Inhibition of SGK1 potentiates the anticancer activity of PI3K inhibitor in NSCLC cells through modulation of mTORC1, p­ERK and ß­catenin signaling.

Non-small cell lung cancer (NSCLC) is one of the deadliest types of cancer with poor prognosis, accounting for 85% of all lung cancer cases. The phosphoinositide 3-kinase (PI3K) signaling pathway is most frequently altered in NSCLC; nonetheless, targeting this pathway yields limited success primarily because of drug-induced resistance. PI3K-independent activation of serum and glucocorticoid-induced kinase 1 (SGK1) is responsible for development of resistance to PI3K/AKT inhibitors in breast cancer. The present study investigated potential of inhibiting SGK1 activity for the potentiation of PI3K inhibitor activity in NSCLC cell lines using in vitro anti-proliferation assays, protein expression profiling using western blotting and cell cycle analysis. The findings revealed that combined inhibition of PI3K/AKT and SGK1 resulted in synergistic anticancer activity, with increased apoptosis, DNA damage and cell cycle arrest in G1 phase. Furthermore, high SGK1 protein expression in NSCLC cell lines was associated with increased resistance to PI3K inhibitors. Therefore, enhanced SGK1 expression may serve as a marker to predict therapeutic response to PI3K/AKT inhibitors. Profiling of downstream signaling proteins demonstrated that, at the molecular level SGK1-mediated sensitization of NSCLC cell lines to PI3K inhibitors was achieved via inhibition of mTORC1 signaling. Increased sensitivity of NSCLC cell lines was also mediated by other oncogenic pathways, such as Ras/MEK/ERK and Wnt/ß-catenin signaling.

PAK4 inhibition significantly potentiates Gemcitabine activity in PDAC cells via inhibition of Wnt/ß-catenin, p-ERK/MAPK and p-AKT/PI3K pathways.

Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most difficult to treat cancers. Gemcitabine is still the standard of care treatment for PDAC but with modest survival benefit and well reported resistance. Here we explored potential of inhibiting p21 activated kinase 4 (PAK4), a downstream protein of KRAS oncogenic pathway, in combination with Gemcitabine in PDAC cells. PAK4 inhibition by KPT-9274 led to significant potentiation of Gemcitabine activity in PDAC cells, with an increase in apoptosis, DNA damage and cell cycle arrest. At molecular level, PAK4 inhibition dose dependently inhibited Gemcitabine-induced ß-catenin, c-JUN and Ribonucleotide Reductase subunit 2 (RRM2) levels. PAK4 inhibition further inhibited levels of phosphorylated ERK (p-ERK); Gemcitabine-induced phosphorylated AKT (p-AKT), phosphorylated and total c-Myc. These results suggest possible role of ß-catenin, p-ERK and p-AKT, key effector proteins of Wnt/ß-catenin, MAPK and PI3K pathways respectively, in sensitisation of Gemcitabine activity with PAK4 inhibition. Our data unravel probable molecular mechanisms behind combination of PAK4 inhibition with Gemcitabine to counter PDAC, which may be unequivocally proved further with knock down of PAK4. Our findings provide a strong rationale to exploit the combination therapy of Gemcitabine and PAK4 inhibitor for PDAC at pre-clinical and clinical levels.

Dual PI3Kδγ inhibition demonstrates potent anticancer effects in diffuse large B-cell lymphoma models: Discovery and preclinical characterization of LL-00084282.

Phosphoinositide 3-kinase (PI3K) pathway mediates key signaling events downstream to B-cell receptor (BCR) for survival of mature B-cells, and overexpression or overactivation of PI3Kδ is crucial for B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL). Small molecule PI3Kδγ inhibitors, with a known potential to reduce activated B-cell (ABC)-DLBCL transformation, form an important class of therapeutics approved for follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL). In this study, we describe discovery of a potent, selective and efficacious dual PI3Kδγ inhibitor, LL-00084282, having a differentiated efficacy profile in human ABC- and germinal center B-cell (GCB)-DLBCL cell lines. LL-00084282 displayed high potency and superior PI3Kδγ engagement with excellent selectivity over other PI3K isoforms at both IC50/90 concentrations in biochemical and cell-based assays. In contrast to selective PI3Kδ inhibitors, LL-00084282 showed superior and potent anticancer activity in both ABC- and GCB-DLBCL cell lines. LL-00084282 demonstrated in-vivo efficacy in OCI-Ly10 and SU-DHL-6 xenografts with good tolerability. Furthermore, LL-00084282 inhibited pro-inflammatory cytokine secretion and reduced basophil activation in human PBMCs, showing potential implications in immunoinflammatory conditions. Good pharmacokinetic properties in higher species and desirable efficacy profile highlights potential of this novel PI3Kδγ inhibitor for further clinical evaluation in DLBCL patients.

LL-00066471, a novel positive allosteric modulator of α7 nicotinic acetylcholine receptor ameliorates cognitive and sensorimotor gating deficits in animal models: Discovery and preclinical characterization.

α7 nicotinic acetylcholine receptor (α7 nAChR) is an extensively validated target for several neurological and psychiatric conditions namely, dementia and schizophrenia, owing to its vital roles in cognition and sensorimotor gating. Positive allosteric modulation (PAM) of α7 nAChR represents an innovative approach to amplify endogenous cholinergic signaling in a temporally restricted manner in learning and memory centers of brain. α7 nAChR PAMs are anticipated to side-step burgeoning issues observed with several clinical-stage orthosteric α7 nAChR agonists, related to selectivity, tolerance/tachyphylaxis, thus providing a novel dimension in therapeutic strategy and pharmacology of α7 nAChR ion-channel. Here we describe a novel α7 nAChR PAM, LL-00066471, which potently amplified agonist-induced Ca2+ fluxes in neuronal IMR-32 neuroblastoma cells in a α-bungarotoxin (α-BTX) sensitive manner. LL-00066471 showed excellent oral bioavailability across species (mouse, rat and dog), low clearance and good brain penetration (B/P ratio > 1). In vivo, LL-00066471 robustly attenuated cognitive deficits in both procognitive and antiamnesic paradigms of short-term episodic and recognition memory in novel object recognition task (NORT) and social recognition task (SRT), respectively. Additionally, LL-00066471 mitigated apomorphine-induced sensorimotor gating deficits in acoustic startle reflex (ASR) and enhanced antipsychotic efficacy of olanzapine in conditioned avoidance response (CAR) task. Further, LL-00066471 corrected redox-imbalances and reduced cortico-striatal infarcts in stroke model. These finding together suggest that LL-00066471 has potential to symptomatically alleviate cognitive deficits associated with dementias, attenuate sensorimotor gating deficits in schizophrenia and correct redox-imbalances in cerebrovascular disorders. Therefore, LL-00066471 presents potential for management of cognitive impairments associated with neurological and psychiatric conditions.

Discovery of a Potent and Selective PI3Kδ Inhibitor (S)-2,4-Diamino-6-((1-(7-fluoro-1-(4-fluorophenyl)-4-oxo-3-phenyl-4H-quinolizin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile with Improved Pharmacokinetic Profile and Superior Efficacy in Hematological Cancer Models.

PI3Kδ inhibitors have been approved for B-cell malignancies like CLL, small lymphocytic lymphoma, and so forth. However, currently available PI3Kδ inhibitors are nonoptimal, showing weakness against at least one of the several important properties: potency, isoform selectivity, and/or pharmacokinetic profile. To come up with a PI3Kδ inhibitor that overcomes all these deficiencies, a pharmacophoric expansion strategy was employed. Herein, we describe a systematic transformation of a "three-blade propeller" shaped lead, 2,3-disubstituted quinolizinone 11, through a 1,2-disubstituted quinolizinone 20 to a novel "four-blade propeller" shaped 1,2,3-trisubstituted quinolizinone 34. Compound 34 has excellent potency, isoform selectivity, metabolic stability across species, and exhibited a favorable pharmacokinetic profile. Compound 34 also demonstrated a differentiated efficacy profile in human germinal center B and activated B cell-DLBCL cell lines and xenograft models. Compound 34 qualifies for further evaluation as a candidate for monotherapy or in combination with other targeted agents in DLBCLs and other forms of iNHL.

Discovery of Novel, Potent, Brain-Permeable, and Orally Efficacious Positive Allosteric Modulator of α7 Nicotinic Acetylcholine Receptor [4-(5-(4-Chlorophenyl)-4-methyl-2-propionylthiophen-3-yl)benzenesulfonamide]: Structure-Activity Relationship and Preclinical Characterization.

The discovery of a series of thiophenephenylsulfonamides as positive allosteric modulators (PAM) of α7 nicotinic acetylcholine receptor (α7 nAChR) is described. Optimization of this series led to identification of compound 28, a novel PAM of α7 nicotinic acetylcholine receptor (α7 nAChR). Compound 28 showed good in vitro potency, with pharmacokinetic profile across species with excellent brain penetration and residence time. Compound 28 robustly reversed the cognitive deficits in episodic/working memory in both time-delay and scopolamine-induced amnesia paradigms in the novel object and social recognition tasks, at very low dose levels. Additionally, compound 28 has shown excellent safety profile in phase 1 clinical trials and is being evaluated for efficacy and safety as monotherapy in patients with mild to moderate Alzheimer's disease.

5-HT6 receptor agonist and antagonist modulates ICV-STZ-induced memory impairment in rats.

RATIONALE AND OBJECTIVES: 5-HT6 receptors are mainly expressed in brain areas associated with learning and memory. Several studies have reported procognitive effects of both 5-HT6 agonist and antagonists. However, the exact mechanism 5-HT6 receptor modulation has not been properly studied especially in the context of cholinergic functions, cerebral blood flow (CBF), brain-derived neural factor (BDNF), oxidative stress, and behavioral changes. METHODS: In the present study, memory impairment was induced in albino Wistar rats by two doses of intracerebroventricular (ICV) injection of streptozotocin (STZ, 3 mg/kg) on first and third day. These rats were evaluated in a battery of behavioral tasks after 14 days from the first day of ICV-STZ. RESULTS: Significant memory impairment was seen when ICV-STZ induced rats are assessed by Morris water maze, novel object recognition, social recognition, and passive avoidance tests. There was a significant reduction in CBF, increased oxidative stress (MDA, GSH, and ROS), acetylcholinesterase (AChE) activity, and a decrease in BDNF. Treatment with selective 5-HT6 agonist EMD-386088 (5 mg/kg) and antagonist SB-399885 (10 mg/kg) prevented ICV-STZ-induced memory impairment when assessed by behavioral tests. Treatment with 5-HT6 ligands significantly prevented the change in CBF and BDNF. Further, protected from MDA and ROS and decreasing GSH in the brain compared to ICV-STZ rats. The rice in brain AChE activity was normalized by both ligands. The changes in locomotor activity by EMD-386088 and SB-399885 treatment were negligible. CONCLUSION: The findings in this study support the therapeutic potential of 5-HT6 receptor ligands in the treatment of cognitive dysfunction.

5-HT6 Receptor Agonist and Antagonist Against ß-Amyloid-Peptide-Induced Neurotoxicity in PC-12 Cells.

Beta-amyloid peptide (Aß) induced neurotoxicity is considered as a hallmark of the pathogenesis of Alzheimer's disease (AD). The present study demonstrated the neuroprotective role of 5-HT6 receptors against Aß-induced neurotoxicity in PC-12 cells. The 5-HT6 receptor agonist EMD-386088 and antagonist SB-399885 were used as pharmacological tools. The NMDA receptor antagonist, memantine, was used as reference standard. The Aß25-35 (50 µM) induced apoptosis, increased reactive oxygen species (ROS) generation and impaired neurite outgrowth in PC-12 cells. Pre-treatment with 10 µM EMD-386088 and SB-399885 had significantly protected neuronal cell death by maintaining higher cell viability through attenuation of intracellular ROS. Further, both compounds significantly prevented Aß25-35-induced impairment in neurite outgrowth in PC-12 cells. Similarly, memantine prevented Aß25-35-induced neurotoxicity in PC-12 cells. These findings suggest that 5-HT6 receptor ligands have protected neurons from Aß25-35 induced toxicity by reducing ROS and through prevention of impairment in neurite outgrowth. Therefore, 5-HT6 receptor could be an important disease-modifying therapeutic target for AD.