Selective Src Kinase Inhibitor Research Articles

Functionalized 2-Amino-4H-1-Benzopyran-4-yl Phosphonate Scaffolds: Synthesis, Anticancer Evaluation and Computational Studies

Prostate, breast, and lung cancers are various human cancers that have been associated with SRC kinase mutations. Therefore, the development of potent and selective SRC kinase inhibitors is an exciting area of interest for researchers. In this study, we synthesized a series of 2-amino-4H-1-benzopyran-4-yl phosphonate derivatives through a one-pot reaction involving appropriately substituted salicylaldehydes, malononitrile, and diethyl phosphite, with meglumine as the catalyst. We conducted in vitro screening of these compounds for their anticancer activity against human prostate cancer (DU-145), breast cancer (MCF-7), and lung cancer (A549) cell lines using the MTT assay. Notably, compounds 4a, 4j, 4k, and 4l exhibited promising anticancer activity. Molecular docking studies of these compounds supported their potential as therapeutic agents for SRC kinase inhibition. Specifically, compounds 4a, 4b, 4c, 4d, and 4e demonstrated strong ΔG binding affinities ranging from −7.9 to −7.4 kcal/mol. These findings suggest that these molecules hold promise for cancer treatment.

Medicinal Chemistry Perspectives on Recent Advances in Src Kinase Inhibitors as a Potential Target for the Development of Anticancer Agents: Biological Profile, Selectivity, Structure-Activity Relationship.

The physiological Src proto-oncogene is a protein tyrosine kinase receptor that served as the essential signaling pathway in different types of cancer. Src kinase receptor is divided into different domains: a unique domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain, and a regulatory tail, which runs from the N-terminus to the C-terminus. Src kinase inhibitors bind in the kinase domain and are activated by phosphorylation. The etiology of cancer involved various signaling pathways and Src signaling pathways are also involved in those clusters. Although the dysregulation of Src kinase resulted in cancer being discovered in the late 19th century it is still considered a cult pathway because it is not much explored by different medicinal chemists and oncologists. The Src kinase regulated through different kinase pathways (MAPK, PI3K/Akt/mTOR, JAK/STAT3, Hippo kinase, PEAK1, and Rho/ROCK pathways) and proceeded downstream signaling to conduct cell proliferation, angiogenesis, migration, invasion, and metastasis of cancer cells. There are numerous FDA-approved drugs flooded the market but still, there is a huge demand for the creation of novel anticancer drugs. As the existing drugs are accompanied by several adverse effects and drug resistance due to rapid mutation in proteins. In this review, we have elaborated about the structure and activation of Src kinase, as well as the development of Src kinase inhibitors. Our group also provided a comprehensive overview of Src inhibitors throughout the last two decades, including their biological activity, structure-activity relationship, and Src kinase selectivity. The Src binding pocket has been investigated in detail to better comprehend the interaction of Src inhibitors with amino acid residues. We have strengthened the literature with our contribution in terms of molecular docking and ADMET studies of top compounds. We hope that the current analysis will be a useful resource for researchers and provide glimpse of direction toward the design and development of more specific, selective, and potent Src kinase inhibitors.

Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) invitro in normal human lung fibroblasts; 2) invivo in bleomycin and recombinant Ad-TGF-β (adenovirus transforming growth factor-β) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-β-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-β, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

SAR Probing of KX2-391 Provided Analogues With Juxtaposed Activity Profile Against Major Oncogenic Kinases.

Tirbanibulin (KX2-391, KX-01), a dual non-ATP (substrate site) Src kinase and tubulin-polymerization inhibitor, demonstrated a universal anti-cancer activity for variety of cancer types. The notion that KX2-391 is a highly selective Src kinase inhibitor have been challenged by recent reports on the activities of this drug against FLT3-ITD mutations in some leukemic cell lines. Therefore, we hypothesized that analogues of KX2-391 may inhibit oncogenic kinases other than Src. A set of 4-aroylaminophenyl-N-benzylacetamides were synthesized and found to be more active against leukemia cell lines compared to solid tumor cell lines. N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-4-chlorobenzamide (4e) exhibited activities at IC50 0.96 µM, 1.62 µM, 1.90 µM and 4.23 µM against NB4, HL60, MV4-11 and K562 leukemia cell lines, respectively. We found that underlying mechanisms of 4e did not include tubulin polymerization or Src inhibition. Such results interestingly suggested that scaffold-hopping of KX2-391 may change the two main underlying cytotoxic mechanisms (Src and tubulin). Kinase profiling using two methods revealed that 4e significantly reduces the activities of some other potent oncogenic kinases like the MAPK member ERK1/2 (>99%) and it also greatly upregulates the pro-apoptotic c-Jun kinase (84%). This research also underscores the importance of thorough investigation of total kinase activities as part of the structure-activity relationship studies.

Results of a Randomized, Double-Blinded, Placebo-Controlled, Phase 2.5 Study of Saracatinib (AZD0530), in Patients with Recurrent Osteosarcoma Localized to the Lung.

Purpose Osteosarcoma is a rare cancer and a third of patients who have completed primary treatment will develop osteosarcoma recurrence. The Src pathway has been implicated in the metastatic behavior of osteosarcoma; about 95% of samples examined express Src or have evidence of downstream activation of this pathway. Saracatinib (AZD0530) is a potent and selective Src kinase inhibitor that was evaluated in adults in Phase 1 studies. The primary goal of this study was to determine if treatment with saracatinib could increase progression-free survival (PFS) for patients who have undergone complete resection of osteosarcoma lung metastases in a double-blinded, placebo-controlled trial. Patients and Methods. Subjects with recurrent osteosarcoma localized to lung and who had complete surgical removal of all lung nodules were randomized within six weeks after complete surgical resection. Saracatinib, or placebo, was administered at a dose of 175 mg orally, once daily, for up to thirteen 28-day cycles. Results Thirty-seven subjects were included in the analyses; 18 subjects were randomized to receive saracatinib and 19 to receive placebo. Intent-to-treat analysis demonstrated a median PFS of 19.4 months in the saracatinib treatment group and 8.6 months in the placebo treatment group (p=0.47). Median OS was not reached in either arm. Conclusions Although saracatinib was well tolerated in this patient population, there was no apparent impact of the drug in this double-blinded, placebo-controlled trial on OS, and Src inhibition alone may not be sufficient to suppress metastatic progression in osteosarcoma. There is a suggestion of potential clinical benefit as evidenced by longer PFS in patients randomized to saracatinib based on limited numbers of patients treated.

Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation.

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.

Effect of Concurrent Src Kinase Inhibition with Short-Duration Hypothermia on Ca2+/Calmodulin Kinase IV Activity and Neuropathology after Hypoxia-Ischemia in the Newborn Swine Brain

Background: Hypoxia-ischemia (HI) results in increased activation of Ca2+/calmodulin kinase IV (CaM kinase IV) mediated by Src kinase. Therapeutic hypothermia ameliorates neuronal injury in the newborn. Hypothesis: Inhibition of Src kinase concurrently with hypothermia further attenuates the hypoxia-induced increased activation of CaM kinase IV compared with hypothermia alone. Design/Methods: Ventilated piglets were exposed to HI, received saline or a selective Src kinase inhibitor (PP2), and were cooled to 33°C. Neuropathology, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations, and CaM kinase IV activity were determined. Results: The neuropathology mean score (mean ± SD) was 0.4 ± 0.43 in normoxia-normothermia (p < 0.05 vs. hypoxia-normothermia), 3.5 ± 0.89 in hypoxia-normothermia (p < 0.05 vs. normoxia-normothermia), 0.7 ± 0.73 in hypoxia-hypothermia (p < 0.05 vs. normoxia-normothermia), and 0.5 ± 0.70 in normoxia-hypothermia (p < 0.05 vs. hypoxia-normothermia). The CaM kinase IV activity in cerebral tissue (pmol Pi/mg protein/min; mean ± SD) was 2,002 ± 729 in normoxia-normothermia, 1,704 ± 18 in normoxia-hypothermia, 6,017 ± 2,510 in hypoxia-normothermia, 4,104 ± 542 in hypoxia-hypothermia (p < 0.05 vs. normoxia-hypothermia), and 2,165 ± 415 in hypoxia-hypothermia with PP2 (p < 0.05 vs. hypoxia-hypothermia). The hypoxic groups with and without hypothermia or Src kinase inhibitor were comparable in the levels of ATP and PCr, indicating that they were similar in their degree of energy failure prior to treatments. Hypothermia or Src kinase inhibitor (PP2) did not restore the ATP and PCr levels. Conclusions: Hypothermia and Src kinase inhibition attenuated apoptotic cell death and improved neuropathology after hypoxia. The combination of short-duration hypothermia with Src kinase inhibition following hypoxia further attenuates the increased activation of CaM kinase IV compared to hypothermia alone in the newborn swine brain.

Cytochrome P450 Mediated Bioactivation of Saracatinib.

Saracatinib is a highly selective Src kinase inhibitor against all Src kinase family members and has demonstrated anticancer effects in preclinical models. Unfortunately, it has shown multiple adverse effects during its clinical trials, along with time-dependent inhibition of P450 enzymes. The major objective of this study was to identify reactive metabolites of saracatinib in vitro and in vivo. Four oxidative metabolites (M1-M4) were detected in rat and human liver microsomal incubation systems after exposure to saracatinib. An ortho-quinone-derived reactive metabolite existing as a GSH conjugate (M5) was found in microsomes fortified with GSH as a trapping agent. The formation of the metabolites detected was NADPH dependent. Metabolites M2-M4 were also observed in bile and urine of rats given saracatinib, and M5 was only detected in bile. Inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the primary enzyme responsible for the metabolic activation of saracatinib. The metabolism study facilitates the understanding of correlation between saracatinib-induced hepatotoxicity and bioactivation.

Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles

Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the "gatekeeper" kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

Pharmacological investigations on possible role of Src kinases in neuroprotective mechanism of ischemic postconditioning in mice

The present study has been undertaken to investigate the possible role of Src Kinases in a neuroprotective mechanism of ischemic postconditioning in mice. Bilateral carotid artery occlusion for 12 min followed by reperfusion for 24 h produced a significant increase in cerebral infarct size and neurological severity score along with impairment of memory and motor coordination. Ischemic postconditioning involving three episodes of 10 s carotid artery occlusion with intermittent reperfusion of 10 s proceeding ischemic insult of 12 min, produced a significant decrease in cerebral infarct size and neurological severity score along with reversal of ischemia-reperfusion induced impairment of memory and motor coordination. Ischemic postconditioning induced neuroprotective effects were significantly attenuated by pre-treatment of selective Src Kinase inhibitors SU-6656 (4 mg/kg i.p.) and PP1 (0.2 mg/kg i.p.). It may be concluded that the neuroprotective effect of ischemic postconditioning probably involves activation of Src Kinase pathway.

A randomized, double-blinded, placebo-controlled, multi-institutional, cross-over, phase II.5 study of saracatinib (AZD0530), a selective Src kinase inhibitor, in patients with recurrent osteosarcoma localized to the lung.

TPS10591 Background: Osteosarcoma is a rare cancer and 33% of patients who have completed primary treatment will recur. The Src pathway has been implicated in the metastatic behavior of several tumors including osteosarcoma where 95% of samples express Src or have evidence of downstream activation of this pathway. Saracatinib (AZD0530) is a potent and selective Src kinase inhibitor. The recommended phase II dose in adults was found to be 175mg daily. The primary goal of this study is to determine if treatment with Saracatinib can increase progression free survival (PFS) for patients who have undergone complete resection of metastatic osteosarcoma nodules in the lung. Secondary goals are evaluation of overall survival, time to treatment failure, and evaluation of several biological correlatives. Methods: This is a multi-institutional, phase II.5, placebo-controlled study with an accrual goals of 88 randomized patients. Patients between 15 and 75 years, with histological confirmation of recurrent osteosarcoma, localized to the lung, who have potential for complete surgical resection, are eligible for enrollment. After complete resection, patients are randomized to treatment with saracatinib or placebo, of a daily oral dose of 175 mg, continuously for up to 1 year or until progression. Patients who recur in the lung while on-study and who are amenable to complete surgical resection will be un-blinded. Those patients who received placebo may have the option to undergo surgical resection. If fully resected, they will be offered therapy with saracatinib under the same treatment guidelines as above. As of January 2013, 38 patients have enrolled and 32 patients met the criteria to be randomized and began oral therapy with either saracatinib or placebo. An interim analysis is planned after 40 patients have been randomized. Clinical trial information: NCT00752206.

1686P - SRC Inhibition as a Method to Overcome Temozolomide Resistance in Melanoma

ABSTRACT Background Malignant melanoma is resistant to chemotherapeutic agents. Src signalling plays a role in many malignancies including melanoma. Src is important in cell adhesion regulation, invasiveness and activating other downstream cellular pathways. Src activity is elevated in melanoma and has potential to provide a treatment target for melanoma. Temozolomide is an oral alkylating prodrug. Dasatinib is a dual Src/Abl kinase inhibitor with antiproliferative activity. AZD0530 is a selective oral Src kinase inhibitor. Methods We examined the anti-proliferative effects of dasatinib and AZD0530 alone and in combination with temozolomide (TMZ) in melanoma cell lines - MALME, HT144 parent and temozolomide resistant cell lines MALME-TMZ and HT144-TMZ. MALME-TMZ and HT144-TMZ were produced by the pulse method. IC50 and combination proliferation assays were performed using an acid phosphatase-based colourimetric assay. Results IC50 (50% inhibitory concentrations ± SD) values for TMZ were 338 ±25 µM in HTI44 and 490 ±19 µM in HT144-TMZ. IC50 values for TMZ were 306 ±29 µM in MALME and 515 ±45 µM in MALME-TMZ. Dasatinib was more effective as a single agent in HT144-TMZ (71 ± 8% growth inhibition at 1 µM) and MALME-TMZ (60 ± 6% growth inhibition at 1 µM) as compared to MALME (14 ± 10% inhibition at 1 µM) and HTI44 (0 ± 3% inhibition at 1 µM). The combination of dasatinib/TMZ was more effective than TMZ alone in HT144 and HT144-TMZ. AZD0530 increased proliferation in HT144 (25 ± 8% at 1 µM) and HT144-TMZ (35 ± 9% at 1 µM) as a single agent. AZD0530 caused growth inhibition as a single agent in MALME-TMZ (18 ± 6% inhibition at 1 µM) and MALME (4 ± 2% at 1 µM). The combination of AZD0530 and TMZ had no effect in HT144 or HT144-TMZ while AZD0530 combined with TMZ was more effective than TMZ alone in MALME and MALME-TMZ. Conclusion Dasatinib improved response to TMZ in both the chemotherapy naive and resistant cell line models tested (HT144, HT144-TMZ, MALME, MALME-TMZ). AZD0530 improved response to TMZ in one of the models only– MALME and MALME-TMZ. These in vitro results support further preclinical evaluation of Src Kinase inhibitors in combination with TMZ, and other DNA-targeting agents, particularly in TMZ-refractory melanoma. Disclosure All authors have declared no conflicts of interest.

Mechanism of caspase-9 activation during hypoxia in the cerebral cortex of newborn piglets: The role of Src kinase

We have previously shown that hypoxia results in increased activation of caspase-9 in the cerebral cortex of newborn piglets. The present study tests the hypothesis that the increased activation of caspase-9 during hypoxia is mediated by Src kinase. To test this hypothesis a highly selective Src kinase inhibitor PP2 [IC50 5nm] was administered to prevent caspase-9 activation during hypoxia. Cytosolic fraction from the cerebral cortical tissue was isolated and the activation of caspase-9 was documented by the expression of active caspase-9 and the activity of caspase-9 and caspase-3. Piglets were divided into: normoxic (Nx, n=5), hypoxic (Hx, n=5) and hypoxic-treated with Src inhibitor (Hx-PP2). Hypoxia was induced by decreasing FiO2 to 0.07 for 60min. PP2 was administered (0.4mg/kg, i.v.) 30min prior to hypoxia. ATP and phosphocreatine (PCr) levels were determined to document cerebral tissue hypoxia. Activity of caspase-9 and caspase-3 were determined spectrofluorometrically using specific fluorogenic substrates. Expression of active caspase-9 was determined by Western blot using active caspase-9 antibody. Caspase-9 activity (nmoles/mgprotein/h) was 1.40±0.12 in Nx, 2.12±0.11 in Hx (p<0.05 vs Nx) and 1.61±0.14 in Hx-PP2 (p<0.05 vs Hx). Active caspase-9 expression (OD×mm2) was 42.3±8.3 in Nx, 78.9±11.0 in Hx (p<0.05 vs Nx) and 41.2±7.6 in Hx-PP2 (p<0.05 vs Hx). Caspase-3 activity (nmoles/mgprotein/h) was 4.11±0.1 in Nx, 6.51±0.1 in Hx (p<0.05 vs Nx) and 4.57±0.7 in Hx+PP2 (p<0.05 vs Hx). Active caspase-3 expression (OD×mm2) was 392.1±23.1 in Nx, 645.0±90.3 in Hx (p<0.05 vs Nx) and 329.7±51.5 in Hx-PP2 (p<0.05 vs Hx). The data show that pretreatment with Src kinase inhibitor prevents the hypoxia-induced increased expression of active caspase-9 and the activity of caspase-9. Src kinase inhibitor also prevented the hypoxia-induced increased activation of caspase-3, a consequence of caspase-9 activation. We conclude that the hypoxia-induced activation of caspase-9 is mediated by Src kinase. We propose Src kinase-dependent tyrosine phosphorylation (Tyr154) in the active site domain of caspase-9 is a potential mechanism of caspase-9 activation in the hypoxic brain.

Phase I study of saracatinib (AZD0530) in combination with paclitaxel and/or carboplatin in patients with solid tumours

Background:As a prelude to combination studies aimed at resistance reversal, this dose-escalation/dose-expansion study investigated the selective Src kinase inhibitor saracatinib (AZD0530) in combination with carboplatin and/or paclitaxel.Methods:Patients with advanced solid tumours received saracatinib once-daily oral tablets in combination with either carboplatin AUC 5 every 3 weeks (q3w), paclitaxel 175 mg m−2 q3w, paclitaxel 80 mg m−2 every 1 week (q1w), or carboplatin AUC 5 plus paclitaxel 175 mg m−2 q3w. The primary endpoint was safety/tolerability.Results:A total of 116 patients received saracatinib 125 (N=20), 175 (N=44), 225 (N=40), 250 (N=9), or 300 mg (N=3). There were no clear dose-related trends within each chemotherapy regimen group in number or severity of adverse events (AEs). However, combining all groups, the occurrence of grade ⩾3 asthenic AEs (all causality) was dose-related (125 mg, 10% 175 mg, 20% ⩾225 mg, 33%), and grade ⩾3 neutropenia occurred more commonly at doses ⩾225 mg. There was no evidence that saracatinib affected exposure to carboplatin or paclitaxel, or vice versa. Objective responses were seen in 5 out of 44 patients (11%) receiving carboplatin plus paclitaxel q3w, and 5 out of 24 (21%) receiving paclitaxel q1w.Conclusion:Saracatinib doses up to 175 mg with paclitaxel with/without carboplatin showed acceptable toxicity in most patients, and are suitable for further trials.

SU-6656, a Selective Src Kinase Inhibitor, Attenuates Mecamylamine-Precipitated Nicotine Withdrawal Syndrome in Mice

Src kinase is reported to regulate neuronal nicotinic acetylcholine receptor activity, which is among the principal receptor systems acted upon by nicotine. Src kinase is documented to mediate the pathogenesis of substance dependence. Therefore, the present study has been designed to investigate the effect of SU-6656, selective src kinase inhibitor, on the development of nicotine dependence in a mouse model of mecamylamine-induced nicotine withdrawal syndrome. Our experimental protocol consisted of administration of nicotine (2.5 mg/kg, subcutaneously), 4 times daily for 7 days. In order to precipitate nicotine withdrawal, mice were given 1 injection of mecamylamine (3 mg/kg, intraperitoneally), 1 hr after the last nicotine injection on the test day (Day 8). Behavioral observations were made for a period of 30 min immediately after mecamylamine treatment. Withdrawal syndrome was quantitated in terms of a composite withdrawal severity score (WSS), and withdrawal syndrome-related anxiety was assessed by elevated plus maze test results. SU-6656 markedly and dose dependently (p < .01) attenuated mecamylamine-induced experimental nicotine withdrawal syndrome in mice measured in terms of WSS and anxiety score. Thus, it is suggested that src kinase is involved in the development of nicotine dependence-induced precipitation of its withdrawal syndrome and thus may serve as a viable pharmacological target to tackle the problem of nicotine addiction.

4-Aryl-4H-Chromene-3-Carbonitrile Derivatives: Evaluation of Src Kinase Inhibitory and Anticancer Activities

Src kinase mutations and/or overexpression have been implicated in the development of a number of human cancer including colon, breast, and lung cancers. Thus, designing potent and selective Src kinase inhibitors as anticancer agents is a subject of major interest. A series of 4-aryl substituted derivatives of 2-amino-7-dimethylamino-4H-chromene-3-carbonitrile were synthesized using one-pot reaction of appropriate substituted aromatic aldehydes, malononitrile, and 3-(dimethylamino)phenol in the presence of piperidine. All 23 compounds were evaluated for inhibition of Src kinase and cell proliferation in human colon adenocarcinoma (HT-29) and leukemia (CCRF-CEM) cell lines. Among the tested compounds, 2-chlorophenyl- (4c), 3-nitrophenyl- (4h), 4-trifluoromethyphenyl- (4i), and 2,3-dichlorophenyl- (4k) substituted chromenes showed Src kinase inhibitory effect with IC(50) values of 11.1-18.3 µM. Compound 4c was relatively selective against Src (IC(50) = 11.1 µM), when compared with selected kinases, epidermal growth factor receptor (EGFR, IC(50) > 300 µM), C-terminal Src kinase (Csk, IC(50) = 101.7 µM), and lymphocyte-specific protein tyrosine kinase (Lck, IC(50) = 46.8 µM). 3-Chlorophenyl substituted thiazole (4v) and 2-chlorophenylsubstituted thiazole (4u) chromene derivatives inhibited the cell proliferation of HT-29 and CCRF-CEM by 80% and 50% respectively, at a concentration of 50 µM. The data indicate that 4H-chromene-3-carbonitrile scaffold has the potential to be optimized further for designing more potent Src kinase inhibitors and/or anticancer lead compounds.

Abstract 3581: Characterization of human breast cancer cells with acquired resistance to the EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib

Abstract The epidermal growth factor receptor (EGFR) and the ErbB-2 receptor are co-expressed in a subset of breast carcinomas that show a more aggressive phenotype. The dual EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib has been recently approved for the treatment of metastatic breast cancer patients whose tumors overexpress ErbB-2. We investigated the mechanisms of acquired resistance to lapatinib in breast cancer cells. For this purpose, we used as model the SK-Br-3 human breast carcinoma cell line that express high levels of ErbB-2, and moderate levels of EGFR and ErbB-3. We selected lapatinib-resistant SK-Br-3 cells by stepwise dose escalation of lapatinib. After six months of selection, we isolated SK-Br-3 cells that were able to grow in 1 μM lapatinib. The IC50 of lapatinib for parental SK-Br-3 cells was 140 nM, whereas the IC50 for the lapatinib-resistant SK-Br-3 derived cell line (Lap-R) was &amp;gt; 4 μM. Treatment with lapatinib significantly inhibited the activation of EGFR, ErbB-2 and ErbB-3 in both parental and Lap-R SK-Br-3 cells. However, following treatment with lapatinib, Lap-R cells showed a persistent activation of AKT and MAPK, which in contrast were completely inhibited in parental SK-Br-3 cells, as measured by both western blotting and a Luminex-based phosphoprotein array. In addition, treatment with lapatinib produced a slight activation of c-Src in parental cells, whereas significant levels of c-Src activation were detected in Lap-R cells cultured in presence of lapatinib. Lap-R cells also showed higher levels of expression of the chemokine receptor CXCR-4 than parental cells. In agreement with these findings, Lap-R cells showed a significantly higher ability to invade a matrigel-coated membrane as compared with parental SK-Br-3 cells, as measured with a Boyden-chamber based colorimetric invasion assay. Preliminary findings demonstrate that inhibition of c-Src with the selective src kinase inhibitor saracatinib produces a more significant inhibition of Lap-R cell invasion as compared with parental SK-Br-3 cells. Taken together, these findings suggest that activation of c-Src signalling might play a role in acquired resistance to lapatinib in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3581. doi:10.1158/1538-7445.AM2011-3581

Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder

Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 μM), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca 2+] i transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 μM), a tyrosine phosphatase inhibitor, significantly augmented the ACh-induced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion.

Involvement of src-kinase activation in ischemic preconditioning induced protection of mouse brain

AimsTo investigate the role of src-kinase in ischemic preconditioning induced reversal of ischemia and reperfusion induced cerebral injury in mice. Main methodsBilateral carotid artery occlusion of 17min followed by reperfusion for 24h was employed to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining using both by volume and by weight methods differently. Memory was evaluated using elevated plus maze test. Rota rod test was employed to assess motor incoordination. Key findingsBilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1min and reperfusion of 1min (ischemic preconditioning) prevented markedly ischemia–reperfusion-induced cerebral injury measured in terms of infarct size (38.5±1.3% and 38.5±2.9% mean infarct of control animals was reduced to 24.3±1.2% and 23.5±1.8% of the preconditioning groups respectively), loss of memory (72.2±3.6 mean transfer latency time of control animals was reduced to 25.6±5.2 of the preconditioning group respectively) and motor coordination (78.3±17.6s mean falling down latency time of control animals was increased to a mean value of 180.9±6.5s of the preconditioning groups respectively). SU6656 (2mg/kg, ip) and PP1 (0.1mg/kg, ip), highly selective src-kinase inhibitors, attenuated this neuroprotective effect of ischemic preconditioning. SignificanceTherefore, neuroprotective effect of ischemic preconditioning may be due to src-kinase linked mechanism.

PH006, a novel and selective Src kinase inhibitor, suppresses human breast cancer growth and metastasis in vitro and in vivo

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC₅₀ of 0.38 μM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.