New research may help develop drugs that target γ-secretase

Inhibition of γ secretase activity is a potential therapeutic strategy
for Alzheimer's disease (AD).
MRK-560 is a selective inhibitor that is more potent against PS1 than PS2, two subtypes of γ secretase-catalyzed subunits, but the underlying mechanisms remain elusive
.

On October 22, 2022, Shi Yigong of Westlake University and Zhou Rui of Tsinghua University jointly published a research paper
online.
The study reported cryo-electron microscopy structures of PS1 and PS2 γ-secretase complexes with and without MRK-560 with an overall resolution of 2.
9-3.
4.

。 MRK-560 occupies the substrate binding site of PS1, but is not visible
in PS2.
The structural comparison found that Thr281 and Leu282 in PS1 were determinants of MRK-560 subtype-dependent sensitivity, and exchange experiments between PS1 and PS2 confirmed this
.
By revealing the mechanism of selective suppression of subtypes of progerin, this work may contribute to the discovery
of future drugs targeting γ-secretase.

Strategies to inhibit γ-secretase activity show serious side effects

01

γ-secretase complexes can cleave a variety of type I transmembrane proteins, such as amyloid precursor protein (APP) and Notch protein
.
APP is initially cleaved by β-secretase to produce carboxy-terminal 99 residue fragments, called APP-C99, and then continuously cleaved by γ-secretase, releasing APP intracellular domains (AICD) and β-amyloid peptides (Aβ)
of varying lengths.
Relatively long Aβ tends to form oligomers and amyloid plaques
in the brain.

The accumulation of amyloid plaque is a hallmark
of AD.
Inhibition of γ-secretase activity has been used in the treatment
of AD.
So far, however, this strategy has not shown benefit; To make matters worse, serious side effects
were observed in clinical trials.
Side effects may be attributed in part to nonspecific inhibition of cleavage of other substrates by γ-secretase, particularly Notch, which plays a crucial role
in the determination of development and cell fate.
Selective inhibition of γ-secretase cleavage of apps on Notch is thought to mitigate side effects
.
However, APP and Notch bind γ-secretase in a similar way, complicating the design of substrate-selective γ-secretase inhibitors (GSIs
).

There is a priority association between the MRK-560 and the PS1

02

To verify the inhibitory effect of MRK-560, an isotype-selective inhibitor for PS1 and PS2, the research team reconstructed the in vitro lysis assay
using purified PS1 or PS2 complexes.
The production of Aβ40 (the main lysate in Aβs of different lengths) and AICD (whose cleavage reflects the endopeptidase activity of γ secretase) was measured
with AlphaLISA and Western blot, respectively.

Consistent with previous studies, MRK-560 showed more effective inhibition
of the PS1 complex.
The MRK-560 of the integrated circuit PS1 or PS2 complex is estimated to be 33±2 nM or 173±24 nM
, respectively.
In the presence of 200 nM MRK-560, AICD produced by the PS1 complex is completely inhibited
.
In contrast, AICD produced by the PS2 complex can still be detected
even in the presence of 10 μM MRK-560.

Upon binding to MRK-560, PS1 underwent substantial conformational changes
compared to the ligand-free state.
Similar to other reported GSIs, MRK-560 binds to induce the formation of two β and the movement
of PAL loops.
The cytosolic sequence after TM6 folds to TM6a, which is connected to TM6 by a flexible linker and bends
towards the inhibitor binding site.
PS2M adopts almost the same conformation as the apo complex and lacks the extended characteristics
of anti-parallel β sheet and TM6a.
Parallel structural analysis showed that there was a preferential association
between MRK-560 and PS1 compared to PS2.

Next, the researchers examined the structure to determine the molecular basis
of MRK-560 isoform selectivity.
MRK-560 forms three hydrogen bonds (H bonds)
with Asp385, Leu432 and Leu282 of PS1.
Among them, Asp385 is one of the catalytic residues of PS1, Leu432 precedes PAL motif and Leu282 is located in the intermediate circuit
between TM6a and β1 of PS1 。 The difluorobenzyl (DFB) and trifluoromethanesulfonamide (TFMS) groups of MRK-560 interact with Val272, Thr281, Lys380, Gly382, Ala431 and Pro433, and chlorophenyl (CP) groups interact with the side chains of Val379, Leu381, Leu418, Thr421, Leu422 and Ala434
.

To investigate whether these two residues were responsible for subtype-dependent sensitivity to MRK-560, the researchers generated PS1 and PS2 variants, swapped residues at these two locations, and examined their response
to MRK-560 inhibition.
A total of six variants were generated, including single residual exchange and double residue exchange of Thr281/Leu282 in PS1 versus Pro287/Ile288 in PS2
.
The response of MRK-560 to six complexes containing PS1/PS2 mutants is examined in an in vitro lysis assay, each purified to homogeneity
.

Research significance

03

Studies have shown that MRK-560 reduces the production
of Aβ in the brain.
Importantly, it does not induce Notch-related side effects
in wild-type mouse models.
In this work, the researchers determined the cryo-EM structure of the MRK-560-treated PS1/PS2 complex to elucidate the molecular basis
of MRK-560 isotype specificity.

MRK-560 occupies the substrate binding site of PS1, but is not visible
in PS2.
The structural comparison found that Thr281 and Leu282 in PS1 were determinants of MRK-560 subtype-dependent sensitivity, and exchange experiments between PS1 and PS2 confirmed this
.
By revealing the selective inhibition mechanism of subtypes of progerin, this research work may contribute to the discovery
of future drugs targeting γ-secretase.