A single dose of the γ-secretase inhibitor semagacestat alters the cerebrospinal fluid peptidome in humans

Abstract

Background: In Alzheimer's disease, beta-amyloid peptides in the brain aggregate into toxic oligomers and plaques, a process which is associated with neuronal degeneration, memory loss, and cognitive decline. One therapeutic strategy is to decrease the production of potentially toxic beta-amyloid species by the use of inhibitors or modulators of the enzymes that produce beta-amyloid from amyloid precursor protein (APP). The failures of several such drug candidates by lack of effect or undesired side-effects underscore the importance to monitor the drug effects in the brain on a molecular level. Here we evaluate if peptidomic analysis in cerebrospinal fluid (CSF) can be used for this purpose.

Methods: Fifteen human healthy volunteers, divided into three groups, received a single dose of placebo or either 140 mg or 280 mg of the γ-secretase inhibitor semagacestat (LY450139). Endogenous peptides in CSF, sampled prior to administration of the drug and at six subsequent time points, were analyzed by liquid chromatography coupled to mass spectrometry, using isobaric labeling based on the tandem mass tag approach for relative quantification.

Results: Out of 302 reproducibly detected peptides, 11 were affected by the treatment. Among these, one was derived from APP and one from amyloid precursor-like protein 1. Nine peptides were derived from proteins that may not be γ-secretase substrates per se, but that are regulated in a γ-secretase-dependent manner.

Conclusions: These results indicate that a CSF peptidomic approach may be a valuable tool both to verify target engagement and to identify other pharmacodynamic effects of the drug. Data are available via ProteomeXchange with identifier PXD003075.

Trial registration: NCT00765115 , registered 30/09/2008.

Figures

Fig. 1

Study design. CSF sampled at several time points following oral administration of semagacestat was subjected to labeling using TMT reagents 128-131. The combined labeled samples from each participant (TMT 6-plex set) were centrifuged through a 30 kDa molecular weight cut-off filter. The flow-through, containing the endogenous peptide fraction, was analyzed by LC-MS. The relative change in concentration of each identified peptide was calculated from the TMT reporter ion signals. CSF cerebrospinal fluid, TMT tandem mass tag, LC-MS liquid chromatography-mass spectrometry

Fig. 2

β-amyloid 22-28. The peptide EDVGSNK, constituting fragment 22-28 of β-amyloid within APP. a annotated MS2 spectrum. b Relative abundance of the peptide after semagacestat treatment. In the 280 mg dosage group the concentration of the peptide decreased to a minimum of 42 % at 9 h (p = 0.0035), while in the 140 mg dosage group the minimum relative abundance was 13 % at 12 h (p = 0.36). Graphed data are medians with median absolute deviations. APP amyloid precursor protein

Fig. 3

APL1β17. The peptide DELAPAGTGVSREAVSG, constituting fragment APL1β17 from amyloid-like protein 1 (APLP1). a Annotated MS2 spectrum. b Relative abundance of the peptide after semagacestat treatment. In the 280 mg dosage group the concentration of the peptide decreased to a minimum of 32 % at 12 h (p = 0.0018), and in the 140 mg group the minimum relative concentration was 15 % at 18 h (p = 0.078). Graphed data are medians with median absolute deviations

Fig. 4

Transmembrane location of Aβ22-28 and APLP1β17. Location of the peptides (a) Aβ22-28 and (b) APLP1β17 relative to the transmembrane region of their respective protein sequences. The γ-secretase cleavage sites in APP are indicated with arrows. APP amyloid precursor protein