The presenilin containing ␥-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. ␥-Secretase catalyzes the final step in the generation of A␤ 40 and A␤ 42 peptides from APP. Amyloid ␤-peptides (A␤ 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 A␤ 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 ␥-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits A␤ production with low nanomolar potency in cellular and cell-free assays of ␥-secretase function, and displaces a tritiated analog of GSI-953 from enriched ␥-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 A␤ levels, and a reversal of contextual fear-conditioning deficits that are correlated with A␤ load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dosedependent changes in plasma A␤ levels, confirming pharmacodynamic activity of GSI-953 in humans.This research was supported by Wyeth Research. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

No abstract

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.

No abstract

No abstract

. 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 ϭ 4.9 Ϯ 0.6 and 11.0 Ϯ 0.5 M for large-and small-diameter neurons, respectively. Loperamideinduced 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 M 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 M 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 loperamideinduced 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.

No abstract

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 (IC 50 values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K d (254 nM) was similar to the IC 50 (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.Plasminogen activator inhibitor 1 (PAI-1) is a rapidly acting inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (Dellas and Loskutoff, 2005). PAI-1 is a member of the serpin class of serine protease inhibitors that characteristically produce SDS-stable complexes with their cognate protease targets (Silverman et al., 2001). Formation of the acyl-enzyme adduct between PAI-1 and the protease involves initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational changes that trap the protease in a covalent complex (Olson et al., 2001). Two other conformation states of PAI-1 are known. First, the acyl-enzyme adduct between PAI-1 and tPA (or uPA) can be hydrolyzed to form cleaved (inactive) PAI-1 and regenerate active plasminogen activator (PA) (Declerck et al., 1992). Second, active PAI-1 can undergo a spontaneous large conformation change that gives rise to an inactive (latent) state of the inhibitor (Levin and Santell, 1987;Mottonen et al., 1992).PAI-1 plays a pivotal role in a myriad of physiological processes that involve activation of plasminogen (Dellas and Loskutoff, 2005). High ...