J. Phillip Kennedy

Novel Selective Allosteric Activator of the M1 Muscarinic Acetylcholine Receptor Regulates Amyloid Processing and Produces Antipsychotic-Like Activity in Rats

Recent studies suggest that subtype-selective activators of M1/M4 muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimers disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M1 receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1′-2-methylbenzyl)-1,4′-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M1 through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M1 by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M2 and M4. TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Aβ production in vitro. Together, these data suggest that selective activation of M1 may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimers disease.

A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning

M1 muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M1 receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M1 receptor affinity for acetylcholine. We found that activation of the M1 receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M1 receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimers disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M1 receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimers disease patients. Together, these studies provide compelling evidence that M1 receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M1 mAChR subtype may ameliorate impairments in cognitive function.

Centrally Active Allosteric Potentiators of the M4 Muscarinic Acetylcholine Receptor Reverse Amphetamine-Induced Hyperlocomotor Activity in Rats

Previous clinical and animal studies suggest that selective activators of M1 and/or M4 muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimers disease. However, highly selective centrally penetrant activators of either M1 or M4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M4 mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M4. VU0152099 and VU0152100 had no agonist activity but potentiated responses of M4 to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M4 reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M4 plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M4 may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

Loss of Phosphatase and Tensin Homologue Deleted on Chromosome 10 Engages ErbB3 and Insulin-Like Growth Factor-I Receptor Signaling to Promote Antiestrogen Resistance in Breast Cancer

Knockdown of the tumor suppressor phosphatase Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) with shRNA in three estrogen receptor (ER)-positive breast cancer cell lines resulted in increased phosphatidylinositol-3 kinase (PI3K) and AKT activities, resistance to tamoxifen and fulvestrant, and hormone-independent growth. PTEN knockdown induced the up-regulation of ER transcriptional activity in MCF-7 cells but decreased ER protein levels and transcriptional activity in T47D and MDA-361 cells. Tamoxifen and fulvestrant treatment inhibited estradiol-induced ER transcriptional activity in all shPTEN cell lines but did not abrogate the increased cell proliferation induced by PTEN knockdown. PTEN knockdown increased basal and ligand-induced activation of the insulin-like growth factor-I (IGF-I) and ErbB3 receptor tyrosine kinases, and prolonged the association of the p85 PI3K subunit with the IGF-I receptor (IGF-IR) effector insulin receptor substrate-1 and with ErbB3, implicating PTEN in the modulation of signaling upstream of PI3K. Consistent with these data, PTEN levels inversely correlated with levels of tyrosine-phosphorylated IGF-IR in tissue lysate arrays of primary breast cancers. Inhibition of IGF-IR and/or ErbB2-mediated activation of ErbB3 with tyrosine kinase inhibitors restored hormone dependence and the growth inhibitory effect of tamoxifen and fulvestrant on shPTEN cells, suggesting that cotargeting both ER and receptor tyrosine kinase pathways holds promise for the treatment of patients with ER+, PTEN-deficient breast cancers.

Application of combinatorial chemistry science on modern drug discovery.

The science that established combinatorial chemistry as important new discipline in the 1980s and 1990s has re-emerged and infiltrated every sub-discipline within modern drug discovery and has profound impact. The application of combinatorial chemistry science has revolutioned high-throughput screening paradigms, chemical lead optimization, library purification, and post-purification sample handling, as well as in vitro and in vivo drug metabolism and pharmacokinetic assays. Although no longer in the spotlight and heralded as the savior of the drug industry, “combinatorial chemistry” is alive and well; in fact, combinatorial chemistry science is more prevalent and wide-spread than ever before.

Inhibition of Mammalian Target of Rapamycin Is Required for Optimal Antitumor Effect of HER2 Inhibitors against HER2-Overexpressing Cancer Cells

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2+ mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells. Conclusions: Inhibition of PI3K and mTOR are required for the growth-inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. (Clin Cancer Res 2009;15(23):7266–76)

Insulin reveals Akt signaling as a novel regulator of norepinephrine transporter trafficking and norepinephrine homeostasis

Noradrenergic signaling in the CNS plays an essential role in circuits involving attention, mood, memory, and stress as well as providing pivotal support for autonomic function in the peripheral nervous system. The high-affinity norepinephrine (NE) transporter (NET) is the primary mechanism by which noradrenergic synaptic transmission is terminated. Data indicate that NET function is regulated by insulin, a hormone critical for the regulation of metabolism. Given the high comorbidity of metabolic disorders such as diabetes and obesity with mental disorders such as depression and schizophrenia, we sought to determine how insulin signaling regulates NET function and thus noradrenergic homeostasis. Here, we show that acute insulin treatment, through the downstream kinase protein kinase B (Akt), significantly decreases NET surface expression in mouse hippocampal slices and superior cervical ganglion neuron boutons (sites of synaptic NE release). In vivo manipulation of insulin/Akt signaling, with streptozotocin, a drug that induces a type 1-like diabetic state in mice, also results in aberrant NET function and NE homeostasis. Notably, we also demonstrate that Akt inhibition or stimulation, independent of insulin, is capable of altering NET surface availability. These data suggest that aberrant states of Akt signaling such as in diabetes and obesity have the potential to alter NET function and noradrenergic tone in the brain. Furthermore, they provide one potential molecular mechanism by which Akt, a candidate gene for mood disorders such as schizophrenia and depression, can impact brain monoamine homeostasis.

Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part I: Development of the first highly selective M5 PAM

This Letter describes a chemical lead optimization campaign directed at VU0238429, the first M(5)-preferring positive allosteric modulator (PAM), discovered through analog work around VU0119498, a pan G(q) mAChR M(1), M(3), M(5) PAM. An iterative library synthesis approach delivered the first selective M(5) PAM (no activity at M(1)-M(4) @ 30microM), and an important tool compound to study the role of M(5) in the CNS.

Synthesis and SAR of analogues of the M1 allosteric agonist TBPB. Part I. Exploration of alternative benzyl and privileged structure moieties

This Letter describes the first account of the synthesis and SAR, developed through an iterative analogue library approach, of analogues of the highly selective M1 allosteric agonist TBPB. With slight structural changes, mAChR selectivity was maintained, but the degree of partial M1 agonism varied considerably.

Synthesis and Structure–Activity Relationships of Allosteric Potentiators of the M4 Muscarinic Acetylcholine Receptor

J. Phillip Kennedy[a],+, Thomas M. Bridges[b],+, Patrick R. Gentry[b], John T. Brogan[a], Alexander S. Kane[b], Carrie K. Jones[b],[c], Ashley E. Brady[b], Jana K. Shirey[b], P. Jeffrey Conn [Prof.][b], and Craig W. Lindsley [Prof.]*,[a],[b] [a] Department of Chemistry, Vanderbilt University 12475 MRB4, Nashville, TN 37232-6600 (USA) [b] Vanderbilt Program in Drug Discovery, Department of Pharmacology Vanderbilt University Medical Center 12475 MRB4, Nashville, TN 37232-6600 (USA)