Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r2 = 0.56) between the respective IC50s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

Robert L. Martone, Hua Zhou, Kevin Atchison, Thomas Comery, Jane Z. Xu, Xinyi Huang, Xioahai Gong, Mei Jin, Anthony Kreft, Boyd Harrison, Scott C. Mayer, Suzan Aschmies, Cathleen Gonzales, Margaret M. Zaleska, David R. Riddell, Erik Wagner, Peimin Lu, Shaiu-Ching Sun, June Sonnenberg-Reines, Aram Oganesian, Karissa Adkins, Michael Leach, David W. Clarke, Donna Huryn, Magid Abou-Gharbia, Ronald Magolda, Jonathan Bard, Glen Frick, Sangeeta Raje, S. Bradley Forlow, Carrie Balliet, Michael E. Burczynski, Peter H. Reinhart, Hong I. Wan, Menelas N. Pangalos, J. Steven Jacobsen*

Gamma secretase is responsible for the intramembraneous cleavage of the Alzheimer's Precursor Protein (APP), the Notch receptor, and several other substrates. While inhibition of this protease results in potentially therapeutic reductions in the neurotoxic Abeta peptide, severe side effects might result from inhibiting Notch processing. We report a novel thiophene sulfonamide gamma-secretase inhibitor, GSI-953, that selectively inhibits cleavage of APP while sparing Notch processing. In vitro assays of Abeta production and Notch function, measurements of Abeta levels in plasma and brain of Tg2576 mouse, and assessments of cognitive function using the contextual fear conditioning model are described, as well as human plasma Abeta levels and initial biomarker data. This compound inhibits Abeta production with low nM potency in vitro in cellular and cell-free assays. Cellular assays of Notch cleavage reveal that this compound is >15-fold selective for the inhibition of APP cleavage. In the Tg2576 transgenic mouse, this compound causes a robust reduction in brain and plasma Abeta levels and reverses memory deficits that are correlated with Abeta load. A lowering of plasma Abeta levels in human demonstrates target engagement. These data demonstrate that GSI-953 is a potent and selective gamma-secretase inhibitor with potential for therapeutic utility in Alzheimer's Disease. For these reasons, GSI-953 has been advanced into human clinical trials.

Blocking the synthesis of the AD pathogen beta amyloid peptide (Αβ) through inhibition of APP processing by gamma secretase (GS) is a promising therapeutic strategy that may provide the first disease-modifying anti-Alzheimer's drug (DMAAD). In animal models, GSI-953 is a Notch-sparing gamma secretase inhibitor (GSI) and this compound has subsequently entered clinical trials. Our objective was to characterize the relative affinity of GSI-953 for binding to GS and compare it to the potency of GS inhibition. A cell-free GS binding assay was developed employing membranes isolated from human neuroblastoma SY5Y cells and the tritiated GSI-953 analog TGSI (5-chloro-N-[(1S,2R)–4,4,4-trifluoro-1-(hydroxyl[3H2]methyl)–2-methylbutyl]thiophene-2-sulfonamide). GSI-953 and benchmark GSIs (DAPT, LY411575, LY450139, DuPont E, L-685458, a BMS sulfonamide and an Amgen sulfonamide) were profiled in this assay for their ability to displace the radiolabeled GSI-953 analog. For comparison to GS binding affinity, the GSIs were also profiled for inhibition of Αβ synthesis in a cellular assay (hAPPCHO cells). GSI-953 competitively displaced TGSI from the GS complex in the cell-free binding assay (IC50=8 nM) and its affinity for GS was comparable to its GSI potency in the cellular assay (EC50Αβ42=15 nM). The stereospecific nature of both the binding to and inhibition of GS by GSI-953 was confirmed by the low affinity (IC50>10,000 nM) and GSI activity (EC50Αβ42>30,000 nM) observed for the enantiomer of GSI-953. Benchmark GSIs DAPT (IC50=29 nM), LY411575 (IC50=3 nM), LY450139 (IC50=26 nM), DuPont E (IC50<10 nM), a BMS sulfonamide (IC50=57 nM) and an Amgen sulfonamide (IC50=986 nM) were able to competitively displace TGSI from GS at concentrations comparable to their EC50s for inhibition of Αβ synthesis. The transition state inhibitor L-685458 was only able to partially displace TGSI. GSI-953 and benchmark GSIs (except L-685458) can competitively displace TGSI from GS suggesting that these GSIs may bind to the same site. Among the GSIs profiled, a good correlation was observed between GS binding affinity and GSI potency.

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 Alzheimer's disease.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.

Gamma secretase is responsible for the intramembraneous cleavage of the Alzheimer's Precursor Protein (APP), the Notch receptor, and several other substrates. While inhibition of this protease results in potentially therapeutic reductions in the neurotoxic Abeta peptide, severe side effects might result from inhibiting Notch processing. We report a novel thiophene sulfonamide gamma-secretase inhibitor, GSI-953 thatselectively inhibits cleavage of APP while sparing Notch processing. In vitro assays of Abeta production and Notch function: measurements of Abeta levels in plasma and brain of Tg2576 mouse, and assessments of cognitive function using the contextual fear conditioning model. This compound inhibits Abeta production with low nM potency in vitro in cellular and cell-free assays. Cellular assays of Notch cleavage reveal that this compound is >15-fold selective for the inhibition of APP cleavage. In the Tg2576 transgenic mouse, this compound causes a robust reduction in brain and plasma Abeta levels and reverses memory deficits that are correlated with Abeta load. These data demonstrate that GSI-953 is a potent and selective gamma-secretase inhibitor with potential for therapeutic utility in Alzheimer's disease. For these reasons, GSI-953 has been advanced into human clinical trials.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons (Ih) and cardiac (If) cells. As such, the identification and characterization of novel blockers of HCN channels is important to enable the dissection of their function in vivo. Using a new IonWorks HT electrophysiology assay with human HCN1 and HCN4 expressed stably in cell lines, four HCN channel blockers are characterized. Two blockers known for their activity at opioid/Ca2+ channels and K+ channels, loperamide and CP-339,818 (respectively), are described to block HCN1 more potently than HCN4. The known HCN blocker ZD7288 was also found to be more selective for HCN1 over HCN4, while the HCN blocker DK-AH269 was equipotent on HCN4 and HCN1. Partial replacement of the intracellular Cl− with gluconate reduced the potency on both channels, but to varying degrees. For both HCN1 and HCN4, ZD7288 was most sensitive in lower Cl− solutions, while the potency of loperamide was not affected by the differing solutions. The block of HCN1 for all compounds was voltage-dependent, being relieved at more negative potentials. The voltage-dependent, Cl− dependent, HCN1 preferring compounds described here elaborate on the current known pharmacology of HCN channels and may help provide novel tools and chemical starting points for the investigation of HCN channel function in natively expressing systems.

An asymmetric synthesis of alpha-amino acids with novel beta-branched side chains has been implemented. The syntheses feature a p-toluenesulfinylimine induced chiral Strecker approach and were found to be applicable to the introduction of both aliphatic and aromatic beta-branched sidechains for preparation of previously unknown alpha-amino acids.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC50 values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophoretagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K-d (254 nM) was similar to the IC50 (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min ( sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog ( producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.