The Interaction Between Gamma-Secretase And Its Inhibitors And Modulators – Structural Insights

Gamma-secretase is a four subunit, 20-pass transmembrane aspartyl protease that has sustained a great deal of scientific scrutiny due to its role in disease: Gamma-secretase cleaves amyloid precursor protein, catalyzing the formation of amyloid plaques, which contribute to Alzheimer’s disease (AD) pathogenesis, and it also cleaves Notch, the abnormal signaling of which can lead to cancer. Gamma-secretase has been reported to process over 90 other substrates, all of which are type 1 transmembrane proteins. As a result of gamma-secretase’s enzymatic promiscuity and role in many pivotal cellular processes, it is a difficult target for drug development. Gamma-secretase inhibitors (GSIs), which pan-inhibit gamma-secretase so that it cannot cleave any of its substrates, are poor candidates for AD therapy as they cause toxicity. Still, GSIs are promising candidates for cancer. Gamma-secretase modulators (GSMs), which reduce formation of the amyloidogenic A?42 without affecting Notch signaling, are promising therapeutics for AD, as they avoid the Notch-associated toxicities seen with GSI treatment. In order to develop safe and effective therapies for AD and cancer we must gain a deeper understanding of gamma-secretase biology and the mechanism of action of GSIs/GSMs. To understand the process of gamma-secretase activation we developed CBAP-BPyne, a clickable photoaffinity probe that inhibits not only gamma-secretase activity, but also endoproteolysis, an event required for formation of an active gamma-secretase complex. We found that CBAP-BPyne specifically labels PS1-NTF and signal peptide peptidase (SPP). Endoproteolysis is not well characterized and CBAP-BPyne is a valuable tool with which to further explore its mechanism. To provide insight into the mechanism of action of GSIs/GSMs, we studied the impact of these compounds on the active sites of their target enzyme, gamma-secretase, and on an off-target enzyme, SPP. We found that GSIs/GSMs impact the active site architecture of not only gamma-secretase, but also SPP, suggesting that they may lead to a change in SPP activity and function, potentially causing toxicity in the clinic. Furthermore, we identified the binding site of BMS-708163, a clinically relevant GSI, on gamma-secretase, marking the first time that a GSI/GSM was mapped onto its target with high precision