Seven years after semagacestat failed in Phase 3 clinical trials for Alzheimer’s disease, researchers led by Masayasu Okochi at Osaka University, Japan, offer a new reason why. In the October 3 Cell Reports, the scientists claim that the γ-secretase inhibitor, developed by Eli Lilly & Co. to reduce Aβ in the brain, traps accumulating Aβ peptides within cells. Because γ-secretase activity typically correlates with release of Aβ42 into the extracellular space, researchers had assumed that semagacestat’s ability to drive down Aβ secretion reflected its block of the secretase. Now the authors suggest the drug instead alters a previously unknown function of γ-secretase, i.e., translocation of Aβ across cell membranes. They caution against using secreted Aβ to measure γ-secretase activity and suggest semagacestat failed because it did not work as expected. Bart De Strooper, director of the U.K. Dementia Research Institute, considers the paper a wake-up call for the field (see full comment below). Researchers at Lilly declined to comment for this article.
- Researchers call semagacestat a “pseudo” γ-secretase inhibitor.
- The drug allows Aβ and other APP peptides to accumulate in cells.
- Semagacestat traps peptides in cell membranes.
“The study is important because it gives us new clues about the mechanism of γ-secretase activities,” said Lucía Chávez Gutiérrez at VIB/KU Leuven, Belgium. “To understand Alzheimer’s disease we need to look at all Aβ products generated, not just those that are released by cells.”
First co-authors Shinji Tagami and Kanta Yanagida decided to look inside and outside cells for γ-secretase products. They exposed neuron-like cells derived from human stem cells to 2 μM semagacestat—the highest concentration of the drug reported in spinal fluid in a Phase 1 clinical trial. As expected, secreted Aβ levels fell, while the β-secretase carboxy-terminal fragment of Aβ precursor protein (βAPP-CTF) rose, as determined by immunoprecipitation and western blot. Surprisingly, however, various Aβ species accumulated within the cells, including Aβ40, Aβ43, and Aβ46. “At first we thought we made a mistake,” said Okochi. “But no matter how many times we repeated it, we got the same result.”
Sema Surprise. Cleaving βAPP-CTFs (top), γ-secretase releases Aβ and peptides 3-6 amino acids long. Transition-state analogs, called true-GSIs by the authors, reduce production and secretion of these γ-byproducts (bottom left). Paradoxically, semagacestat drives up Aβ within cells (bottom right). (Courtesy of Tagami et al., Cell Reports.)
The researchers then surveyed the collection of Aβ peptides, and the 3-6 amino-acid peptides γ-secretase clips off, in carboxypeptidase fashion, as it trims longer Aβ fragments (Okochi et al., 2013; Takami et al., 2009; Sep 2016 news). They used high-performance liquid chromatography and mass spectrometry. The scientists also examined neuroblastoma SH-SY5Y cells, human embryonic kidney (HEK) cells, and HEK cells whose Aβ levels were jacked up due to expression of APP KM670/671NL carrying the Swedish mutation, which readily undergoes β-secretase cleavage.
The researchers found many 3-6 amino acid peptides within the various cells, seven of which shot up after semagacestat treatment: VVI (Aβ38-41), IAT (Aβ40-43), TVI (Aβ42-45), VIV (Aβ43-46), VIT (Aβ45-48), ITL (Aβ46-49), and VITL (Aβ45-49) (see image above). Because these small peptides could, in theory, come from hundreds of other proteins by chance, Tagami and Yanagida confirmed they were products of γ-secretase by testing for them in HEK/APP KM670/671NL cells lacking presenilins 1 and 2, key components of the γ-secretase complex. As expected, these cells harbored very low levels of the tiny peptides, and their concentrations remained unchanged after semagacestat treatment.
The researchers also found that related γ-secretase inhibitors RO4929097, MK-0752, and avagacestat increased the small peptides as well as intracellular Aβ40, Aβ42/43, and Aβ45/46. These types of inhibitor may bind allosteric sites on presenilin 1 (Svedružić et al., 2013). However, yet another compound, L685,458, mimics the catalytic transition state, and it decreased both the tiny peptides and intracellular Aβ40-46. The authors concluded that whereas L685,458 acts as a true γ-secretase inhibitor (GSI), semagacestat and related compounds are pseudo inhibitors.
To test their findings in vivo, the researchers fed three 30 mg/kg of semagacestat at 12-hour intervals to PS1 I213T knock-in mice (Nakano et al., 1999) overexpressing APP KM670/671NL. They then measured Aβ in whole brain extracts by immunoprecipitation followed by western blot, and also measured the tiny peptides by mass spec. The researchers chose these knock-in mice, which produce more Aβ42 than wild-type, because they were readily available. Although most small peptides showed up at similar levels in the brains of treated versus untreated knock-ins, the two major ones, VIV (Aβ43-46) and ITL (Aβ46-49), increased in semagacestat-treated mice, as did Aβ1-x peptides ranging in size from 43 to 46 amino acids.
Next, the researchers examined γ-secretase activity in cell-free assays. When they mixed affinity-purified γ-secretase and βAPP-CTF, then added 10 μM of semagacestat, the drug blocked generation of the small, 3-6 amino acid peptides almost completely. But when they included a crude membrane fraction, activity fell by only 50 percent. Chávez Gutiérrez said the conformation and activity of γ-secretase, as well as its interaction with semagacestat, may vary depending on its association with cell membranes. In addition, the authors discovered more small peptides trapped in the membranes in the semagacestat-treated assays than in the controls, suggesting the drug retards the release of γ-products from membranes.
Okochi and Tagami acknowledge they do not fully understand their findings. They think γ-secretase executes two functions: its well-known protease activity and a newly proposed translocator function that ferries Aβ peptides from the membrane to the extracellular space. How semagacestat interferes with each of these remains unclear, they noted, but translocation inhibition could be particularly harmful since it results in accumulation of Aβ peptides inside cells. “This study should be taken seriously. Longer Aβ peptides are potentially more toxic than shorter ones,” said Chávez Gutiérrez.
De Strooper wrote that the work is a technical tour de force. “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 γ-secretase inhibitor that targets the active site of the enzyme,” he wrote. Still, he agreed that the data are difficult to interpret. “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. … 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.”
Pinpointing where in the cell γ-secretase cuts APP and facilitates the exit of Aβ peptides may shed light on this question, he added. A recent study indicates the location of γ-secretase within cells, which varies depending on the presenilin protein associated with the γ-secretase complex, determines the ratio of Aβ42 to Aβ40 peptides produced and their intracellular versus extracellular fate (Jun 2016 news).
Lili Zhang at Aquinnah Pharmaceuticals in Cambridge, Massachusetts, also said the findings were difficult to explain. Zhang led a group at Schering-Plough (before it merged with Merck) that profiled semagacestat extensively, because it was a major competitor in her efforts to develop γ-secretase inhibitors and modulators. “An obvious alternative interpretation of their data is that the processing of intracellular Aβ involves additional protease activit(ies),” she wrote to Alzforum (full comment below). When semagacestat blocks γ-secretase, other proteases may take over the task of proteolyzing the accumulated βAPP-CTF substrate, she hypothesized. Semagacestat would have no effect on presenilin knockout cells, as the authors observed, because, with no γ-secretase to inhibit, no acute accumulation of substrate would occur.
Zhang also noted the transition-state analogs that appear to act as “true” GSIs may be blocking not only γ-secretase, but other proteases that might also generate Aβ peptides. Indeed, pharmacological data from at least one such inhibitor, L685,458, suggests it targets aspartyl proteases more broadly than do non-transition analog inhibitors (Clarke et al., 2006). “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,” Zhang wrote. She also noted that others have reported γ-secretase-independent processing of intracellular Aβ (Wilson et al., 2002).
What do the findings say about the failed semagacestat Phase 3 IDENTITY trials (Aug 2010 news)? The authors suggested the semagacestat-induced intracellular buildup of Aβ peptides helps explain the worsening of dementia, but Alex Roher, Banner Health System, Phoenix, who reported enhanced accumulation of Aβ in the brain of a patient treated with semagacestat, was not so sure (Roher et al., 2014). “The results should be seriously considered, but the paradigm is far too distant from the complexity of the human brain,” he said.
The IDENTITY trials had other shortcomings (De Strooper, 2014; Feb 2015 news). Semagacestat likely never reached levels high or steady enough within the brain to significantly engage γ-secretase, whereas in the periphery, levels were sufficient to block cleavage of potentially dozens of γ-secretase substrates, including Notch, causing side effects. “I do agree that the semagacestat trial did not truly test the amyloid hypothesis, but for reasons different from those suggested by this paper,” wrote Zhang.
What are the study’s implications going forward? “It should revive interest in the γ-secretases,” wrote De Strooper, who believes they are still the best validated drug target for treating AD (Voytyuk et al., 2017).
Okochi and Chávez Gutiérrez, who share De Strooper’s enthusiasm about γ-secretases as therapeutic targets, noted that the field has been looking beyond inhibitors. “When semagacestat and other GSIs were developed, we had very limited knowledge of γ-secretase mechanisms,” said Chávez Gutiérrez. “I’m now more in favor of stabilizers of γ-secretase.” Based on her recent findings, she thinks chemical chaperones that steady the enzyme’s interaction with amyloid peptides could promote the generation of shorter fragments that are less toxic (Szaruga et al., 2017; July 2017 news). Okochi is also interested in modifiers that would enhance the complete digestion of Aβ peptides.
Okochi and Tagami want to test additional secretase inhibitors for unexpected intracellular effects. “If pharma asks us to check, we are very willing to do so,” Okochi said. Their ultra-performance liquid chromatography/mass spec system can measure about 20 small peptides at once with high resolution and sensitivity. They also want to characterize γ-secretase’s translocator function. “No one has proposed before that a translocator malfunction could be involved in AD, but it might be a good target,” he said.—Marina Chicurel
- γ-Secretase Gives Up Secrets About Its Cleavage
- Lodged in Late Endosomes, Presenilin 2 Churns Out Intraneuronal Aβ
- Lilly Halts IDENTITY Trials as Patients Worsen on Secretase Inhibitor
- Semagacestat Failure Analysis: Should γ-Secretase Remain a Target?
- sAPP Binds GABA Receptor, and More News on APP
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