Background: Potent GSMs have been identified that lower A␤42; however, the mechanism of modulation is not well understood. Results:The photoaffinity probe E2012-BPyne specifically labels PS1-NTF at a unique site. Conclusion: Acid and imidazole GSMs bind to distinct sites on PS1-NTF and are differentially affected by L458. Significance: Our results provide evidence for multiple binding sites within ␥-secretase that confer specific modulatory effects.

LY-450139 is a ␥-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-␤ (A␤) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A␤ responses to LY-450139 in the guinea pig, a nontransgenic model that has an A␤ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma A␤ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A␤ levels at early time points, with return to baseline within hours. Higher doses inhibited A␤ levels in all compartments at early time points, but elevated plasma A␤ levels at later time points. To determine whether this phenomenon occurs under steadystate drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma A␤ was significantly inhibited at 10 -30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A␤ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A␤ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the ␥-secretase complex, eliciting A␤ lowering at high concentrations but A␤ elevation at low concentrations.The pathological accumulation of amyloid-␤ peptide into dense core plaques in the brains of Alzheimer's disease patients is the ultimate target of multiple disease-modifying drug discovery efforts. One strategy that has entered the clinic is the use of a ␥-secretase inhibitor to reduce central A␤ production. Preclinically, multiple ␥-secretase inhibitors have demonstrated central and peripheral A␤-lowering activity in transgenic mouse lines overexpressing human mutant amyloid precursor protein (Dovey et al., 2001;Cirrito et al., 2003;Lanz et al., 2003Lanz et al., , 2004Wong et al., 2004;, as well as nontransgenic species (Anderson et al., 2005;Best et al., 2006;El Mouedden et al., 2006). Whereas acute treatment of old, plaque-bearing mice should have little immediate impact on plaque load (insoluble A␤), these inhibitors have been shown to inhibit A␤ in CSF (Lanz et al., 2003;Barten et al., 2005) and interstitial fluid (Cirrito et al., 2003) similarly in both plaque-free and plaque-bearing mice. In addition, plasma A␤ has been shown to be reduced similarly by ␥-secretase inhibition in both young and old Tg2576 mice (Lanz et al., 2003;Barten et al., 2005). These findings indicate that despite the presence or absence of insoluble A␤ plaques, these compounds had similar potency in reducing soluble, secreted A␤ in young and old transgenic mice.The ability of plasma and CSF A␤ to track pharmacologic...

Aβ42 is believed to play a causative role in Alzheimer’s disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.

The “Notch-sparing” γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer’s disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate due to its reported Notch-sparing activity for the inhibition of Aβ production over Notch cleavage. We now report that BMS-708,163 binds directly to PS1-NTF, and that binding can be competed by other pan-GSIs, but not by γ-secretase modulators (GSMs). Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a non-selective γ-secretase inhibitor.

Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-)5 vs 1.5 x 10(-)6 M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S100b, both proteins were treated with 1 M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD-GCAP could not be a modified form of S100b. Hydroxylamine also broke down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.

The membrane guanylate cyclase in retinal rod outer segments (ROS-GC) is known to be negatively regulated by calcium; when the calcium concentration is reduced below the dark-adapted level of about 500 nM, the enzyme is activated by a soluble protein. We now report that the enzyme is also positively regulated by calcium; a novel soluble protein is identified and purified from bovine retina which activates ROS-GC, with half-maximal activation occurring at 2-5 microM calcium. The activation is dose-dependent, and at its maximum, cyclase is stimulated up to 25-fold. The activator has a molecular mass of about 40 kDa and is a multimer of a 6-7 kDa peptide.

No abstract

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel ␥-secretase inhibitor that reduces amyloid-␤ (A␤) production with an in vitro IC 50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T-and B-cell maturation in an in vitro thymocyte assay with an EC 50 of 2.1 M. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma A␤ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma A␤. To further characterize A␤ dynamics in brain, CSF, and plasma in relation to drug exposure and Notchrelated toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in A␤, and the magnitude and duration of A␤ lowering exceeded those of the reductions in B-cell endpoints. Other ␥-secretase inhibitors have shown high potency at elevating A␤ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in A␤11-40 and A␤1-43 at doses that potently inhibited A␤1-40 and A␤1-42. PF-3084014, like previously described ␥-secretase inhibitors, preferentially reduced A␤1-40 relative to A␤1-42. Potency at A␤ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.Amyloid-␤ (A␤) peptide is the primary component of senile plaques (Glenner and Wong, 1984) and is the protein product of a gene [amyloid precursor protein (APP)] whose mutation can result in early-onset Alzheimer's disease. The intersection of this genetic and pathologic evidence has led to a strong focus on A␤ as a major culprit in the etiology of Alzheimer's disease. A number of compounds have advanced to the clinic with the goal of either reducing production of this peptide (e.g., ␤-or ␥-secretase inhibitors) or increasing its clearance from the brain (e.g., A␤ vaccines or monoclonal antibodies). Of these approaches, ␥-secretase has yielded the greatest diversity of chemical tools that enable the study of A␤ pharmacodynamics in animal models and humans. Bioavailable small-molecule inhibitors of ␥-secretase from various chemical series have been shown to rapidly reduce A␤ levels in brain, cerebrospinal fluid (CSF), and plasma from wild-type mice (Yohrling et al., 2007), rats (Best et al., 2005;El Mouedden et al., 2006;Lanz and Schachter, 2006), guinea pigs (Anderson et al., 2005;, and multiple muArticle, publication date, and citation information can be found at