting ponatinib-induced cardiotoxicity. RESULTS: Proteomic analysis revealed that ponatinib activated the ISR in cardiac cells. We identified GCN2 (general control nonderepressible 2) as the eIF2α (eukaryotic translation initiation factor 2α) kinase responsible for relaying mitochondrial stress signals to trigger the primary ISR effector-ATF4 (activating transcription factor 4), upon ponatinib exposure. Mechanistically, ponatinib treatment exerted inhibitory effects on ATP synthase activity and reduced its expression levels resulting in ATP deficits. Perturbed mitochondrial function resulting in ATP deficits then acts as a trigger of GCN2-mediated ISR activation, effects that were negated by nicotinamide mononucleotide, an NAD+ precursor, supplementation. Genetic inhibition of ATP synthase also activated GCN2. Interestingly, we showed that the decreased abundance of ATP also facilitated direct binding of ponatinib to GCN2, unexpectedly causing its activation most likely because of a conformational change in its structure. Importantly, administering an ISR inhibitor protected human induced pluripotent stem cell-derived cardiomyocytes against ponatinib. Ponatinib-treated mice also exhibited reduced cardiac function, effects that were attenuated upon systemic ISRIB administration. Importantly, ISRIB does not affect the antitumor effects of ponatinib in vitro. CONCLUSIONS: Neutralizing ISR hyperactivation could prevent or reverse ponatinib-induced cardiotoxicity. The findings that compromised ATP production potentiates GCN2-mediated ISR activation have broad implications across various cardiac diseases. Our results also highlight an unanticipated role of ponatinib in causing direct activation of a kinase target despite its role as an ATP-competitive kinase inhibitor.

Recent advances in metabolism and toxicity of tyrosine kinase inhibitors.

Small molecule tyrosine kinase inhibitors (TKIs) are widely used as anticancer drugs approved by U.S. FDA. However, the toxicities of TKIs to multiple organs have greatly limited their clinical applications. The metabolism of TKIs generates several potentially toxic metabolites in vivo, that can disturb the endogenous metabolism as well as cellular function, leading to organ damage. Therefore, it is essential to identify the toxic metabolites and elucidate the underlying mechanism of TKI-induced toxicity. Metabolomics is a powerful tool for the identification of the xenobiotic metabolites and metabolic derangement associated with xenobiotic exposure, that is helpful to understand the toxicity of TKIs. The study using metabolomics approach has revealed that the reactive metabolites/intermediates (e.g., N-oxide metabolite, primary amine metabolite, 1,4-benzoquinone intermediate) and adducts with glutathione, cysteine and mercapturic acid can be derived from TKIs. Fourteen metabolic pathways could be affected following the TKI treatment, including lipid metabolism, bile acid metabolism, and gut microbiota-related pathway. Modulation of xenobiotic receptor signaling, inhibition of xenobiotic metabolism, and supplementation of endogenous metabolites are potential strategies to protect against TKI-induced toxicity. In this review, studies on the metabolism of TKIs and the alterations of endogenous metabolism are discussed, and the potential preventions against TKI-induced toxicity are summarized.

Calcium-activated chloride channel is involved in the onset of diarrhea triggered by EGFR tyrosine kinase inhibitor treatment in rats.

EGFR tyrosine kinase inhibitors (TKIs) are mainly used to treat non-small cell lung cancer; however, adverse effects such as severe diarrhea represent a major obstacle towards the continuation of EGFR-TKIs therapy. Chloride channels, which control the fluid flow in the intestinal lumen, are proposed as an important target to remediate EGFR-TKIs-induced diarrhea, but the mechanism remains unclear. The aim of this study was to clarify the mechanism underlying EGFR-TKIs-induced diarrhea with a particular focus on the role of intestinal chloride channels. Here, we show that osimertinib-treated rats exhibit diarrhea and an increase in fecal water content without showing any severe histopathological changes. This diarrhea was attenuated by intraperitoneal treatment with the calcium-activated chloride channel (CaCC) inhibitor CaCCinh-A01. These findings were confirmed in afatinib-treated rats with diarrhea. Moreover, treatment with the Japanese traditional herbal medicine, hangeshashinto (HST), decreased fecal water content and improved fecal appearance in rats treated with EGFR-TKIs. HST inhibited the ionomycin-induced CaCC activation in HEK293 cells in patch-clamp current experiments and its active ingredients were identified. In conclusion, secretory diarrhea induced by treatment with EGFR-TKIs might be partially mediated by the activation of CaCC. Therefore, blocking the CaCC could be a potential new treatment for EGFR-TKI-induced diarrhea.

A novel monovalent FGFR1 antagonist: Preclinical safety profiles in rodents and non-human primates.

Blocking Fibroblast Growth Factor Receptor 1 (FGFR1) is an attractive therapeutic option for treatment of cancer subtypes with amplification and over-expression of FGFR1. Selective targeting of FGFR1 can be achieved using an antibody-based approach, as small molecule inhibitors may not discriminate between FGFR1, 2, 3 and 4 due to their highly homologous kinase domain. However, development of classical bivalent FGFR1 directed antibodies has failed due to non-tolerated body weight decreases in preclinical species. M6123 is a novel IgG-like monovalent FGFR1 specific binder with enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) effector function and inhibits tumor growth significantly in FGFR1-dependent human xenograft models without reduced body weight in tumor-bearing mice. Toxicology studies reported here characterized the safety profile of M6123 in mouse, rat, and monkey. There were significant differences among animal species under similar M6123 exposure levels. Rats showed metastatic mineralization with an imbalance in serum phosphate at low doses, while mineralization was not found in mice or monkeys, even though hyperphosphatemia was detected in mice. Subtle differences in calcium/phosphate homoeostasis feedback loops may trigger the susceptibility to mineralization among animal species; nevertheless, the exact mechanism remains unknown. Monkeys showed marked, but reversible, decreases in peripheral blood NK cells and neutrophils. The latter was associated with considerably increased neutrophilic infiltrates in the liver sinusoids and red pulp of the spleen. These effects in monkeys are likely related to the enhanced ADCC activity of M6123. Overall, M6123 showed a superior safety profile in animals compared to bivalent FGFR1 antagonists or pan-FGFR inhibitors.

Dasatinib can Impair Left Ventricular Mechanical Function But May Lack Proarrhythmic Effect: A Proposal of Non-clinical Guidance for Predicting Clinical Cardiovascular Adverse Events of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors are known to clinically induce various types of cardiovascular adverse events; however, it is still difficult to predict them at preclinical stage. In order to explore how to better predict such drug-induced cardiovascular adverse events, we tried to develop a new protocol by assessing acute electrophysiological, cardiohemodynamic, and cytotoxic effects of dasatinib in vivo and in vitro. Dasatinib at 0.03 and 0.3 mg/kg was intravenously administered to the halothane-anesthetized dogs for 10 min with an interval of 20 min between the dosing (n = 4). Meanwhile, that at 0.1, 0.3, and 1 µM was cumulatively applied to the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (n = 7). In the dogs, the low and high doses provided peak plasma concentrations of 40 ± 5 (0.08) and 615 ± 38 ng/mL (1.26 µM), respectively. The low dose decreased the heart rate, impaired the left ventricular mechanical function, and prolonged the ventricular effective refractory period. The high dose prolonged the repolarization period, induced hemorrhagic tendency, and increased plasma cardiac troponin I level in addition to enhancement of the changes observed after the low dose, whereas it neither affected the cardiac conduction nor induced ventricular arrhythmias. In the hiPSC-CMs, dasatinib prolonged the repolarization and refractory periods like in dogs, while it did not induce apoptotic or necrotic process, but that it increased the conduction speed. Clinically observed major cardiovascular adverse events of dasatinib were observed qualitatively by currently proposed assay protocol, which may become a useful guide for predicting the cardiotoxicity of new tyrosine kinase inhibitors.

Carnitine Supplementation Attenuates Sunitinib-Induced Inhibition of AMP-Activated Protein Kinase Downstream Signals in Cardiac Tissues.

This study has been initiated to investigate whether sunitinib (SUN) alters the expression of key genes engaged in mitochondrial transport and oxidation of long chain fatty acids (LCFA), and if so, whether these alterations should be viewed as a mechanism of SUN-induced cardiotoxicity, and to explore the molecular mechanisms whereby carnitine supplementation could attenuate SUN-induced cardiotoxicity. Adult male Wister albino rats were assigned to one of the four treatment groups: Rats in group 1 received no treatment but free access to tap water for 28 days. Rats in group 2 received L-carnitine (200 mg/kg/day) in drinking water for 28 days. Rats in group 3 received SUN (25 mg/kg/day) in drinking water for 28 days. Rats in group 4 received the same doses of L-carnitine and SUN in drinking water for 28 days. Treatment with SUN significantly increased heart weight, cardiac index, and cardiotoxicity enzymatic indices, as well as severe histopathological changes. Moreover, SUN significantly decreased level of adenosine monophosphate-activated protein kinase (AMPKα2), total carnitine, adenosine triphosphate (ATP) and carnitine palmitoyltransferase I (CPT I) expression and significantly increased acetyl-CoA carboxylase-2 (ACC2) expression and malonyl-CoA level in cardiac tissues. Interestingly, carnitine supplementation resulted in a complete reversal of all the biochemical, gene expression and histopathological changes-induced by SUN to the control values. In conclusion, data from this study suggest that SUN inhibits AMPK downstream signaling with the consequent inhibition of mitochondrial transport of LCFA and energy production in cardiac tissues. Carnitine supplementation attenuates SUN-induced cardiotoxicity.

Bioactivities of Geranium wallichianum Leaf Extracts Conjugated with Zinc Oxide Nanoparticles.

This study attempts to obtain and test the bioactivities of leaf extracts from a medicinal plant, Geranium wallichianum (GW), when conjugated with zinc oxide nanoparticles (ZnONPs). The integrity of leaf extract-conjugated ZnONPs (GW-ZnONPs) was confirmed using various techniques, including Ultraviolet-visible spectroscopy, X-Ray Diffraction, Fourier Transform Infrared Spectroscopy, energy-dispersive spectra (EDS), scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy. The size of ZnONPs was approximately 18 nm, which was determined by TEM analysis. Additionally, the energy-dispersive spectra (EDS) revealed that NPs have zinc in its pure form. Bioactivities of GW-ZnONPs including antimicrobial potentials, cytotoxicity, antioxidative capacities, inhibition potentials against α-amylase, and protein kinases, as well as biocompatibility were intensively tested and confirmed. Altogether, the results revealed that GW-ZnONPs are non-toxic, biocompatible, and have considerable potential in biological applications.

Report- Antioxidant, cytotoxicity, protein kinase inhibition and antibacterial activities of Fragaria x ananassa leaves.

Fragaria × ananassa leaves extracts prepared in different solvents were subject for antioxidative, cytotoxicity, protein kinase inhibition and antibacterial activities. The extracts showed varying activities depending upon solvent used for extraction. Combined effect of methanol and ethyl acetate showed maximum antioxidant and reducing power potential (207.65±6µg AAE/mg and 88.58±20µg AAE/mg, respectively). Maximum DPPH (2,2-diphenyl-1-picryl hydrazyl) free radical scavenging activity was calculated by when methanol: chloroform and acetate fractions were used (87.68% and 86.88% inhibition, respectively). Total phenolics varied from 186 to 1.91µg AAE/mg while total flavonoids also significantly varied among the extracts. The extracts also showed significant activities against brine shrimp larvae and bacterial strains tested. The study concludes that Fragaria × ananassa leaves can be a good source for isolation of active phytochemicals to be used in different industries.

Discovery of GDC-0853: A Potent, Selective, and Noncovalent Bruton's Tyrosine Kinase Inhibitor in Early Clinical Development.

Bruton's tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.

The immune system as a chronotoxicity target of the anticancer mTOR inhibitor everolimus.

The circadian timing system controls many biological functions in mammals including xenobiotic metabolism, detoxification, cell proliferation, apoptosis and immune functions. Everolimus is a mammalian target of rapamycin inhibitor, whose immunosuppressant properties are both desired in transplant patients and unwanted in cancer patients, where it is indicated for its antiproliferative efficacy. Here we sought whether everolimus circadian timing would predictably modify its immunosuppressive effects so as to optimize this drug through timing. C57BL/6J mice were synchronized with light-dark 12h:12h, with L onset at Zeitgeber Time (ZT) 0. Everolimus was administered orally to male (5 mg/kg/day) and female mice (15 mg/kg/day) at ZT1, during early rest span or at ZT13, during early activity span for 4 weeks. Body weight loss, as well as hematological, immunological and biochemical toxicities, were determined. Spleen and thymus were examined histologically. Everolimus toxicity was less severe following dosing at ZT13, as compared to ZT1, as shown with least body weight inhibition in both genders; least reductions in thymus weight both in males (p < 0.01) and females (p < 0.001), least reduction in female spleen weight (p < 0.05), and less severe thymic medullar atrophy both in males (p < 0.001) and females (p < 0.001). The mean circulating counts in total leukocytes, total lymphocytes, T-helper and B lymphocytes displayed minor and non-significant changes following dosing at ZT13, while they were decreased by 56.9% (p < 0.01), 45.5% (p < 0.01), 43.1% (p < 0.05) and 48.7% (p < 0.01) after everolimus at ZT1, respectively, in only male mice. Chronotherapy of everolimus is an effective way to increase the general tolerability and decrease toxicity on the immune system.

Identification of Atuveciclib (BAY†1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer.

Selective inhibition of exclusively transcription-regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY-958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer.

Melatonin reverses H-89 induced spatial memory deficit: Involvement of oxidative stress and mitochondrial function.

Oxidative stress and mitochondrial dysfunction play indispensable role in memory and learning impairment. Growing evidences have shed light on anti-oxidative role for melatonin in memory deficit. We have previously reported that inhibition of protein kinase A by H-89 can induce memory impairment. Here, we investigated the effect of melatonin on H-89 induced spatial memory deficit and pursued their interactive consequences on oxidative stress and mitochondrial function in Morris Water Maze model. Rats received melatonin (50 and 100µg/kg/side) and H-89(10µM) intra-hippocampally 30min before each day of training. Animals were trained for 4 consecutive days, each containing one block from four trials. Oxidative stress indices, including thiobarbituric acid (TBARS), reactive oxygen species (ROS), thiol groups, and ferric reducing antioxidant power (FRAP) were assessed using spectrophotometer. Mitochondrial function was evaluated through measuring ROS production, mitochondrial membrane potential (MMP), swelling, outer membrane damage, and cytochrome c release. As expected from our previous report, H-89 remarkably impaired memory by increasing the escape latency and traveled distance. Intriguingly, H-89 significantly augmented TBARS and ROS levels, caused mitochondrial ROS production, swelling, outer membrane damage, and cytochrome c release. Moreover, H-89 lowered thiol, FRAP, and MMP values. Intriguingly, melatonin pre-treatment not only effectively hampered H-89-mediated spatial memory deficit at both doses, but also reversed the H-89 effects on mitochondrial and biochemical indices upon higher dose. Collectively, these findings highlight a protective role for melatonin against H-89-induced memory impairment and indicate that melatonin may play a therapeutic role in the treatment of oxidative- related neurodegenerative disorders.

Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

Transarterial Chemoembolization Using Sorafenib in a Rabbit VX2 Liver Tumor Model: Pharmacokinetics and Antitumor Effect.

PURPOSE: To investigate feasibility, safety, and effect of transarterial chemoembolization using sorafenib on degree of tumor necrosis in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: New Zealand White rabbits (n = 20) with a VX2 tumor were divided into two groups; one group was treated with hepatic arterial administration of 0.5 mL ethiodized oil alone (Lipiodol; Guerbet, Aulnay-sous-Bois, France) (transarterial embolization with Lipiodol [TAE-L] group), and one group was treated with 0.5 mL ethiodized oil plus 10 mg sorafenib (transarterial embolization with sorafenib [TAE-S] group). Liquid chromatography tandem mass spectrometry was used to measure sorafenib concentration in peripheral blood and tissue. Hepatic enzymes, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1α (HIF-1α) were measured at 0, 24, and 72 hours after treatment. Histopathologic examination was performed to evaluate extent of tumor necrosis and normal parenchymal damage. RESULTS: Serum sorafenib concentration peaked at 2 hours after treatment. The mean tissue concentration was 406.8 times greater than the serum concentration. Aspartate aminotransferase and alanine aminotransferase levels were significantly elevated in the TAE-S group at 24 hours after treatment. Serum VEGF and HIF-1α concentrations were not significantly different between the TAE-L and TAE-S groups. Hepatic parenchymal damage was more severe in the TAE-S group. Mean fraction of tumor necrosis after treatment was significantly greater in the TAE-S group. CONCLUSIONS: Transarterial chemoembolization using sorafenib resulted in a high intrahepatic concentration of sorafenib. The degree of tumor necrosis was significantly greater in the TAE-S group compared with the TAE-L group, but more severe toxicity of normal liver tissue also occurred.

Discovery of novel Bcr-Abl inhibitors targeting myristoyl pocket and ATP site.

Bcr-Abl plays an essential role in the pathogenesis and development of chronic myeloid leukaemia (CML). Inhibition of Bcr-Abl has great potential for therapeutic intervention in CML. In order to obtain novel and potent Bcr-Abl inhibitors, twenty seven 4,6-disubstituted pyrimidines were synthesized and evaluated herein. The biological results indicated that four compounds of them (C4, C5, C16, and C23) were potent Bcr-Abl inhibitors which were comparable to positive control. Moreover, C4 and C5 displayed promising antiproliferative activity against K562 cells. The results suggested that these 4,6-disubstituted pyrimidines could serve as promising leads for further optimization of Bcr-Abl inhibitors.

Discovery andw biological evaluation of novel 6,7-disubstituted-4-(2-fluorophenoxy)quinoline derivatives possessing 1,2,3-triazole-4-carboxamide moiety as c-Met kinase inhibitors.

A series of 6,7-disubstituted-4-(2-fluorophenoxy)quinoline derivatives possessing 1,2,3-triazole-4-carboxamide moiety were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase and five typical cancer cell lines (A549, H460, HT-29, MKN-45 and U87MG). Most compounds showed moderate to excellent antiproliferative activity. In this study, a promising compound 34, with a c-Met IC50 value of 1.04nM, was identified as a multitargeted receptor tyrosine kinase inhibitor. The SAR analyses indicated that compounds with halogen group, especially fluoro group, at 4-position on the phenyl ring (moiety B) have potent antitumor activity, and methylation on the 5-atom linker played an important role in the c-Met enzymatic activity.

Substrate activity screening with kinases: discovery of small-molecule substrate-competitive c-Src inhibitors.

Substrate-competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small-molecule substrates for any protein kinase were discovered, as well as the first substrate-competitive inhibitors of c-Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate-competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP-competitive inhibitors.

Phenotypic profiling of Raf inhibitors and mitochondrial toxicity in 3D tissue using biodynamic imaging.

The existence of phenotypic differences in the drug responses of 3D tissue relative to 2D cell culture is a concern in high-content drug screening. Biodynamic imaging is an emerging technology that probes 3D tissue using short-coherence dynamic light scattering to measure the intracellular motions inside tissues in their natural microenvironments. The information content of biodynamic imaging is displayed through tissue dynamics spectroscopy (TDS) but has not previously been correlated against morphological image analysis of 2D cell culture. In this article, a set of mitochondria-affecting compounds (FCCP, valinomycin, nicardipine, ionomycin) and Raf kinase inhibitors (PLX4032, PLX4720, GDC, and sorafenib) are applied to multicellular tumor spheroids from two colon adenocarcinoma cell lines (HT-29 and DLD-1). These were screened by TDS and then compared against conventional image-based high-content analysis (HCA). The responses to the Raf inhibitors PLX4032 and PLX4720 are grouped separately by cell line, reflecting the Braf/Kras difference in these cell lines. There is a correlation between TDS and HCA phenotypic clustering for most cases, which demonstrates the ability of dynamic measurements to capture phenotypic responses to drugs. However, there are significant 2D versus 3D phenotypic differences exhibited by several of the drugs/cell lines.

Discovery and optimization of novel 4-phenoxy-6,7-disubstituted quinolines possessing semicarbazones as c-Met kinase inhibitors.

A novel series of N(1)-(3-fluoro-4-(6,7-disubstituted-quinolin-4-yloxy)phenyl)-N(4)-arylidenesemicarbazide derivatives were synthesized and evaluated for their c-Met kinase inhibition and cytotoxicity against A549, HT-29, MKN-45 and MDA-MB-231 cancer cell lines in vitro. Several potent compounds were further evaluated against three other cancer cell lines (U87MG, NCI-H460 and SMMC7721). Most of compounds tested exhibited moderate to excellent activity. The studies of SARs identified the most promising compound 28 (c-Met IC50=1.4nM) as a c-Met kinase inhibitor. In this study, a promising compound 28 was identified, which displayed 2.1-, 3.3-, 48.4- and 3.6-fold increase against A549, HT-29, U87MG and NCI-H460 cell lines, respectively, compared with that of Foretinib.

A combination strategy to inhibit Pim-1: synergism between noncompetitive and ATP-competitive inhibitors.

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.