Derek Cecil Cole

Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin

The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

Neuropharmacological profile of novel and selective 5-HT6 receptor agonists: WAY-181187 and WAY-208466.

One of the most recently identified serotonin (5-hydroxytryptamine (5-HT)) receptor subtypes is the 5-HT6 receptor. Although in-depth localization studies reveal an exclusive distribution of 5-HT6 mRNA in the central nervous system, the precise biological role of this receptor still remains unknown. In the present series of experiments, we report the pharmacological and neurochemical characterization of two novel and selective 5-HT6 receptor agonists. WAY-181187 and WAY-208466 possess high affinity binding (2.2 and 4.8 nM, respectively) at the human 5-HT6 receptor and profile as full receptor agonists (WAY-181187: EC50=6.6 nM, Emax=93%; WAY-208466: EC50=7.3 nM; Emax=100%). In the rat frontal cortex, acute administration of WAY-181187 (3–30 mg/kg, subcutaneous (s.c.)) significantly increased extracellular GABA concentrations without altering the levels of glutamate or norepinephrine. Additionally, WAY-181187 (30 mg/kg, s.c.) produced modest yet significant decreases in cortical dopamine and 5-HT levels. Subsequent studies showed that the neurochemical effects of WAY-181187 in the frontal cortex could be blocked by pretreatment with the 5-HT6 antagonist, SB-271046 (10 mg/kg, s.c.), implicating 5-HT6 receptor mechanisms in mediating these responses. Moreover, the effects of WAY-181187 on catecholamines were attenuated by an intracortical infusion of the GABAA receptor antagonist, bicuculline (10 μM), confirming a local relationship between 5-HT6 receptors and GABAergic systems in the frontal cortex. In the dorsal hippocampus, striatum, and amygdala, WAY-181187 (10–30 mg/kg, s.c.) elicited robust elevations in extracellular levels of GABA without producing similar effects on concentrations of norepinephrine, serotonin, dopamine, or glutamate. In contrast to these brain regions, WAY-181187 had no effect on the extracellular levels of GABA in the nucleus accumbens or thalamus. Additional studies showed that WAY-208466 (10 mg/kg, s.c.) preferentially elevated cortical GABA levels following both acute and chronic (14 day) administration, indicating that neurochemical tolerance does not develop following repeated 5-HT6 receptor stimulation. In hippocampal slice preparations (in vitro), 5-HT6 receptor agonism attenuated stimulated glutamate levels elicited by sodium azide and high KCl treatment. Furthermore, in the rat schedule-induced polydipsia model of obsessive compulsive disorder (OCD), acute administration of WAY-181187 (56–178 mg/kg, po) decreased adjunctive drinking behavior in a dose-dependent manner. In summary, WAY-181187 and WAY-208466 are novel, selective, and potent 5-HT6 receptor agonists displaying a unique neurochemical signature in vivo. Moreover, these data highlight a previously undescribed role for 5-HT6 receptors to modulate basal GABA and stimulated glutamate transmission, as well as reveal a potential therapeutic role for this receptor in the treatment of some types of anxiety-related disorders (eg OCD).

Discovery of Begacestat, a Notch-1-Sparing γ-Secretase Inhibitor for the Treatment of Alzheimer's Disease

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimers disease.

Inhibition of the Signal Transducer and Activator of Transcription-3 (STAT3) Signaling Pathway by 4-Oxo-1-Phenyl-1,4-Dihydroquinoline-3-Carboxylic Acid Esters

The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester 4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the 7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC 50 of 170 nM. Compound 8 also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation in K562 cells.

Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of 4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.

Identification and Characterization of Acidic Mammalian Chitinase Inhibitors

Acidic mammalian chitinase (AMCase) is a member of the glycosyl hydrolase 18 family (EC 3.2.1.14) that has been implicated in the pathophysiology of allergic airway disease such as asthma. Small molecule inhibitors of AMCase were identified using a combination of high-throughput screening, fragment screening, and virtual screening techniques and characterized by enzyme inhibition and NMR and Biacore binding experiments. X-ray structures of the inhibitors in complex with AMCase revealed that the larger more potent HTS hits, e.g. 5-(4-(2-(4-bromophenoxy)ethyl)piperazine-1-yl)-1H-1,2,4-triazol-3-amine 1, spanned from the active site pocket to a hydrophobic pocket. Smaller fragments identified by FBS occupy both these pockets independently and suggest potential strategies for linking fragments. Compound 1 is a 200 nM AMCase inhibitor which reduced AMCase enzymatic activity in the bronchoalveolar lavage fluid in allergen-challenged mice after oral dosing.

Discovery and initial optimization of 5,5'-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S(1) to the S(3) pocket.

Synthesis and PKCθ inhibitory activity of a series of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles

The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCtheta. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC(50) value of 7.5nM for the inhibition of PKCtheta.

Synthesis and PKCθ inhibitory activity of a series of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles

A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCtheta were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC(50) value of 18nM for the inhibition of PKCtheta.