David Anthony Barda

Metabotropic glutamate 2 receptor potentiators: receptor modulation, frequency-dependent synaptic activity, and efficacy in preclinical anxiety and psychosis model(s)

RationaleTo increase subtype selectivity and provide a novel means to alter receptor function, we discovered and characterization potentiators for the metabotropic glutamate 2 receptor (mGlu2).Methods and resultsA class of 3-pyridylmethylsulfonamides (e.g., 3-MPPTS; 2,2,2-trifluoro-N-[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were found to be potent, subtype-selective potentiators of human and rat mGlu2. The sulfonamides increased agonist potency in functional assays but did not displace orthosteric radiolabeled antagonist or agonist binding to cloned mGlu2 receptors. Rather, the modulators increased the affinity of most of the orthosteric agonists including glutamate, DCG-IV (2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine), and LY354740 (1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate). In striatal brain slices, LY354740 inhibited evoked excitatory postsynaptic potentials (EPSPs) equally well following either a low- (0.06 Hz) or high (4 Hz)-frequency stimulation of corticostriatal afferents. In contrast, the mGlu2 potentiator cyPPTS (2,2,2-trifluoro-N-[3-(cyclopentyloxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) inhibited striatal EPSPs only at higher frequencies of stimulation (2 and 4 Hz). Several sulfonamides including 4-MPPTS, 4-APPES (N-[4-(4-carboxamidophenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride monohydrate) and/or CBiPES N-[4′-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride) were tested in mGlu2/3 agonist-sensitive rodent model(s) of anxiety and psychosis. As seen with LY354740, both 4-MPPTS and 4-APPES were efficacious in a rat fear-potentiated startle paradigm. Likewise in mice, CBiPES attenuated a stress-induced hyperthermia and PCP-induced hyperlocomotor activity. Furthermore, CBiPES mediated alteration in PCP-induced hyperlocomotor activity was sensitive to mGlu2/3 antagonist pretreatment.ConclusionsTaken together, the data indicate mGlu2 receptor potentiators have a unique use-dependent effect on presynaptic glutamate release, and show efficacy in several mGlu2/3-sensitive animal models of psychiatric disorders.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1s role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe. Mol Cancer Ther; 14(9); 2004–13. ©2015 AACR.

Discovery of LY2457546: a multi-targeted anti-angiogenic kinase inhibitor with a novel spectrum of activity and exquisite potency in the acute myelogenous leukemia-Flt-3-internal tandem duplication mutant human tumor xenograft model

SummaryLY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2–7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drugs half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344–56. ©2016 AACR.

Abstract B248: Characterization and preclinical development of LCI‐1, a selective and potent Chk1 inhibitor in phase 1 clinical trials

Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti‐cancer therapies. Ionizing radiation, DNA cross‐linkers, topoisomerase inhibitors and anti‐metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI‐1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell‐based experiments LCI‐1 inhibited doxorubicin‐dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI‐1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism‐dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI‐ 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1‐dependent G2M checkpoint, HT‐29 cells (mutant p53) treated with LCI‐1 were more sensitive to killing by gemcitabine then were LCI‐1 treated HCT116 cells (wild‐type p53). In vivo, LCI‐1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI‐1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI‐1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI‐1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.

Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1, a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2, an alkyne containing version of Compound 1, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

Abstract B229: TBKI kinase inhibition blocks RANTES secretion and exhibits minimal tumor growth inhibition in oncogenic Ras-driven tumor models.

TANK-binding kinase 1 (TBK1) is a non-canonical IKK family member and plays a critical role in innate immunity by modulating cytokine production, interferon, and NF-kB signaling. It is recently reported that TBK1 directly engages Akt survival signaling to support oncogenic Ras-driven transformation. TBK1 is also identified as a synthetic lethal partner in KRas mutant NSCLC through systematic RNA interference. In this study, we have characterized LSN3090729, a 4-aryl-2-aminopyrimidine derivative as a selective TBK1 kinase inhibitor. Biochemical and cellular analyses demonstrate that LSN3090729 is a potent TBK1 kinase inhibitor, and selectively inhibits TBK1 based on in vitro activities in biochemical assays developed with a panel of protein kinases. In Panc-1, a pancreatic tumor cell line with KRas mutation, LSN3090729 inhibits EGF-induced phosphorylation of AKT at both Thr308 and Ser473 sites. Pharmacokinetic analysis shows that LSN3090729 has an over 70% of oral bioavailability with an acceptable half life in rodents. In a mouse pharmacology model, LSN3090729 blocks LPS-induced RANTES secretion in a dose-dependent manner with 67%, 79%, and 90% inhibition at 10, 30, and 100 mg/kg, respectively. LSN3090729 is assessed for its anti-proliferation activities in vitro in a panel of tumor cells with KRas mutation or other genetic background. The sensitivity of these tumor cells to LSN3090729 in two dimensional proliferation or three dimensional soft agar growth assays appears not correlated with status of KRas mutation. In xenograft models of HCT116 and Panc-1, both with a KRas mutation, treatment of LSN3090729 exhibits minimal anti-tumor growth activities, suggesting that a combination approach might be required for TBK1 kinase inhibition to be effective in cancer settings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B229. Citation Format: Robert Van Horn, Tinggui Yin, Xiaoyi Zhang, Chunping Yu, Youyan Zhang, Xue-Qian Gong, Sean Buchanan, Xiang S. Ye, William McMillen, David Barda, Sheng-Bin Peng. TBKI kinase inhibition blocks RANTES secretion and exhibits minimal tumor growth inhibition in oncogenic Ras-driven tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B229.