[N-methyl-3H3]AZ10419369 binding to the 5-HT1B receptor: in vitro characterization and in vivo receptor occupancy.

Radiotracers suitable for positron emission tomography studies often serve as preclinical tools for in vivo receptor occupancy. The serotonin 1B receptor (5-HT(1B)) subtype is a pharmacological target used to discover treatments for various psychiatric and neurological disorders. In psychiatry, 5-HT(1B) antagonists may provide novel therapeutics for depression and anxiety. We report on the in vitro and in vivo evaluation of tritiated 5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylicacid (4-morpholin-4-yl-phenyl)-amide ([N-methyl-(3)H(3)]AZ10419369), a potent 5-HT(1B) radiotracer. [N-methyl-(3)H(3)]-AZ10419369 showed saturable single-site high-affinity in vitro binding (guinea pig, K(d) = 0.38 and human, K(d) = 0.37) to guinea pig or human 5-HT(1B) receptors in recombinant membranes and high-affinity (K(d) = 1.9 nM) saturable (B(max) = 0.099 pmol/mg protein) binding in membranes from guinea pig striatum. When [N-methyl-(3)H(3)]AZ10419369 was administered to guinea pigs by intravenous bolus, the measured radioactivity was up to 5-fold higher in brain areas containing the 5-HT(1B) receptor (striatum/globus pallidus, midbrain, hypothalamus, and frontal cortex) compared with the cerebellum, the nonspecific binding region. Specific uptake peaked 30 min after injection with slow dissociation from target regions, as suggested by the in vitro binding kinetic profile. Pretreatment with 6-fluoro-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid [4-(4-propionyl-piperazin-1-yl)-phenyl]-amide (AZD1134) and 2-aminotetralin (AR-A000002), 5-HT(1B)-selective ligands, inhibited [N-methyl-(3)H(3)]AZ10419369-specific binding in a dose-dependent manner. In the guinea pig striatum, AZD1134 (ED(50) = 0.017 mg/kg) occupies a greater percentage of the 5-HT(1B) receptors at a lower administered dose than AR-A000002 (ED(50) = 2.5 mg/kg). In vivo receptor occupancy is an essential component to build binding-efficacy-exposure relationships and compare novel compound pharmacology. [N-methyl-(3)H(3)]AZ10419369 is a useful preclinical tool for investigating 5-HT(1B) receptor occupancy for novel compounds targeting this receptor.

Novel small molecule inhibitors of caspase-3 block cellular and biochemical features of apoptosis.

Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury. Its interest as a therapeutic target has led many to pursue the development of inhibitors. To date, only one series of nonpeptidic inhibitors have been described, and these have limited selectivity within the caspase family. Here we report the properties of a series of anilinoquinazolines (AQZs) as potent small molecule inhibitors of caspase-3. The AQZs inhibit human caspase-3 with Ki values in the 90 to 800 nM range. A subset of AQZs are equipotent against caspase-6, although most lack activity against this isoform and caspase-1, -2, -7, and -8. The AQZs inhibit endogenous caspase-3 activity toward a cell permeable, exogenously added substrate in staurosporine-treated SH-SY5Y cells. The AQZs reduce biochemical and cellular features of apoptosis that are thought to be a consequence of caspase-3 activation including DNA fragmentation, TUNEL staining, and the various morphological features that define the terminal stages of apoptotic cell death. Moreover, the AQZs also inhibit apoptosis induced by nerve growth factor withdrawal from differentiated PC12 cells. Thus, the AQZs represent a new and structurally novel class of inhibitors, some of which selectively inhibit caspase-3 and will thereby allow evaluation of the role of caspase-3 activity in various cellular models of apoptosis.