γ-Secretase is an intramembrane aspartyl protease that is important in regulating normal cell physiology via cleavage of over 100 transmembrane proteins, including Amyloid Precursor Protein (APP) and Notch family receptors. However, aberrant proteolysis of substrates has implications in the progression of disease pathologies, including Alzheimer’s disease (AD), cancers, and skin disorders. While several γ-secretase inhibitors have been identified, there has been toxicity observed in clinical trials associated with non-selective enzyme inhibition. To address this, γ-secretase modulators have been identified and pursued as more selective agents. Recent structural evidence has provided an insight into how γ-secretase inhibitors and modulators are recognized by γ-secretase, providing a platform for rational drug design targeting this protease. In this study, docking- and pharmacophore-based screening approaches were evaluated for their ability to identify, from libraries of known inhibitors and modulators with decoys with similar physicochemical properties, γ-secretase inhibitors and modulators. Using these libraries, we defined strategies for identifying both γ-secretase inhibitors and modulators incorporating an initial pharmacophore-based screen followed by a docking-based screen, with each strategy employing distinct γ-secretase structures. Furthermore, known γ-secretase inhibitors and modulators were able to be identified from an external set of bioactive molecules following application of the derived screening strategies. The approaches described herein will inform the discovery of novel small molecules targeting γ-secretase.

Background: The PRESENILIN genes (PSEN1, PSEN2) encoding for their respective proteins have critical roles in many aspects of Alzheimer’s disease (AD) pathogenesis. The PS2V transcript of PSEN2 encodes a truncated protein and is upregulated in AD brains; however, its relevance to AD and disease progression remains to be determined. Objective: Assess transcript levels in postmortem AD and non-AD brain tissue and in lymphocytes collected under the Australian Imaging Biomarker and Lifestyle (AIBL) study. Methods: Full length PSEN2 and PS2V transcript levels were assessed by quantitative digital PCR in postmortem brain tissue (frontal cortex and hippocampus) from control, AD, frontotemporal dementia (FTD), and Lewy body dementia (LBD). Transcript levels were also assessed in lymphocytes obtained from the Perth subset of the AIBL study (n = 160). Linear regression analysis was used to assess correlations between transcript copy number and brain volume and neocortical amyloid load. Results: PS2V levels increased in AD postmortem brain but PS2V was also present at significant levels in FTD and LBD brains. PS2V transcript was detected in lymphocytes and PS2V/PSEN2 ratios were increased in mild cognitive impairment (p = 0.024) and AD (p = 0.019) groups compared to control group. Increased ratios were significantly correlated with hippocampal volumes only (n = 62, β= –0.269, p = 0.03). Conclusion: Taken together, these results suggest that PS2V may be a marker of overall neurodegeneration.

The aim was to assess tacrine hydrochloride (THA) as an inhibitor of rat hepatic oxidative enzymes. A model of hepatic microsome oxidative metabolism was established using antipyrine (AP) incubated with NADPH. AP and its metabolites, 3-hydroxymethyl antipyrine (HMA). 4-hydroxy antipyrine (OHA) and norantipyrine (NORA) were measured by high performance liquid chromatography (HPLC). Aliquots of 200, 400 and 600 microg/ml antipyrine were incubated with the microsomal preparation alone, with 20 microg/ml cimetidine or with 40, 80 or 200 microg/ml THA. Cimetidine inhibited HMA production by 35-38% (P<0.001) and OHA production by 49-52% (P<0.001). Incubation with the 3 concentrations of THA inhibited HMA production by 17%, 24% and 41% (P<0.001) and OHA production by 52%, 55% and 79%, respectively (P<0.001). NORA was identifiable when antipyrine was incubated with NADPH alone, but could not be identified after incubation with either cimetidine or THA. This study has shown that THA causes the inhibition of AP metabolism to HMA, OHA and possibly NORA. We suggest THA is an inhibitor of three different hepatic microsomal cytochrome P-450 enzyme sub-families.

Presenilin homologues in the γ-secretase complex play a pivotal role in substrate binding and processing, impacting β-amyloid (Aβ) peptide generation in Alzheimer's disease. We conducted a molecular simulation study to determine substrate preferences between presenilin-1 (PS1) and presenilin-2 (PS2) γ-secretase enzymes for amyloid precursor protein (APP) and Notch1 processing. Using homology modelling, we generated PS1- and PS2-γ-secretase models bound to substrates in the Aβ40 and Aβ42 generation pathways and Notch1 S3 and S4 cleavage site substrates. Metadynamics simulations and binding free energy calculations were used to explore conformational ensembles and substrate preferences. PS2-γ-secretase exhibited increased conformational flexibility and preferential binding energy for initiating the Aβ42 pathway compared to PS1-γ-secretase. Additionally, Notch1 exhibits a preference for binding to PS2-γ-secretase over PS1-γ-secretase. This study provides valuable insights into the conformational dynamics of γ-secretase bound to different substrates within a cleavage pathway, improving our understanding of substrate processivity. The findings highlight the importance of considering both PS1- and PS2-γ-secretase in structure-based drug design efforts, with implications for stabilizing or destabilizing specific states during APP processing.