A γ-secretase inhibitor that doesn’t block γ-secretase may seem like a bit of an oxymoron, but there are some compounds, including several non-steroidal anti-inflammatory drugs (NSAIDs), that appear to do just that. These γ-secretase modulators (GSMs) work by binding not the enzyme but the substrate, which, in the case of amyloid precursor protein (APP), leads to a shift in proteolytic cleavage that favors production of shorter, less toxic Aβ fragments. New evidence supports this “substrate targeting” effect and demonstrates how it might work. In the August 2 PNAS online, researchers led by Gerd Multhaup at the Free University of Berlin, Germany, report that GSMs prevent formation of APP dimers, which are more likely to be cleaved at the 42 position. The strength of the dimerization inhibition correlates with the Aβ42-lowering potency of these GSMs, a good indication that the modulators work by keeping APP in monomeric form. “This seems to be an important mechanism for some NSAIDS such as sulindac sulfide and indomethacin,” said Multhaup, though he agreed that other γ-secretase inhibitors may directly target presenilin, the catalytic subunit of γ-secretase.
Researchers led by Todd Golde, then at the Mayo Clinic, Jacksonville, Florida, and Eddie Koo at the University of California, San Diego, initially reported that GSMs, including some NSAIDs, block Aβ production by binding to APP (see ARF related news story on Kukar et al., 2008). “It is nice to see another group supporting our similar findings,” said Golde in an interview with ARF, but he cautioned that the dimerization hypothesis, first posited by Multhaup and colleagues (see Munter et al., 2007), has some challenges. “You can see how a compound that binds to APP dimerization motifs would interfere with dimerization, but that doesn’t necessarily mean that that is the mechanism that results in shifts in Aβ cleavage,” he said. Golde sees “inverse” modulators as a conundrum. His group showed that small tweaks to the molecular structure can turn GSMs from molecules that block Aβ42 production into compounds that promote it (see, e.g., ARF related news story on Kukar et al., 2005). “If you have compounds that lower Aβ42 by disrupting dimerization, then you would predict that compounds that raise Aβ42 would do the opposite [i.e., strengthen the dimer],” said Golde. “That could happen, but given the small difference in the structure of the compounds, it is highly unlikely.”
Part of the challenge in proving the dimerization theory lies in measuring it. APP is a large transmembrane protein that does not lend itself to the study of its intermolecular shenanigans. Multhaup and colleagues used a chimera instead. They coupled transmembrane residues 29-42 of APP (part of the Aβ sequence) to ToxR, a bacterial membrane protein. ToxR is a transcriptional activator that only works as a homodimer (see Langosch et al., 1996). In the chimera, transcriptional activation then becomes a readout of dimerization of the APP transmembrane domain.
First author Luise Richter and colleagues found that suldinac sulfide and indomethacin not only bind the Aβ sequence in a lipid-free environment, but also dose-dependently reduced activity of the ToxR-APP chimera. The authors also made eight derivatives of suldinac sulfide that had varying effects on Aβ42 secretion when added to Chinese hamster ovary cells expressing human APP and γ-secretase. Interestingly, those that reduced Aβ42 production most were also best able to block APP dimerization.
How do these compounds interfere with APP interactions? Multhaup and colleagues previously showed that a GxxxG motif in the Aβ sequence of APP is important for dimerization, and that mutations in that sequence can lower Aβ42 production (see Munter et al., 2010). Using a molecular modeling approach, Richter found that the GSMs bound to a flat interface formed when the four glycines, in two α helices, come together. The model explains why some derivatives of suldinac sulfide do not act as good γ-secretase modulators. The model predicts that suldinac sulfoxide, a poor GSM, repulses the acyl group of glycine 33 and does have a strong anti-dimerization effect.
While blocking dimerization is not the only way GSMs can work—the authors found one compound that fails to inhibit the ToxR chimer assay but does reduce Aβ42 production—it may be a good strategy to adopt for drug discovery, suggest the authors, because it could yield drugs with high specificity, since APP is the only γ-secretase substrate with three consecutive GxxxG motifs where drugs could bind. “Our results suggest a unique strategy including a high-throughput screen for the identification and for the development of new compounds with optimized pharmacological characteristics superior to well-known GSMs,” conclude the authors.—Tom Fagan
- Surprise! Some γ-Secretase Modulators Work by Targeting APP
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