. Semagacestat Is a Pseudo-Inhibitor of γ-Secretase. Cell Rep. 2017 Oct 3;21(1):259-273. PubMed.


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  1. This paper in Cell Reports is a wake-up call for the field. It should revive interest in the γ-secretases, a field in Alzheimer’s research that got a severe blow some years ago when the failure of the semagacestat trial was announced (Doody et al., 2013). That trial marked the end of γ-secretase research in companies. As a consequence, fundamental research on these fascinating enzymes also fell out of fashion and it became very hard to get research funds to study them. This is short-sighted and a mistake (De Strooper, 2014). Luckily, a few stubborn academic groups continued to try to better understand the biology, structure, function, and pharmacological properties of this fascinating family of enzymes. The research group of Okochi and colleagues is one of them, and their new paper should revive interest in the γ-secretases/presenilins also from a pharmaceutical point of view. They expose flaws in the underlying pharmacology of semagacestat and suggest that the field jumped to conclusions with regard to the implications of the failed trial for the amyloid hypothesis. Arguably, γ-secretases remain by far the clinically best-validated targets for drug development in the fight against Alzheimer’s disease available at this moment (see our recent review where we explain why γ-secretases are better-validated drug targets than β-secretase: Voytuck et al., 2017). 

    Okochi and collaborators’ report is a technical tour de force. They provide a systematic analysis in different experimental conditions of all the intermediary peptides that are generated during the consecutive cleavages of APP by γ-secretase and provide a unique tool to assess this important, and still not fully understood, aspect of γ-secretase function. They show that semagacestat exerts unexpected and paradoxical effects on these intermediary peptides that are different from the effects of real loss of function of presenilin or from a real γ-secretase inhibitor that targets the active site of the enzyme. As the effect of semagacestat on the initial epsilon endopeptidase cleavage and on the accumulation of these peptides is divergent, the explanation for the semagacestat effect must be complex.

    The authors suggest that γ-secretase is responsible for the turnover/removal of the tri- and pentapeptides and that semagacestat affects that clearance mechanism, resulting in the paradoxical accumulation of these peptides in the cell. They find also in mouse models that semagacestat causes the accumulation of Aβ43-x peptides. I would have loved to see dose-response curves and time-course experiments to understand better where this pool of peptides is coming from and how they are degraded. One would expect that the high doses of semagacestat (as used in the paper) would block the generation of these peptides since the initial cleavage at the epsilon site is blocked. It is very hard to see how effects on γ-secretase alone could explain this accumulation. Can the authors exclude cellular effects on γ-secretase distribution (Sannerud et al., 2016) that are involved in the accumulation of these peptide pools? In follow-up work we certainly need to get a better understanding of where those peptides are located in the cell: Are they in the γ-secretase complex or in the membrane compartment around the complex or in specific subcellular compartments? How is γ-secretase involved in the removal of these peptides from that compartment?

    An important message of the paper is that the field has jumped to conclusions when interpreting the failure of the semagacestat trial. I made that point before (De Strooper, 2014). From the Doody et al. paper it was already clear that semagacestat had aberrant effects on γ-secretase: In blood measurements it actually increased the Aβ42 to Aβ40 ratio, which a classical inhibitor would not do. In my opinion, the drug failed mainly because of pharmacokinetic reasons, however. The dosage of semagacestat was such that side effects were maximized and therapeutic effects minimized. The result was that the steady-state levels of Aβ in the brain were not affected at all (De Strooper, 2014). This trial never tested the amyloid hypothesis in a serious way. On top of this criticism, the work of Okochi et al. now clarifies the unknown effects of semagacestat on alternate peptides that may have pathological effects on their own. 


    . A phase 3 trial of semagacestat for treatment of Alzheimer's disease. N Engl J Med. 2013 Jul 25;369(4):341-50. PubMed.

    . Lessons from a failed γ-secretase Alzheimer trial. Cell. 2014 Nov 6;159(4):721-6. PubMed.

    . Modulation of γ- and β-Secretases as Early Prevention Against Alzheimer's Disease. Biol Psychiatry. 2017 Aug 10; PubMed.

    . Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool. Cell. 2016 Jun 30;166(1):193-208. Epub 2016 Jun 9 PubMed.

    View all comments by Bart De Strooper
  2. This is a comprehensive study that further shows the complexity of the processing of APP and Aβ. The authors have used several different cell lines and a mouse model to show that the γ-secretase inhibitor (GSI) semagacestat may not act as previously believed.

    The obvious question is if the findings in the paper can be translated to humans. For example, Bateman and colleagues have shown that semagacestat lowers the production of CNS Aβ in a dose-dependent manner. We have shown that treatment with the GSIs semagacestat and avagacestat increases the levels of CSF Aβ1-15/16 while Aβ1-34 decreases. In another set of experiments we showed that cells treated with the GSI DAPT (at a high dosage) greatly increased Aβ1-15/16, while all longer Aβ species from Aβ1-17 up to Aβ1-42 disappeared (even though many of the peptides cannot be substrates for γ-secretase). However, we don’t know what happened with the intracellular Aβ.    

    In conclusion, there are still many questions left to fully understand the generation of Aβ and the involved enzymes.

    View all comments by Erik Portelius
  3. Based on the observations that semagacestat inhibited only secreted Aβ but not intracellular or Aβ byproducts, Tagami, et al. suggested that semagacestat is only a “pseudo” γ-secretase inhibitor (GSI). However, an obvious alternative interpretation of their data is that the processing of intracellular Aβ involves additional protease activit(ies) other than γ-secretase. L-684,458, labeled as a “true GSI” in this paper, and semagacestat represent transition state and non-transition state GSIs, respectively. The distinct pharmacological profiles have been described many years ago (Clarke et al., 2006). The fact that the Bmax of L-684,458 is twofold of that for non-transition state GSIs suggests that L-684,458 binds to additional aspartyl proteases in addition to γ-secretase. L-684,458 blocks broader protease activity than non-transition state GSIs. I disagree with the conclusion of this paper and believe semagacestat is a potent, selective GSI that blocks both APP and Notch processing in vitro and in vivo.

    I do agree that the semagacestat trial did not truly test the amyloid hypothesis, but for reasons different from that suggested by this paper. The pharmacological property of semagacestat, combined with poor brain penetration and dose-limiting toxicity, resulted in a lack of target engagement, i.e., there was no significant CSF Aβ reduction in patients treated with the drug.     

    View all comments by Lili Zhang
  4. This is a very interesting study lending additional support for the hypothesis that increased intracellular Aβ is a major culprit of neurodegeneration and cognitive decline, while extracellular pool of Aβ plays relatively minor role. One obvious conclusion of the study is that future cell-based assays in screening for Aβ-lowering drugs should include quantification of intracellular pool of AβA as their readout. It will be interesting to see if semagacestat causes intracellular accumulation of Aβ by affecting the function of intracellular Aβ-degrading proteases (i.e., insulin-degrading enzyme, IDE) through their direct inhibition or blocking their access to Aβ by impairing their trafficking.

    View all comments by Igor Kurochkin
  5. This is a very important paper, showing the unexpected result that semagacestat causes the accumulation of longer Aβ species and short peptide byproducts that are produced by the sequential carboxy-terminal trimming activity of γ-secretase.

    The paper highlights that we still have not fully understood the complex processing of C99 by γ-secretase at the mechanistic level. Now, with these findings by Tagami et al., it is getting even more complex. The data suggest that semagacestat is not inhibiting the enzyme but rather altering its processivity, leading to an accumulation of longer Aβ species such as Aβ46 and Aβ43. It is possible that C99 could perhaps not bind anymore if Aβ46 and Aβ43 would not be released from γ-secretase, leading to pseudoinhibition and reduction of Aβ and AICD as a secondary event. Likewise, byproducts that are not leaving the membrane potentially could also be inhibitory if they stay bound to the enzyme or alternatively if they flood the membrane and thus compete with C99 binding. It is possible that in particular the accumulation of intracellular long Aβ may add to the likely membrane-toxicity of uncleaved C99 that accumulates in the presence of semagacestat, although the amount of long Aβ, such as Aβ46, seems very minor as compared to that of C99.

    Interestingly, earlier studies also showed that the γ-secretase inhibitor DAPT causes the accumulation of longer Aβ45/46 inside cells, now indicating pseudoinhibition of this compound as well (Zhao et al., 2004; Qi-Takahara et al., 2005). However, it remains puzzling that in cell-free γ-secretase cleavage assays with CHAPSO-solubilized enzyme accumulation of such longer Aβ species in the presence of DAPT has not been observed. Phosphatidylcholine membranes are typically added in such assays that could take up accumulating longer Aβ peptides (Kakuda et al., 2006). However, DAPT clearly inhibits the formation of AICD in these assays as well as the carboxy-terminal processing to Aβ (Kakuda et al., 2006), and so does semagacestat (Chávez-Gutiérrez et al., 2012). 

    Why the short-peptide byproducts can accumulate in the membrane also remains mysterious. Tagami et al. now propose a new additional “pump-like” activity function of γ-secretase that releases the byproducts from the membrane, with which semagacestat interferes. However, intuitively, one would assume that the affinity of the 3–5 amino acid peptide byproducts will be so low that they should be released immediately into the hydrophilic space after they are generated, especially as we know from the structural studies that the active site of γ-secretase is in a solvent exposed cavity.

    Clearly, more studies are now needed to understand how inhibitors such as semagacestat cause the observed effects. 


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    View all comments by Harald Steiner
  6. First, we would like to thank all the scientists and interviewees who made comments and gave their opinions on our study. These are very precious rewards for our efforts.

    In our previous Cell Reports paper (Okochi et al., 2013), we had started to confront the contradictory increase of γ-by-products by semagacestat. These are the 3–5 amino-acid peptides consecutively clipped off by γ-secretase. After the paper was accepted, I wondered where to go next, and visited Yasuo Ihara because I needed his group’s help to measure γ-by-products (Takami et al., 2009). Of course, both of us thought that the semagacestat story would be interesting, but very tough stuff to explore. However, we decided to stick with it, because we felt that the new findings looked most mysterious. I must mention, Yasuo's stubbornness helped us move forward.

    There are many issues yet to be addressed, as noted by commenters. We do not know how toxic the intracellular, probably membrane-bound, Aβ is for neurons, and the same is true for γ-by-products. I strongly agree that there are still many uncovered secrets about the functions of presenilin/γ-secretase. Differing from the common approach in this field, I believe it is possible that we still have not acquired all essential knowledge about γ-secretase, which is key to developing AD therapeutics. This may be the reason why developing therapeutics based on the Aβ hypothesis has not worked yet.

    Based on the results of our paper, we think that, when PS/γ-secretase is knocked down, no other enzyme can take its place. Although our observations are limited, it seems to me that when PS/γ-secretase function becomes insufficient in cells, γ-by-products start to accumulate intramembranously, and Aβ tends not to be liberated from membrane. Isn’t it rather important that these membrane-bound Aβ and/or γ-by-products can stay there stably, depending on some condition related to γ-secretase?

    Needless to say, the transmembrane domain of βAPP is insoluble. Once the transmembrane domain is chopped up to tripeptides, these become water-soluble, but the processing occurs in hydrophobic conditions. They seem to be able to stay put amidst a hydrophobic environment, according to our data. One may think that γ-by-products are amphipathic, but, I think this is not exactly the case, since γ-by-products in hydrophobic and hydrophilic phases are clearly separated. This indicates that liberation of γ-by-products from hydrophobic conditions to hydrophilic conditions requires some kind of catalytic activity, otherwise the liberation to hydrophilic space at a low-energy state fraction would occur automatically.

    The concept of γ-by-products gives us new insight into the major γ-by-product: Aβ. For example, we usually do not pay attention to whether γ-by-products and Aβ are found in soluble or insoluble space. However, how can we be so certain that γ-by-products and Aβ (secretable forms, like Aβ40) are automatically liberated from the membrane and transferred to the soluble space? We may have to start thinking whether soluble forms of Aβ are really the only culprits triggering AD pathology. It is important to study whether membrane-bound stable Aβ (and γ-by-products) play any roles.

    We hope our study will motivate many researchers in this field to study novel functions of this fascinating enzymatic complex that we proposed. Of course, we remain more than willing to collaborate in studies that will explore whether any kinds of compounds acting on γ-secretase (or other compounds unrelated to the enzyme) cause accumulation of γ-by-products or not. Thank you again for your kind interest in our study. 


    . γ-secretase modulators and presenilin 1 mutants act differently on presenilin/γ-secretase function to cleave Aβ42 and Aβ43. Cell Rep. 2013 Jan 31;3(1):42-51. PubMed.

    . gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.

    View all comments by Masayasu Okochi

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  1. Semagacestat, a Pseudo γ-Secretase Inhibitor?