16 September 2011. The γ-secretase complex continues to tantalize researchers seeking to treat Alzheimer’s disease. Since this enzyme finishes the job of snipping APP into Aβ, interfering with it could lower amyloid and perhaps delay disease onset. Even as γ-secretase inhibitors have faced setbacks due to side effects (see, e.g., ARF related conference story), γ-secretase modulators (GSMs) have gained luster as a potentially safer approach. But exactly how GSMs work and even to what protein they bind remain burning questions, and it appears the answers may be different for different classes of GSMs. In the September 6 Journal of Biological Chemistry, researchers led by Harald Steiner and Christian Haass at Ludwig-Maximilians University, Munich, Germany, and Karlheinz Baumann at F. Hoffmann-La Roche Ltd., Basel, Switzerland, report that their second-generation GSMs bind directly to the N-terminal end of presenilin, the enzymatically active protein in the γ-secretase complex. However, only some GSMs from other groups show the same behavior in their assay. The authors first presented this data last July at the 2011 Alzheimer’s Association International Conference in Paris, France. It follows similar reports at AD/PD 2011 in Barcelona, Spain, from several groups who also showed that their second-generation GSMs directly bind presenilin (see ARF related news story).
The findings contrast with the sometimes-confusing literature on first-generation GSMs. These molecules belong to the class of non-steroidal anti-inflammatory drugs (NSAIDs) and include such compounds as sulindac sulfide and flurbiprofen. Some researchers have reported that NSAID-based GSMs lower Aβ by acting on the substrate, APP, rather than on the enzyme complex (see ARF related news story on Kukar et al., 2008 and ARF related news story on Richter et al., 2010). However, a later study questioned this finding (see Beel et al., 2009), and other papers suggest these GSMs may act directly on presenilin (see ARF related news story on Lleó et al., 2004; Eriksen et al., 2003; Takahashi et al., 2003; and Beher et al., 2004).
Second-generation, higher-potency GSMs, under development by several groups, are structurally distinct from NSAID-based compounds, and may have different targets. For example, TorreyPines Therapeutics, Inc., which has since folded, reports that its second-generation compounds primarily bound to the Pen2 protein component of the γ-secretase complex, and to a lesser extent to presenilin-1 (see ARF related news story on Kounnas et al., 2010).
Steiner and colleagues investigated the binding specificity of their second-generation GSMs, RO-02, RO-03, and RO-57. These are bridged aromatic aminopyrimidine-derived compounds that are potent in the low nanomolar range. To identify the molecular target, first author Amelie Ebke created a labeling compound by adding photoactivatable and biotin groups to the end of RO-57. The modified molecule, dubbed RO-57-BpB, was able to lower Aβ in cell culture, albeit with lower affinity than unmodified RO-57. When Ebke and colleagues irradiated the cell culture with UV light, the photosensitive end of RO-57-BpB bound covalently to the target molecule. The authors then pulled down the target using streptavidin, which binds biotin. Importantly, high amounts of RO-57, RO-02, and RO-03 competed with RO-57-BpB and reduced labeling, demonstrating that the modified molecule was binding its normal target. Over several experiments, the authors found that RO-57-BpB pulled down the N-terminal fragment of presenilin-2, and less strongly the N-terminal fragment of presenilin-1, but did not capture any other γ-secretase subunits or APP.
One of the advantages of this system is that it looks at binding in native cellular conditions, rather than in dissociated membranes, Baumann told ARF. Another plus is that the competitive assay confers specificity, he added. Other scientists praised the paper’s methodology. “The biochemistry in this paper is outstanding,” noted Joanna Jankowsky at Baylor College of Medicine, Houston, Texas.
The authors also tested several other classes of second-generation GSMs. They found that two GSMs under development at the pharma company Eisai (E2012 and ES2), which are structurally similar to RO-02, efficiently competed against the labeling molecule, but only one of the two TorreyPines compounds did. A second-generation GSM developed by Merck, GSM-1, competed well for presenilin-2 labeling but only weakly affected presenilin-1, suggesting the binding site differs between the two presenilins. In addition, Ebke and colleagues found that a few first-generation GSMs, including sulindac sulfide and fenofibrate, showed some binding to presenilin. In contrast, various γ-secretase inhibitors did not compete with RO-57-BpB, indicating GSMs do not bind at the enzyme’s catalytic site. This suggests that GSMs act allosterically, i.e., by altering the enzyme’s shape rather than directly interfering with the catalytic site.
The differential results highlight the idea that not all GSMs act in the same way. “Ultimately, I think the truth will be that there will be flavors of GSMs that interact with substrate, substrate and presenilin/γ-secretase, and combinations of these with lipid membrane interactions,” Todd Golde at the University of Florida, Gainesville, wrote to ARF (see full comment below).
The next step is to pin down the exact amino acid residues that second-generation GSMs are binding to, Baumann told ARF. This knowledge would enable researchers to develop molecular models of GSM action and ultimately use that information to design more potent drugs. Jankowsky agreed, telling ARF, “The more we know about where different modulators bind, the more we will understand about the mechanism by which they act. We then can go in and do rational drug design, so that we end up with something that is the third-generation γ-secretase modulator.”—Madolyn Bowman Rogers.
Ebke A, Luebbers T, Fukumori A, Shirotani K, Haass C, Baumann K, Steiner H. Novel gamma-secretase modulators directly target presenilin. J Biol Chem. 2011 Sep 6. Abstract