With rare exceptions the biological role of secretase

With rare exceptions, the biological role of γ-secretase cleavage of substrates other than Notch 1 has been ignored during proof of concept preclinical repurposing studies. For example, in the GSI AD trial, many individuals noted changes in hair color, apparently due to inhibition of tyrosinase, another γ-secretase substrate [101]. In a few studies other Notch paralogs and VEGFR1 have been considered to be targets [70], [102], [103]. Given that tools are not readily available to perform facile, detailed studies on the impact of γ-secretase cleavage on γ-secretase substrates other than APP and Notch 1; it will be important to develop these tools to better understand the biological consequences of GSI based therapies [4]. Indeed, the biology of γ-secretase is complex and our understanding of it suffers from the lamplight effect. As a field we have largely focused only on what we can easily see — cleavage of Notch 1 and APP [104]. Sub-unit composition and subcellular localization of γ-secretase within the target cell may influence both activity on a given substrate and response to a given GSI. Zhao and colleagues have shown that sulfonamide based GSIs selectively inhibit PSEN1 over PSEN2, whereas the GSIs DAPT and L685458 showed minimal selectivity [105]. Similarly, recent data suggest that the GSI MRK-560 preferentially targets PSEN1 over PSEN2 and that this selectivity, at least in mice, increases the tolerability of this GSI [106]. De Strooper and colleagues also show that heterogeneity with respect to the Aph1 subunit is important with respect to viability and overall phenotype of mice, indicating that selective targeting of Aph1b γ-secretase complexes may be less toxic [107]; however, it is not clear whether selective targeting of Aph1b γ-secretase complexes is feasible. Other studies also provide some evidence that various γ-secretase complexes may have differential sensitivity to GSI and different substrate preferences [108], [109], [110]. In many cases it is not clear if substrate preference, differential spatiotemporal PU-H71 patterns of the various γ-secretase subunits, or a combination of these factors, contributes to the phenotype. Outside of AD, the fact that γ-secretase is a heterogeneous activity has largely been ignored.
Avoiding GSI toxicity, substrate selective GSIs and other strategies Given the toxicities associated with inhibition of Notch 1, especially those associated with altered proliferation and maturation of gut epithelium, there has been considerable effort to develop APP selective inhibitors or at least inhibitors that would reduce in vivo toxicity sufficiently to enable significant reduction of Aβ production in the brain [111]. Unfortunately, so called “Notch-sparing” APP selective GSIs that can be shown to preferentially inhibit γ-secretase APP relative to Notch 1 in preclinical studies have not shown reduced toxicity nor increased Aβ lowering in humans [112], [113], [114], [115], [116], [117], [118], [119]. Furthermore, recent studies suggest that one potential issue is that substrate selectivity has not always been assessed in matched assays, and when GSIs inhibitory profiles are examined in matched assays substrate selectivity is absent [117], [120]. Thus, it is possible that the apparent substrate selectivity is really attributable to different in vitro assays. Discrepant IC50 values may arise when using reporter assays for γ-secretase activity that rely on translocation of endogenous or transcription factor tagged intracellular domains of a given substrate. In these assays, γ-secretase cleavage liberates the intracellular domain and enables it to translocate to the nucleus where it activates a reporter gene [121], [122]. It is not clear that these reporter assays accurately reflect effects on cleavage, as there are often large differnces between inhibitor IC50s in the reporter assays versus direct cleavage assays. One thought is that the overexpression of and cleavage of substrate may over-saturate the transcriptional reporter; thus; significant inhibition of cleavage can be observed before the reporter activity is decreased.