A study in this week’s Nature offers unexpected insight into a class of drugs being explored for treatment of Alzheimer disease. These drugs target the activity of γ-secretase, a multimeric enzyme that plays a key role in generating amyloid-β (Aβ), whose abnormal buildup in the brain is thought to underlie AD. Led by Thomas Kukar and Todd Golde at Mayo Clinic College of Medicine, Jacksonville, Florida, a multinational collaboration involving researchers at six institutions reports that certain compounds known to blunt γ-secretase activity may do so without directly contacting the enzyme itself. Instead, these agents stymie γ-secretase by binding its substrate—amyloid precursor protein (APP). What’s more, the drugs tie up a region critical for neurotoxic aggregation of the Aβ peptides that get snipped from APP. Based on these findings, the authors suggest that the search for drugs in AD and other diseases could be greatly widened to include molecules that target substrates of enzymes suspected of being involved in pathology.

The substrate-targeting activity detailed in the Nature report was revealed in studies that extended prior work (Eriksen et al., 2003) showing that certain non-steroidal anti-inflammatory drugs (NSAIDs) could selectively lower levels of Aβ42—the form of Aβ believed to be most neurotoxic—and that they do so independent of their well-known effects on inflammation (Weggen et al., 2001; ARF related news story; ARF related conference story). Intrigue grew when researchers led by Sascha Weggen, then at the University of California, San Diego, reported that those NSAIDs lowered Aβ42 by modulating γ-secretase activity (Weggen et al., 2003), putting them into a class of drugs known as γ-secretase modulators (GSMs). Those findings had AD researchers wondering whether the Aβ-lowering NSAIDs could be safely harnessed to fight AD.

To get a better grip on how GSMs influence Aβ production, lead author Kukar and colleagues set out to identify their molecular targets. Collaborating with Abdul Fauq at Mayo’s chemical synthesis core and Boris Schmidt at Darmstadt Technical University, Germany, the researchers made biotinylated photoprobes from derivatives of two GSMs—one that raises Aβ42 levels—fenofibrate—and one that lowers Aβ42—tarenflurbil (inactive R-enantiomer of the NSAID flurbiprofen) which has been licensed to Myriad Genetics and is undergoing Phase 3 testing as a treatment for mild AD (see ARF related news story and drugs in clinical trials).

The Mayo team expected the GSM photoprobes to label presenilin-1 or some other core component of the γ-secretase complex. “We actually spent a lot of time looking for this interaction and came up with a lot of negative results,” Kukar told the Alzforum, recalling early attempts to detect presenilin-1 labeling in an APP-overexpressing human neuroglioma cell line. Blaming the negative results on limited sensitivity of their GSM photoprobes, Kukar and colleagues sought help from Harvard γ-secretase experts Michael Wolfe and then-postdoc Pat Fraering, who now heads an AD lab at the Swiss Federal Institute of Technology in Lausanne. After Wolfe and Fraering failed to detect labeling of any of the core proteins in a highly purified prep of active γ-secretase, “we went back to the drawing board and thought, what's left? That's when we thought about APP,” Kukar said.

Applying their photoprobes to a batch of recombinant APP, the researchers were delighted to see that APP lit up over a range of concentrations at which the GSMs typically modulate Aβ42 production. To establish the specificity of the GSM-APP interaction, the researchers threw Aβ42-lowering and -raising GSMs into the mix and found that each competed for labeling of APP, whereas a non-GSM NSAID did not. They showed that their photoprobes labeled APP more efficiently than Notch, another γ-secretase substrate.

Using a series of truncation mutants, the researchers mapped the GSM interaction domain to the Aβ region of APP and, furthermore, to an eight-amino-acid stretch of Aβ critical for aggregation. Co-authors Dominic Walsh and colleagues at University College Dublin, Ireland, then applied two Aβ42-raising GSMs and one Aβ42-lowering GSM to cultured CHO cells overexpressing APP and found that all three GSMs decreased Aβ oligomer formation.

“The idea that certain compounds will not only affect the toxic ratio of Aβ peptides generated, but could also lower toxicity by interfering with such oligomer formation, is very attractive indeed,” wrote Bart De Strooper of K.U. Leuven, Belgium, in an e-mail to ARF.

To further establish that binding to APP is, in fact, what enables some GSMs to decrease Aβ42 production, co-authors Edward Koo and colleagues at the University of California, San Diego, replaced a portion of the GSM binding site in APP with the analogous region of human Notch. Cleavage of this chimeric construct was not significantly affected by an Aβ42-lowering or -raising GSM but was inhibited by treatment with a γ-secretase inhibitor, the researchers found.

“This is an astonishing result,” writes Thomas Kodadek, University of Texas Southwestern Medical Center, Dallas, in a commentary accompanying the paper. “There are precious few examples of substrate-targeted enzyme inhibitors in the literature, all of which are peptides rather than drug-like small molecules. If Kukar and colleagues’ findings prove to be relevant to other proteases and their substrates, it would suggest that a completely new set of drug targets exists for the treatment of a variety of diseases.”

As a next step toward optimizing GSMs that show promise for AD drug development, Kukar and colleagues are taking a closer look not only at how the compounds reduce levels of Aβ42 but also at whether they raise levels of Aβ38 and shorter Aβ species. While studies have shown that increased levels of Aβ40 can prevent Aβ42 aggregation in animal models, the jury is still out as to whether shorter Aβ species confer similar protection. Kukar told ARF that his group has unpublished in vitro results suggesting that Aβ30, Aβ36 and Aβ37 might mimic Aβ40 in its ability to prevent Aβ42 aggregation.—Esther Landhuis.

References:
Kukar TL, Ladd TB, Bann MA, Fraering PC, Narlawar R, Maharvi GM, Healy B, Chapman R, Welzel AT, Price RW, Moore B, Rangachari V, Cusack B, Eriksen J, Jansen-West K, Verbeeck C, Yager D, Eckman C, Ye W, Sagi S, Cottrell BA, Torpey J, Rosenberry TL, Fauq A, Wolfe MS, Schmidt B, Walsh DM, Koo EH, Golde TE. Substrate-targeting gamma-secretase modulators. Nature. 2008 June 12;453:925-930. Abstract

Kodadek T. Molecular cloaking devices. Nature. 2008 June 12;453:861-2. Abstract

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  1. The recent report by Kukar et al. provides compelling evidence that some GSMs can modulate γ-secretase activity by binding the APP-CTF. The authors propose that the binding of GSMs to the APP-CTF induces conformational changes in the PS1/γ-secretase when the GSM-bound substrate enters the complex. As pointed out by Golde and colleagues, these exciting new findings nicely support our previous studies (Tesco et al., 2005) showing that the APPV715F substitution in the APP transmembrane domain mimics the effects of Aβ42-lowering NSAIDs, by reducing the ratio of Aβ42:Aβ40. This same substitution affected AICD production and PS1 conformation in a similar way to that of NSAIDs. These data suggested that the Aβ transmembrane domain might serve as a target for γ-secretase modulation.

    Phenylalanine-scanning mutagenesis of the APP transmembrane domain (TMD) had previously shown that APPV715F substitution shifts Aβ cleavage toward the production of the less fibrillogenic species Aβ38, while decreasing levels of Aβ40 and even more dramatically, levels of Aβ42 (Lichtenthaler et al., 1999). We also found that the APPV715F substitution does not affect AICD production, but significantly increases the distance between the N- and C-termini of PS1, as assessed by FLIM analysis. The transmembrane domain of APP has been postulated to adopt an α-helix (Lichtenthaler et al., 1999; Qi-Takahara et al., 2005).

    Since amino acid substitutions in the APP transmembrane domain may change its helical conformation, in the discussion section of our paper, we hypothesized that amino acid substitutions in the TMD of APP can produce a change in PS1 conformation, e.g., the APPV715F substitution induces an Aβ1-38-favoring conformation that shifts γ-secretase cleavage away from Aβ42 production. Accordingly, we suggested that the Aβ42-lowering NSAIDs may not necessarily target the PS1/γ-secretase complex, but instead bind to the transmembrane portion of APP, altering its helical conformation, which, in turn, alters the conformation of the PS1/γ-secretase complex. In the conclusion of our paper we suggested “that an allosteric modulation of γ-secretase shifting Aβ generation toward the less fibrillogenic Aβ1-38 may be achieved by small compounds targeting not only PS1 and/or other components of the γ-secretase complex, but also APP.” The excellent new study from Kukar et al. provides exciting new data clearly supporting this hypothesis.

    References:

    . APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation. J Neurochem. 2005 Oct;95(2):446-56. PubMed.

    . Mechanism of the cleavage specificity of Alzheimer's disease gamma-secretase identified by phenylalanine-scanning mutagenesis of the transmembrane domain of the amyloid precursor protein. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):3053-8. PubMed.

    . Longer forms of amyloid beta protein: implications for the mechanism of intramembrane cleavage by gamma-secretase. J Neurosci. 2005 Jan 12;25(2):436-45. PubMed.

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References

News Citations

  1. Anti-inflammatory Drugs Side-Step COX Cascade to Target Aβ
  2. Stockholm: Not All NSAIDs Are Equally Good When it Comes to Alzheimer’s
  3. News Brief: Flurizan Still in the Running

Therapeutics Citations

  1. Flurizan™

Paper Citations

  1. . NSAIDs and enantiomers of flurbiprofen target gamma-secretase and lower Abeta 42 in vivo. J Clin Invest. 2003 Aug;112(3):440-9. PubMed.
  2. . A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature. 2001 Nov 8;414(6860):212-6. PubMed.
  3. . Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid beta 42 production by direct modulation of gamma-secretase activity. J Biol Chem. 2003 Aug 22;278(34):31831-7. PubMed.
  4. . Biochemistry: Molecular cloaking devices. Nature. 2008 Jun 12;453(7197):861-2. PubMed.
  5. . Substrate-targeting gamma-secretase modulators. Nature. 2008 Jun 12;453(7197):925-9. PubMed.

External Citations

  1. Myriad Genetics

Further Reading

Papers

  1. . Possible mechanisms of action of NSAIDs and related compounds that modulate gamma-secretase cleavage. Curr Top Med Chem. 2008;8(1):47-53. PubMed.
  2. . Substrate-targeting gamma-secretase modulators. Nature. 2008 Jun 12;453(7197):925-9. PubMed.
  3. . Biochemistry: Molecular cloaking devices. Nature. 2008 Jun 12;453(7197):861-2. PubMed.

News

  1. News Brief: Flurizan Still in the Running
  2. Anti-inflammatory Drugs Side-Step COX Cascade to Target Aβ
  3. Stockholm: Not All NSAIDs Are Equally Good When it Comes to Alzheimer’s

Primary Papers

  1. . Substrate-targeting gamma-secretase modulators. Nature. 2008 Jun 12;453(7197):925-9. PubMed.
  2. . Biochemistry: Molecular cloaking devices. Nature. 2008 Jun 12;453(7197):861-2. PubMed.