Targeting the APP-Mint2 Protein-Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation

Peptide-Based Inhibitor Reduces Amyloid ‑β Formation

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Targeting the APP-Mint2 Protein−Protein Interaction with a

Christian R. O. Bartling, Thomas M. T. Jensen, Shawna M. Henry, Anna L. Colliander, Vita Sereikaite,

Marcella Wenzler, Palash Jain, Hans M. Maric, Kasper Harpsøe, Søren W. Pedersen,

Louise S. Clemmensen, Linda M. Haugaard-Kedstro

and Kristian Strømgaard *

m, David E. Gloriam, Angela Ho,

Cite This: J. Am. Chem. Soc. 2021, 143, 891 −901

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sı Supporting Information

ABSTRACT: There is an urgent need for novel therapeutic

approaches to treat Alzheimer’s disease (AD) with the ability to

both alleviate the clinical symptoms and halt the progression of the

disease. AD is characterized by the accumulation of amyloid-β

(

Aβ) peptides which are generated through the sequential

proteolytic cleavage of the amyloid precursor protein (APP).

Previous studies reported that Mint2, a neuronal adaptor protein

binding both APP and the γ-secretase complex, aﬀects APP

processing and formation of pathogenic Aβ. However, there have

been contradicting results concerning whether Mint2 has a

facilitative or suppressive eﬀect on Aβ generation. Herein, we deciphered the APP-Mint2 protein−protein interaction (PPI) via

extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-aﬃnity

peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deﬁcient Mint2 variant and a cell-permeable PPI

inhibitor signiﬁcantly reduced Aβ levels in a neuronal in vitro model of AD. Together, these ﬁndings demonstrate a facilitative role

of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a

promising therapeutic strategy to reduce pathogenic Aβ levels.

INTRODUCTION

modulation of direct binding partners involved in APP

traﬃcking and processing, could therefore increase the chance

of developing a successful AD treatment, which is of increasing

importance in light of a constantly growing patient

■

Amyloid-β (Aβ) peptides, the main component of senile

amyloid plaques, play a key role in Alzheimer’s disease (AD)

pathogenesis. Aβ peptides are generated by the sequential

population.

proteolysis of the amyloid precursor protein (APP), a type I

Munc18-interacting proteins (Mints, also known as X11s)

are a family of adaptor proteins encoded by three distinct

genes (ABPA1, ABPA2, and ABPA3) that produce neuron-

speciﬁc Mint1 (X11α) and Mint2 (X11β) and ubiquitously

transmembrane protein of 695−770 amino acids. In the

pathogenic amyloid cascade, cleavage by an aspartyl protease

(

BACE-1 or β-secretase) generates a soluble APP ectodomain

sAPPβ) and a membrane-bound C-terminal fragment (C99).

−11

Subsequent cleavage of the C99 fragment by γ-secretase

generates the APP intracellular domain (AICD) and Aβ

expressed Mint3 (X11γ).

Mint proteins consist of a

variable isoform-speciﬁc N-terminal region and a conserved C-

terminal region that contains a phosphotyrosine binding

^f^r^a^g^m^eⁿ^t^s^o^f^v^a^rⁱ^o^u^s^l^eⁿ^g^t^h^s^,ⁱⁿ^c^l^u^dⁱⁿ^g^A^β⁴0 and Aβ . The

latter exhibits an increased propensity to form insoluble

aggregates, which are a hallmark of AD pathology.

(

PTB) domain and two tandem PSD-95/discs large/zonula

occludens (PDZ) domains (Figure 1a). The PTB domain of

all three Mint proteins binds the conserved endocytic

YENPTY motif of APP, which is essential for regulating APP

The accumulation of Aβ peptides at synaptic sites has

detrimental eﬀects on synaptic function, which contributes to

the cognitive decline and memory loss associated with AD.

The most advanced anti-Aβ drugs in clinical development are

anti-Aβ immunotherapies promoting clearance of the Aβ

peptide and its aggregates or β-secretase inhibitors intended to

Received: October 8, 2020

Published: January 5, 2021

interfere with the initial step of Aβ formation. However, both

strategies have suﬀered from failure in late-stage clinical

trials, and no disease-modifying AD therapy is yet available.

Alternative strategies to reduce Aβ formation, such as the

2021 American Chemical Society

J. Am. Chem. Soc. 2021, 143, 891−901

Article

relevance to Aβ formation and AD. Eﬀorts have been made to

decipher the physiological role of Mint proteins in Aβ

formation, and numerous knockout and knockdown studies,

17,20,24−30

using mainly in vivo models, have been reported.

However, these studies revealed both suppressive and

facilitative eﬀects of Mint proteins on Aβ formation. The

role of Mint proteins in the formation of pathogenic Aβ

remains controversial since no chemical probe perturbing the

APP-Mint PPI has been reported.

Herein, we characterized the APP-Mint2 interaction with

molecular resolution through extensive substitutional analyses

of both the APP C-terminal peptide ligand (APP_C_‑_t_e_r_m) and the

Mint2 protein. The introduction of backbone amide-to-ester

(

A-to-E) modiﬁcations uncovered an intricate H-bond net-

work at the APP-Mint2 interface, complementing previously

published cocrystal structures. Alanine (Ala) substitutions in

the PTB domain of Mint2 identiﬁed key residues and led to

the generation of an APP binding-deﬁcient Mint2 variant that

reduced Aβ generation in vitro. Furthermore, we performed a

systematic structure−function analysis of the APP C-terminal

peptide which facilitated the development of a proteolytically

stable macrocyclic peptide exhibiting nanomolar aﬃnity

toward Mint2. In a neuronal in vitro model of AD, cell-

permeable variants of the high-aﬃnity C-terminal APP mimetic

peptide demonstrated that modulation of the APP-Mint2

interaction reduces Aβ₄₂levels.

RESULTS AND DISCUSSION

■

Minimal APP Mint2-PARM Binding Sequence and

Semisynthesis of Mint2-PARM Variants. Within the Mint

protein family, Mint2 exerts the greatest eﬀect on Aβ

formation and was therefore selected as the target protein.

The interaction of APP and Mint2 is mediated by the PTB

domain of Mint2 and modulated in an autoinhibited open−

closed fashion by a C-terminal α-helical linker (termed ARM)

Figure 1. Structural overview of the Mint protein family and the

Mint2-APP interaction. (a) Schematic architecture of Mint proteins

illustrating their conserved C-terminal region consisting of a

phosphotyrosine binding (PTB) domain (gray), an α-helical ARM

linker (dark gray), two PSD-95/drosophila discs large/zonula

occludens (ZO-1) (PDZ) domains (light gray), and a variable N-

terminal region. Numbering corresponds to human residues. MI =

Munc-18 interaction domain, CI = CASK interaction domain. (b)

Cartoon representation of the interaction between rat Mint2-PARM

12,31,32

adjacent to the PTB domain.

Consequently, we designed

a human Mint2-PTB-ARM (Mint2-PARM) construct (PARM-

His6; residues 364−570) as a model protein (Figure 1a). To

identify the minimal binding sequence of APP, we systemati-

cally tested N- and C-terminal truncated variants of the human

7-mer APP_C_‑_t_e_r_mpeptide (QNGYENPTYKFFEQMQN;

residues 754−770 in APP ₇ ₇ ₀ ). Using a ﬂ uorescence polar-

ization (FP) assay (SI section Detailed Methods), we

identiﬁed a 12-residue-long peptide, designated as the

(

gray) and APP (residues 754−767; blue). The APP

is binding at

C-term

the interface between the α3-helix and β5-strand of Mint2, forming an

antiparallel β-sheet (N-terminal) followed by a β-turn and a C-

terminal α-helix. The ARM linker (dark gray) is found in an open

conformation enabling APP binding. (c) Structure of the APP peptide

APP_W_Tpeptide (NGYENPTYKFFE; residues 755−766; K =

.0 ± 0.2 μM), as the minimal Mint2-PARM binding sequence

(

Figure S1 and Table S1 ).

(blue sticks) bound to the rat Mint2-PTB domain (gray). The β5-

strand of the PTB domain (gray stick, side chains not depicted) is

shown to highlight the backbone−backbone H-bond network (black

dotted lines) between the Mint2-PTB domain and APP (PDB ID:

The reported X-ray cocrystal structure of Mint2 and the

APP peptide (QNGYENPTYKFFEQ; residues 753−767,

Figure 1b, blue) suggests that an intricate network of backbone

hydrogen bond (H-bond) interactions and side-chain inter-

3SV1).

actions mediates the APP-Mint2 interaction (Figure 1c, dotted

traﬃcking and aﬀects Aβ production. ³⁻¹⁶A number of studies

suggest that the APP-Mint protein−protein interaction (PPI)

is of particular biological signiﬁcance in AD. First, the loss of

each individual Mint isoform delays age-dependent Aβ plaque

lines).

12,14

Therefore, we probed side-chain interactions by

introducing Ala substitutions, whereas backbone H-bond

interactions can only be eﬀectively probed by introducing A-

33−37

to-E substitutions.

While we generated Mint2-PARM Ala

formation in mouse models of AD. Second, Mint proteins

interact via their PDZ domains with presenilin-1, the catalytic

subunit of the γ-secretase complex, thereby promoting APP/

variants through recombinant expression (Table S2), introduc-

ing A-to-E substitutions into the Mint2-PARM domain

required the development of a semisynthesis strategy.

Speciﬁcally, we ﬁrst generated a pseudo-wild-type Mint2-

PARM variant (pPARM-His6; residues 364−570; R455K,

C483A, C501A, C566A) containing three cysteine (Cys) to

Ala substitutions enabling the use of native Ala residues in the

18−21

presenilin-1 colocalization favoring Aβ formation.

Mint proteins are found to be upregulated in AD and are

reported to associate with neuritic plaques.

these ﬁndings indicate that the APP-Mint PPI is of therapeutic

Third,

22,23

Altogether,

J. Am. Chem. Soc. 2021, 143, 891−901

Article

Figure 2. Semisynthesis of Mint2-PARM and eﬀects of A-to-E and Ala mutations in APP and Mint2-PARM. (a) Semisynthetic approach to

introducing A-to-E substitutions in Mint2-PARM. N-terminal fragment PARM (A479-Q570) is fused to a Xa site to generate an N-terminal Cys

by factor Xa cleavage. C-terminal fragment PARM_C(E364-H452) is expressed with a C-terminal intein to generate the required C-terminal

thioester. Semisynthesis is initiated by ligating synthetic peptide fragment PARM_p_e_pto PARM . Next, the Thz group is converted to a free Cys and

PARM_p_e_p₊_Nis ligated to PARM . Semisynthetic Mint2-pPARM is obtained after desulfurization and refolding (Figures S2 −S7). (b) Aﬃnity fold-

change of the APP_W_Tpeptide’s Ala-scan and (c) A-to-E substitutions toward Mint2-PARM obtained in an inhibition FP assay (K ) reported relative

to the APP_W_Tpeptide [K (mutant)/K (APP ) + SEM]. (d) Aﬃnity fold change of the APP peptide binding to Mint2-PARM Ala and (e) A-

C‑term

to-E variants obtained by the saturation FP assay (K ) relative to Mint2-PARM [K (variant)/K (wild type) + SEM]. See Figures S8 and S9 for

FP data.

39,40

native chemical ligation (NCL). Next, we synthesized the same

(SI section Detailed Methods).

It is generally accepted that

version of Mint2-PARM semisynthetically (pPARM ; residues

A-to-E substitutions do not perturb the overall protein

64−570; R455K, C483A, C501A, C566A with no His tag)

structure. However, the H-bond donor ability of the

respective amide-NH is removed and the acceptor capability

of the adjacent carbonyl is reduced, which provides a reﬁned

tool for studying H-bond networks. A-to-E mutations have

successfully been employed to address the signiﬁcance of

backbone H-bonds for folding β-sheet structures and backbone

using a three-fragment expressed protein ligation (EPL)

approach (Figure 2a). Both protein variants (pPARM-His6

and pPARM ) displayed similar secondary structure and

binding aﬃnity for the 17-mer APP _C _‑_t _e _r _m peptide as

recombinantly expressed Mint2-PARM (Figures S2 and S3),

conﬁrming that neither the His6-tag nor the introduced

substitutions aﬀect APP binding. We applied the semisynthetic

strategy to introduce eight A-to-E substitutions (R455κ,

T456τ, I457ι, S458σ, Y459ψ, I460ι, A461α, and D462δ; α-

38,42−44

H-bonds in Aβ peptides.

In total, 21 Mint2-PARM protein variants containing both

Ala and A-to-E substitutions and 17 APP_W_Tpeptide analogues

were generated. We then employed the FP assay to address the

consequences of each substitution on the APP-Mint2

interaction (Figure 2b−e).

Backbone H-Bonds and Side-Chain Contacts Are

Essential to the APP-Mint2 Interaction. Single-point Ala

substitutions in the APP_W_Tpeptide resulted in a >10-fold loss

of a ﬃ nity to Mint2-PARM in 4 out of 12 positions (Figure 2b,

Figure S8a,b , and Table S1 ). The substitution of two residues,

hydroxy amino acids abbreviated with Greek letters) into

Mint2-pPARM_S_S(Tables S3 and S4 ), which were all obtained

with suﬃcient yields and purities (Figures S4 −S7).

Similarly, we introduced A-to-E substitutions in ﬁve

positions of the APP_W_Tpeptide (G756γ, Y757ψ, E758ε,

N759ν, and F764ϕ). The corresponding depsi-peptides were

synthesized on the solid phase using α-hydroxy amino acids

J. Am. Chem. Soc. 2021, 143, 891−901

Journal of the American Chemical Society

FP assay (Figure 3 a and Figure S10 ). To examine whether

Article

Y757A and N759A located in the endocytic YENPTY motif of

APP, resulted in 76-fold and >100-fold decreases in aﬃnity,

respectively. In addition, Ala substitution of residues F764 and

F765 resulted in 14- and 18-fold decreases in aﬃnity,

respectively (Figure 2b). The N-terminal region of the APP

peptide forms an antiparallel β-sheet with the β5 strand of

Mint2-PARM. On the basis of the X-ray cocrystal structure, H-

bond interactions that formed between the APP peptide and

Mint2 were probed by A-to-E substitutions (G756γ, Y757ψ,

E758ε, and N759ν) (Figure 1c). All four A-to-E substitutions

resulted in a reduced binding aﬃnity relative to the APP_W_T

peptide (Figure 2c, Figure S8c,d, and Table S1). Interestingly,

the Y757ψ substitution exhibited a >100-fold loss of aﬃnity to

Mint2-PARM, as did the Y757A substitution, rendering Y757

as a key residue in APP.

with reduced APP binding aﬃnity. Introducing two mutations,

Y459A and F520A, into human Mint2-PARM (hMin-

Y459A/F520A

) resulted in a signiﬁcant 72-fold decrease in the

APP_C_‑_t_e_r_mpeptide binding a ﬃ nity (K = 895 ± 43 μM) in the

In Mint2-PARM, three Ala substitutions (L454A, Y459A,

and F520A) exhibited a >10-fold reduction in the APP_C_‑_t_e_r_m

aﬃnity (Figure 2d, Figure S9, and Table S2). The robust eﬀect

of Y459A substitution in Mint2-PARM is likely a result of

disrupting the hydrophobic interaction with the C atom of

G756 in APP, an interaction also reported for Mint1 (Y418).

Notably, F520A substitution in Mint2-PARM led to a 45-fold

reduction in binding aﬃnity, plausibly explained by contacts

with key residue N759 in APP (Figure S10a). Corresponding

residues in the PTB-domains of Mint1 (F608) and p52 Shc

(

F198) are also reported to be important for peptide

recognition, suggesting that the conserved Phe constitutes an

important mediator of ligand recognition for PTB do-

mains.

3,45

Next, we evaluated the eﬀect of A-to-E substitutions

in Mint2-PARM on the binding to the APP_C_‑_t_e_r_mpeptide. In

general, the introduced substitutions reduced the binding

a ﬃ nity of the APP_C_‑_t_e_r_mpeptide by 2- to 4-fold (Figure 2e and

Table S4 ). However, two A-to-E substitutions, S458σ and

D462δ, resulted in 14- and 25-fold reduced aﬃnities,

respectively. Both residues engage in H-bonds and form the

antiparallel β-sheet with the N-terminal region of the APP_C_‑_t_e_r_m

peptide (Figure 1b,c). Altogether, we identiﬁed two key side

chains (Y757 and N759) and one backbone amide (Y757ψ) in

APP to be critical to the APP-Mint2 interaction. In the Mint2-

PTB domain, we found F520A, Y459A, and one backbone

amide substitution (D462δ) led to a substantial reduction in

aﬃnity to the APP_C_‑_t_e_r_mpeptide (Figure 2d, e).

Compared to the reported cocrystal structure of APP and

Mint2, we observed a number of deviations from the suggested

interactions. First, the side chains of D462 in Mint2 and Y757

in APP are suggested to interact via an H-bond. In our FP

measurements, we detected no eﬀect of the D462A mutation

in Mint2-PARM on the binding of the APP_C_‑_t_e_r_mpeptide

Figure 3. APP binding-deﬁcient rMint2^Y⁴⁶⁰^A^/^F⁵²¹^Avariant reduces

Aβ42 levels in primary mouse neurons. (a) APPC‑term peptide aﬃnity

for selected Mint2-PARM variants. Data are expressed as the mean +

SEM (n = 3). The statistical signiﬁcance was evaluated using one-way

ANOVA with Dunnett’s multiple comparison test, **** P < 0.0001

(

Figure S10 ). (b) Coimmunoprecipitation of APP with the Mint2

antibody from HEK293T cells cotransfected with APP and GFP-

Y460A/F521A

rMint2 or GFP-rMint2

. Western blotting indicates less

Y460A/F521A

APP binding to rMint2

(lane 6) than to GFP-Mint2

(

lane 5). α-Tubulin served as a loading control. (c) Quantiﬁcation of

APP coimmunoprecipitation using GFP-rMint2

and GFP-rMin-

Y460A/F521A

. Data were normalized to GFP-rMint2 and expressed

as the mean + SEM. The statistical signiﬁcance was evaluated with the

Student’s t-test, *** P < 0.001. (n = 4 independent experiments). (d)

Western blot for Mint2, APP, and α-tubulin from neuronal lysates

carrying the APPswe/PS1ΔE9 mutation and infected with the

Y460A/F521A

lentiviral GFP-rMint2

mutant. (e) Aβ ELISA quantiﬁca-

tion of conditioned media from neurons overproducing Aβ shows

(Figure 2d); however, we cannot exclude that the Ala mutation

reduced Aβ levels when neurons were infected with the GFP-

can be compensated for by a water molecule mimicking the H-

bond acceptor of D462. Second, Q767 of APP is proposed to

H-bond to N387 in Mint2, although the truncated APP_W_T

peptide lacking Q767 retains a wild-type binding aﬃnity

Y460A/F521A

rMint2

mutant. Data were normalized to the endogenous

Mint2 control and expressed as the mean + SEM (n = 7 biological

replicates from two independent experiments). The statistical

signiﬁcance was evaluated using the Student’s t-test, *** P < 0.001.

Figure S1 ). Finally, the side chain of the critical N759 residue

hMint2^Y⁴⁵⁹^A^/^F⁵²⁰^Ahas a cellular eﬀect on APP binding, we

in APP is proposed to bind the backbone carbonyls of both

L454 and I457 in Mint2. Here, our A-to-E substitutions

suggest that only the H-bond protruding from I457 of Mint2 is

important, while perturbing the carbonyl of L454 had only

minor eﬀects on the aﬃnity to the APP_C_‑_t_e_r_mpeptide (Figure

produced a full-length GFP-tagged rat Mint2 (GFP-rMint2

)

and corresponding APP binding-deﬁcient variant GFP-

Y460A/F521A

Y459A/F520A

rMint2

(analogous to hMint2

, Figure

S11 ). We conﬁrmed our FP results by cotransfecting

c).

Mutations in the Mint2-PTB Domain Reduce APP

HEK293T with full-length APP and either GFP-rMint2 or

Y460A/F521A

GFP-rMint2

and performing coimmunoprecipitation

Binding and Aβ Formation. We took advantage of the

Mint2-PARM Ala-scan results and designed a Mint2 variant

assays. APP coimmunoprecipitated with both GFP-rMint2

(lane 5, Figure 3b) and GFP-rMint2

Y460A/F521A

(lane 6, Figure

J. Am. Chem. Soc. 2021, 143, 891−901

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Figure 4. Incorporation of ncAAs and evaluation of side chain-to-side chain macrocyclization in the APP_W_Tpeptide. (a) Overview of synthesized

APP_W_Tpeptide variants, including the structure of the introduced amino acids for each position (blue). (b) K values of each APP peptide variant

measured by FP. n.b. indicates nonbinding peptide (i.e., K ≥ 500 μM). Data are expressed as the mean + SEM (n = 3). (c) Structure of side chain-

to-side chain cyclized APP peptide analogues with native residue order (Glu in the i position, dark gray) and (d) inverse residue order (Lys in

the i position, light gray). (e) K values of cyclic APP peptide variants measured by FP. Data are expressed as the mean + SEM (n = 3). (f)

Helical propensity of residues Y762 to E766 from the molecular dynamics simulation of cyclic APP_W_Tpeptide variants 1 and 3−13 (% helical

frames) plotted against the binding aﬃnity (the mean K value) including the trend line (blue).

b); however, GFP-rMint2^Y⁴⁶⁰^A^/^F⁵²¹^Aexhibited a signiﬁcant

5% reduction in APP binding (Figure 3c). Next, we examined

Mint2 plays a facilitative role in Aβ formation in our neuronal

AD model.

the functional eﬀect of an impaired interaction between APP

and Mint2 on Aβ generation. We employed primary cortical

neurons from an established murine AD model with a double

transgene for the expression of mutant APP (APPswe) and

presenilin-1 proteins (PS1ΔE9), resulting in enhanced Aβ

Non-canonical Amino Acids Enhance the Binding of

the APP Peptide to Mint2. Since the APP binding-deﬁcient

Y460A/F521A

rMint2

variant reduced Aβ production in vitro and

previous studies revealed that Mint2 knockout in APPswe/

PS1ΔE9 transgenic mice reduces Aβ formation in vivo, we

hypothesized that a peptide-based inhibitor designed to disrupt

the APP-Mint2 interaction could reduce Aβ generation. As

such, we used the APP_W_Tpeptide as a template for an initial D -

and N -Me-amino acid (D-AA and NMe-AA) scan (Figure S8

and Table S1). D-AAs were tolerated only in the N- and C-

termini of the APP_W_Tpeptide, whereas the N-methylation of

N755 resulted in an improved aﬃnity toward Mint2-PARM.

Notably, the substitution of Y757 with N-MeY757 resulted in a

nonbinding APP_W_Tpeptide analogue, which is in agreement

with the reduced binding of the Y757ψ analogue (Figure 2c).

Next, we prepared a series of APP_W_Tpeptide variants

production. We infected primary neurons with a full-length

Y460A/F521A

GFP-rMint2

lentivirus after 2 days in vitro (2 DIV).

Y460A/F521A

Expression of the GFP-rMint2

variant was conﬁrmed

by Western blot analysis (Figure 3d). We quantiﬁed the eﬀect

on Aβ levels released from primary neurons using an enzyme-

like immunosorbent assay (ELISA) on the neuronal con-

ditioned medium. At 15 DIV, we observed a signiﬁcant 42%

decrease in Aβ₄₂levels in neurons infected with GFP-

Y460A/F521A

rMint2

relative to neurons expressing only endoge-

nous Mint2 (Figure 3e). Together, our results conﬁrm that

APP interacts with the PTB domain of Mint2 and suggest that

J. Am. Chem. Soc. 2021, 143, 891−901

Journal of the American Chemical Society

Article

containing single substitutions of both canonical (cAAs) and

noncanonical amino acids (ncAAs) (Figure 4a). First, we

tested position N759, the most critical residue according to the

Ala scan, and introduced eight diﬀerent amino acids.

Remarkably, all substitutions, even conservative ones such as

human plasma stability assay, resulting in a 4-fold-improved

half-life time (T₁_/₂) over the APP peptide, T₁_/₂= 105 and 26

min, respectively (Figure S12e).

On one hand, the moderately improved aﬃnity might be a

result of the introduced constrain causing a preorganization of

the peptide resembling the bound conformation with a well-

deﬁned helical turn of residues Y762−E766, which reduces the

entropic penalty upon binding. On the other hand, additional

favorable interactions by the formed lactam bridge can also

account for the improved aﬃnity. We performed molecular

dynamics (MD) simulations of cyclized peptides 1 and 3−12

and found a correlation between the degree of helicity in

solution and the observed binding aﬃnities. For the two most

potent cyclic peptides 10 and 12, helical propensity predictions

of 42 and 47%, respectively, were observed. In contrast, for

low-aﬃnity peptides 11 and 13 the molecular dynamics

simulations suggested helical propensities of 6% and less

(Figure 4f). This suggests that ligand preorganization is the

driving force of the improved binding. Altogether, the

systematic lactam macrocyclization scan led to the identi-

ﬁcation of a cyclic APP_W_Tpeptide scaﬀold with improved

binding aﬃnity and proteolytic stability.

N759Q, abrogated the binding to Mint2-PARM (K ≥ 500

μM) (Figure 4b). We therefore consider N759 to be a key

residue in the APP-Mint2 interaction, which is further

supported by its conservation as part of the APP endocytic

NPXY consensus sequence that interacts with the PTB domain

of Mint proteins. Next, we probed the two aromatic residues,

F764 and F765. Individual substitution by Trp had no eﬀect

on the aﬃnity. In contrast, the incorporation of L-3-(1-

naphtyl)alanine (Nal1) at either position improved the aﬃnity

for both variants, and the corresponding double substitution

showed a 4-fold improvement in aﬃnity (K = 1.1 ± 0.3 μM).

We attribute the improved aﬃnity to hydrophobic interactions

with Y524 and F527 in Mint2-PTB located in close proximity

to F764 and F765. Neither L-3-(2-naphtyl)alanine (Nal2) nor

-indanyl-L-glycine (Igl) improved the aﬃnity to the same

extent (Figure 4b).

Finally, the substitution of Y762 to Phe resulted in a 4-fold

increase in aﬃnity (K = 1.0 ± 0.1 μM), while an Y762W

substitution lowered the aﬃnity to Mint2-PARM (Figure 4b

and Table S1).

Taken together, we identiﬁed six positions (N755, Y757,

Y762, F764, F765, and E766) in the APP_W_Tpeptide at which

substitutions to cAAs or ncAAs either improved the aﬃnity or

were expected to improve the proteolytic stability of the

peptide.

Identiﬁcation of a Metabolically Stable, High-Aﬃnity

Mint2 Inhibitor Derived from the APP_W_TPeptide. To

develop a potent and proteolytically stable modulator of the

APP-Mint2 PPI, we selected cyclic peptide 10 (n = 4, m = 1)

as a scaﬀold. Next, we introduced D-N-Me-Ala and D-Glu at

positions 755 and 766, respectively. Finally, ncAA L-Nal1 was

selected for positions 764 and 765. The corresponding peptide,

KSL-221036 (NMe-aGYcyclo-[KNPTYD]Nal1Nal1e-OH, 14),

was generated through SPPS including on-resin cyclization via

orthogonally protected Lys(Aloc) and Asp(All) (Figure 5a).

We found that 14 binds Mint2-PARM with nanomolar aﬃnity

Side Chain-to-Side Chain Cyclization in the APP_W_T

Peptide Improves Aﬃnity and Stability. To improve the

binding and metabolic stability of our peptide ligand, we

introduced side chain-to-side chain lactam cyclizations employ-

ing solid-phase peptide synthesis (SPPS) and on-resin

cyclization (Figure S12a ). According to the reported X-ray

cocrystal structure of APP-Mint2, native residues E758 and

K763 are in close proximity when forming an intramolecular

(K = 53 ± 2 nM) as determined by isothermal titration

calorimetry (ITC). Compared to the APP_W_Tpeptide (K = 2.4

μ M), a 46-fold improved aﬃnity was achieved (Figure 5b and

Figure S13 ). Furthermore, 14 was found to be proteolytically

stable over 24 h (half-life ≥ 1440 min) in human plasma

noncovalent salt bridge (Figure S12b ). We reasoned that

introducing a lactam bridge in this position could confer

conformational constraint and potentially improve the aﬃnity

(Figure 5c), while the APP peptide exhibited a half-life of

<30 min. Finally, we examined the rate of metabolism through

cytochrome P450 enzymes using mouse liver microsomes and

determined the in vitro hepatic clearance of the APP_W_Tpeptide

and 14. We found the APP_W_Tpeptide was cleared fast [CL₍_i_n_t₎

= 15.4 ± 2.5 μL/(mg min)] while 14 was not metabolized over

60 min [CL₍_i_n_t₎= 0.0 ± 0.8 μL/(mg min)] (Figure 5d). This

data demonstrates the design of a peptide using the minimal

binding sequence of the endogenous APP peptide−ligand as a

template. Macrocyclization and the incorporation of selected

ncAAs resulted in the design of KSL-221036 (14), the ﬁrst

reported modulator of the APP-Mint2 PPI exhibiting nano-

molar aﬃnity and suitable stability characteristics for further

studies.

and stability. Indeed, a cyclic analogue of the APP_W_Tpeptide

containing a lactam linkage between E758 and K763 (i to i + 5,

cAPP_E₇₅₈₋_K₇₆₃; 1) exhibited improved aﬃnity compared to the

APP_W_Tpeptide and was also superior to a variant cyclized

between Y762K and E766 (i to i + 4, cAPP_Y₇₆₂₋_E₇₆₆; 2) (Figure

S12b −d ). Next, we prepared a series of cyclic variants of 1 in

which we systematically modiﬁed the lactam bridge by

introducing Asp, Orn, or Dap and reversed the orientation of

the amide bond in the lactam bridge (Figure 4c,d). The aﬃnity

of these 11 peptides (3−13) was highly dependent on the

nature of the introduced macrocyclization (Figure 4e).

In general, macrocyclic peptides with carboxylic acid side

chains in i positions (1 and 3−7; Figure 4c) were characterized

by reduced binding aﬃnities, while peptides with amino side

chains in i positions (8−13, Figure 4d) displayed improved

KSL-221036 (14) Interacts with Mint2 and Reduces

Aβ Levels. To determine whether KSL-221036 (14) binds

full-length Mint2 from primary neuronal lysate, we loaded the

APP_W_Tpeptide and 14 onto epoxy-coated magnetic beads via

an N-terminal Cys-PEG linker (Table S1). Pull-down from the

lysate of primary mouse neurons conﬁrmed that 14 eﬃciently

interacts with endogenous Mint2 at higher aﬃnity (lane 2,

Figure 5e) than for the APP_W_Tpeptide under the same

conditions (lane 3, Figure 5e; see also Figure S14).

aﬃnities relative to the linear APP peptide. Furthermore,

smaller ring sizes (5−7, 11, and 13) resulted in reduced

binding aﬃnities with only peptide 12 (n = 3, m = 1) refuting

this trend. Among the 12 macrocycles, peptide 10 (n = 4, m =

) exhibited the highest aﬃnity (K = 2.2 ± 0.3 μM) (Figure

e). To test whether macrocyclization improved the

To examine the eﬀect of 14 on Aβ production in neurons,

7−57

proteolytic stability, cyclic peptide 1 was tested in an in vitro

we attached cell-penetrating peptide (CPP) TAT

(YGR-

J. Am. Chem. Soc. 2021, 143, 891−901

Article

KKRRQRRR) to the N-terminus of 14 (TAT-14) and the

APP_W_Tpeptide (15, Table S1). TAT is a widely used CPP and

was recently demonstrated to be compatible with late-stage

clinical development in two phase III clinical trials (ESCAPE

and FRONTIER), rendering TAT the most advanced CPP tag

in the clinic. We ﬁrst treated neurons with a concentration

curve of peptide TAT-14 to determine the highest

concentration at which no adverse eﬀect on cell viability was

observed. Using primary neurons and a colorimetric readout of

metabolic activity, peptides 14 (no CPP) and 15 (TAT-

APP_W_T) exhibited no adverse eﬀect while peptide TAT-14

aﬀected cell viability at 20 μ M (75%) but had no measurable

eﬀect at 10 μM (Figure S15 ). To determine whether

modulation of the APP-Mint2 interaction aﬀects Aβ

production, primary cortical neurons (14 DIV) carrying a

double transgene for the expression of mutant APP (APPswe)

and presenilin-1 proteins (PS1ΔE9) were incubated for 24 h

with peptides 14, TAT-14, and 15. ELISA quantiﬁcation of

Aβ from the conditioned medium revealed that TAT-14 (10

μM) led to a 44% reduction of Aβ₄₂levels relative to vehicle

control. In contrast, TAT-APP_W_T(15, 50 μM) and 14 (10 μM,

no CPP) did not aﬀect Aβ₄₂levels. High-aﬃnity γ-secretase

inhibitor DAPT (N-[N-(3,5-diﬂuorophenacetyl)-L-alanyl]-S-

phenyl-glycine tert-butyl ester or LY-374973) served as a

positive control, reducing Aβ levels by 47% (Figure 5f and

Figure S16 ). In addition, we assessed the eﬀect of TAT-14

on cellular integrity by quantifying lactate dehydrogenase

(

LDH) release (a proxy for cytotoxicity) in the same

conditioned medium used for the aforementioned ELISA.

We found low LDH levels for both DAPT- and TAT-14-

treated neurons relative to vehicle control, indicating that the

eﬀect of peptide TAT-14 on Aβ production is not due to

cellular toxicity (Figure 5g; see also Figure S16). Furthermore,

p-value analysis found no signiﬁcant diﬀerence between the

vehicle and TAT-14 (p = 0.2448) or DAPT and TAT-14 (p =

.6525) LDH release. To further support the TAT-mediated

intracellular delivery of 14, we detected TAT immunostaining

in primary neurons incubated with TAT-14 (Figure S17),

indicating that TAT-14 is cell-permeable.

Figure 5. PPI inhibitor of the APP-Mint2 interaction reduces Aβ₄₂

production in the neuronal in vitro model of AD. (a) Schematic

structure of KSL-221036 (14). (b) Representative ITC of titrating

Mint2-PARM with 14; raw heat signature (top) and integrated molar

heat release (bottom). (c) In vitro plasma stability of 14 (blue) and

the APP_W_Tpeptide (black) including buﬀer control for the APP_W_T

peptide (circles); data are expressed as the mean ± SEM (n = 3). (d)

In vitro hepatic clearance of 14 (blue) and the APP_W_Tpeptide (black)

including propranolol control (circles, n = 1); data are expressed as

the mean ± SEM (n = 3). (e) Representative pull down of Mint2

from neuronal cell lysate (15 DIV). Lanes 1−3: expression of Mint2.

Lanes 4−6: knockout of Mint2. The eluent was resolved by SDS-

PAGE and immunoblotted for Mint2 and GAPDH (n = 3). (f) Aβ

ELISA quantiﬁcation from conditioned media of neurons over-

producing Aβ; data are normalized to the vehicle control and

expressed as the mean + SEM (n = 3 independent experiments, n = 10

biological replicates for the vehicle, DAPT, and TAT-14 (10 μM).

Independent experiments are represented by diﬀerent shapes.

Statistical signiﬁcance evaluated using one-way ANOVA with

Dunnett’s multiple comparison test, ***P < 0.001. (g) LDH levels

from conditioned medium of cultured neurons overproducing Aβ

collected after 24 h of treatment with DAPT and TAT-14 (10 μM).

Maximum LDH represents the maximum amount of LDH released

from neurons lysed using detergent. Individual data points in (g)

correspond to data in panel (f) (circles = same assay). Data are

expressed as %LDH normalized to vehicle control and shown as the

mean + SEM. Statistical signiﬁcance was evaluated using one-way

ANOVA with Sidak’s multiple comparison test, ***P < 0.001.

Following the promising Aβ₄₂results of TAT-14, we

investigated the dose−response relationship and found a

weak correlation between the concentration of TAT-14 and

reduced Aβ₄₂levels (Figure S16e ). We reasoned that the

delivery eﬃciency of the cargo potentially depends on the

employed CPP tag; therefore, we synthesized four additional

variants of 14 containing diﬀerent CPPs attached to the N-

terminus: mixTAT-14 (rRrGrKkrK-14), polyArg-14

(

RRRRRRRRR-14), D-SynB3-14 (frrrsyslrr-14), and Mini-

Ap4-14 (cyclo(DLATEPAK(Dap)-14). Next, we determined

the highest concentration at which no adverse eﬀect on cell

viability was observed. Peptide MiniAp4-14 exhibited no

toxicity, while peptides mixTAT-14 and polyArg-14 were

tolerated at concentrations of 5 and 7.5 μM, respectively

(Figure S15). Peptide D-SynB3-14 was poorly soluble and not

further tested. The peptides’ eﬀect on Aβ formation was

evaluated analogously to that of peptide TAT-14. The

quantiﬁcation of Aβ₄₂from the conditioned medium of

primary neurons revealed that incubation with mixTAT-14

(

5 μM) and polyArg-14 (5 μM) reduced Aβ levels by 80 and

4%, respectively. In contrast, incubation with MiniAp4-14 (10

μM) did not aﬀect Aβ production (Figure S16c). Of note, we

detected a small increase in LDH activity following incubation

with mixTAT-14 (5 μM) compared to the vehicle control

J. Am. Chem. Soc. 2021, 143, 891−901

Journal of the American Chemical Society

Figure S16d ). Consequently, polyArg-14, which reduced Aβ₄₂

Article

APP_W_T, peptide 14 exhibited an enhanced aﬃnity to robustly

pull down full-length Mint2 from neuronal lysate. We

conﬁrmed that targeting the Mint2-APP interaction with cell-

permeable variants of 14 (TAT-14 and polyArg-14)

signiﬁcantly reduced Aβ₄₂formation, whereas parent APP_W_T

peptide 15 had no eﬀect on Aβ formation. Peptides TAT-14,

mixTAT-14, and polyArg-14 therefore provide ﬁrst proof-of-

concept evidence that targeting a direct interaction partner of

APP aﬀects its metabolism and Aβ formation. Furthermore,

the comparison of diﬀerent CPP tags indicates that the

selection is critical and aﬀects not only eﬃcacy but also

neuronal viability. Here, the combined analysis of LDH levels

and Aβ₄₂reduction suggests that polyArg-14 might be

preferred over TAT-14 (Figure S16).

production at a lower concentration (5 μM) with minimal

cellular toxicity, is a promising CPP alternative to TAT-14.

Our results indicate that peptide 14 successfully binds Mint2

in vitro with greater aﬃnity than the APP_W_Tpeptide. Further

studies in primary neurons cultured from our AD mouse model

revealed that TAT-14 signiﬁcantly decreases Aβ₄₂production

with minimal toxicity. Further investigation into alternative

CPPs showed that polyArg also provides attractive properties

that facilitate the cellular uptake of peptide ligand 14. The

^o^b^s^e^r^v^a^tⁱ^oⁿ^t^h^a^t^T^A^T^-^A^P^PWT ⁽¹⁵⁾^h^a^sⁿ^o^e^ﬀ^e^c^t^oⁿ^A^β42

formation is in excellent agreement with the pull-down results

and highlights the importance of ligand optimization.

According to the reported eﬀects of Mint knockout on APP

^t^r^a^ﬃ^c^kⁱⁿ^g^aⁿ^d^A^β⁴2 formation, inhibition of the Mint2-APP

PPI by compounds TAT-14 and polyArg-14 could either result

in reduced APP endocytosis or prevent the formation of the

tertiary protein complex comprising APP (C99), Mint2, and

the γ-secretase. Both avenues ultimately result in reduced Aβ

formation. As such, the lowered Aβ levels seen in the in vitro

AD model following treatment with compounds TAT-14 and

polyArg-14 provide evidence that modulation of the APP-

Mint2 PPI might serve as a pharmacological strategy to reduce

pathologic Aβ levels in connection with AD.

Previous work has shown that APP and BACE-1 converge in

56,57

acidic microdomains.

Because the BACE-1 cleavage of

APP is the rate-limiting step in Aβ production, this

convergence in endosomes is a critical initiator of the APP

amyloidogenic cascade. Sullivan et al. reported that the

knockout of Mint proteins resulted in reduced APP

endocytosis and Aβ formation. We suspect that the APP

binding-deﬁcient rMint2

Y460A/F521A

variant and TAT-, mix-

TAT-, and polyArg-14 could have a similar eﬀect. The same

study reported that the knockout of Mint proteins also aﬀects

the internalization of presenilin-1 of the γ-secretase complex

and reduced colocalization with APP.

The impaired binding of APP (C99 fragment) to Mint2 and

presenilin-1 would reduce the probability of γ-site cleavage in

APP and hence Aβ formation. Our results encompass both

observations.

In summary, we demonstrated that targeting the APP-Mint

PPI may present an alternative strategy in the pursuit of new

therapeutic approaches in AD treatment. We believe that the

compounds reported herein will be of help in identifying

mechanisms of Mint2-mediated Aβ formation and enable

future in vivo studies.

CONCLUSIONS

■

In the pursuit of novel strategies to treat AD, pharmacological

approaches modulating direct binding partners involved in

APP traﬃcking and processing are promising alternatives to

currently pursued therapies. Here, the APP-Mint PPI is an

attractive target as the eﬀects of Mint deletions are relatively

mild, and our strategy, which selectively inhibits APP binding

to the PTB domain of Mint proteins, would likely not interfere

with the critical function of Mint proteins in synaptic vesicle

exocytosis, which is mediated through Mint’s other PPI

domains. Although numerous studies have examined the

biological importance of Mint proteins, particularly with regard

to Aβ formation in the context of AD, these studies have

METHODS

been informative but contradictory.

■

Here, we extensively characterized the APP-Mint2 inter-

action at the molecular level by performing comprehensive

mutational scans of the APP-Mint2 interface, covering both

side-chain and backbone interactions. Compared to the

reported structures, we observed distinct diﬀerences and

showed that two backbone alterations, Y757ψ in the APP_W_T

peptide and D462δ in Mint2, had major impacts on the APP-

Mint2 interaction. The introduction of Ala substitutions

demonstrated that the side chains of two residues in APP,

N759, and Y757 and one residue in Mint2, F520, are pivotal to

the APP-Mint2 interaction. These observations led to the

generation of a Mint2 variant containing two distinct

substitutions, Y459A/F520A, which resulted in impaired APP

binding. We showed that reduced APP binding results in

signiﬁcantly reduced Aβ production, which supports a

facilitative role of Mint2 in Aβ formation and suggests that

targeting the APP-Mint2 interaction with a PPI inhibitor is of

potential therapeutic relevance.

Detailed experimental procedures are provided in the Supporting

Information .

Peptide Synthesis. Peptides and depsipeptides were assembled in

a manual or automated manner (LibertyBlue, CEM, Matthews, NC,

USA) on a solid support using Boc- or Fmoc-based chemistry,

puriﬁed by RP-HPLC, and characterized by LCMS and UPLC. When

relevant, side-chain cyclization was performed on resin by the

deprotection of Alloc and Allyl protecting groups, followed by

PyBOP-induced cyclization.

Protein Expression. Human Mint2 constructs were cloned in

pRSET, pET, or pTXB1 vectors, expressed in E. coli Bl21(DE3)pLysS

(Invitrogen), and puriﬁed using HisTrap columns, followed by size-

exclusion chromatography on a HiLoad 16/600 Superdex 75 pg

column or puriﬁcation by reverse-phase chromatography (C4 column,

YMC-Pack-C4, 250 × 20 mm ).

Expressed Protein Ligation. The three-step ligation was

ⁱⁿⁱ^tⁱ^a^t^e^d^b^y^lⁱ^g^a^tⁱⁿ^g^P^A^R^MN ^t^o^t^h^e^t^hⁱ^o^e^s^t^e^r^p^e^p^tⁱ^d^e⁽^P^A^R^MPep

)

fragment. Following buﬀer exchange, the second ligation was

performed using the PARM_Cfragment. Obtained proteins were

puriﬁed using a reverse-phase C4 column (Jupitor, Phenomenex, 250

Using the APP_W_Tpeptide as a template, we developed a

high-aﬃnity, proteolytically stable cyclic peptide 14. We

demonstrated that introducing noncanonical amino acids, N-

methylation, and D-amino acids into a macrocyclic peptide

scaﬀold provided a vast improvement in aﬃnity for Mint2 and

stability relative to the APP_W_Tpeptide. Notably, compared to

10 mm ). After desulfurization, the semisynthetic protein was

refolded into storage buﬀer and characterized by LCMS and UPLC.

Circular Dichroism (CD). Experiments were performed on an

Olis DSM 100 (Olis Inc.) at 15 μM protein concentration, and the

obtained millidegrees of ellipticity (mo) was converted to the mean

residue ellipticity (θ_M_R_E).

J. Am. Chem. Soc. 2021, 143, 891−901

Journal of the American Chemical Society

https://pubs.acs.org/doi/10.1021/jacs.0c10696.

Article

ThermoFluor. ThermoFluor experiments were performed in a 96-

well plate format on a Stratagene Mx3005p qPCR cycler (Agilent)

using SYPRO orange as the dye at 15 μM protein concentration.

Fluorescent Polarization (FP). Saturation binding (for semi-

synthetic and mutated proteins) using 50 nM TAMRA-APP_C_-_t_e_r_m

Data Analysis. All data analysis was performed in GraphPad

Prism 7.0 (GraphPad Inc.), Excel 2010 (Microsoft), and Origin 9.0

(OriginLab). Signiﬁcance was evaluated using the Student’s t test in

the case of two groups. The one-way ANOVA test was used in the

case of three or more groups, combined with either Dunnett’s or

Sidak’s multiple comparison test.

[

(TAMRA)-NNG-QNGYENPTYKFFEQMQN] as a probe was

performed using a Saﬁre2 plate reader (Tecan) and increasing

concentrations of Mint2-PARM. Inhibition experiments were

performed using 50 nM TAMRA-APP_C_-_t_e_r_mand 15 μM Mint2-

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

■

PARM at increasing concentrations of peptide ligands.

Y460A/F521A

Rat Mint2

Cloning. Using a QuikChange II Site-

Directed Mutagenesis Kit (Agilent Technologies), pEGFP-rMin-

Y460A/F521A

was created from rat pEGFP-Mint2 and subcloned into

Synthesis and additional experimental and analytical

details (PDF).

the lentiviral pFUW vector for neuronal expression.

HEK293T Coimmunoprecipitation. Transfected HEK293T cells

were lysed, and protein extracts were incubated with precipitating

antibody and protein A Ultralink resin (Thermo Scientiﬁc).

Precipitated proteins were resolved by SDS-PAGE and immuno-

AUTHOR INFORMATION

Corresponding Author

■

Kristian Strømgaard − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark; orcid.org/0000-0003-2206-

4737 ; Email: kristian.stromgaard@sund.ku.dk

blotted for APP, GFP, and α-tubulin.

Primary Neuron Infection with rMint2

ELISA. Primary neuronal cultures were prepared from newborn mice

Y460A/F521A

and Aβ

Y460A/F521A

and infected with lentivirus for rMint2

. At 15 DIV, neuronal

lysate was collected, resolved by SDS-PAGE, and immunoblotted for

APP, Mint2, and α-tubulin. Conditioned media were collected and

handled according to a Human Aβ₄₂Ultrasensitive ELISA Kit

Authors

Christian R. O. Bartling − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark; Department of Biology, Boston

University, Boston, Massachusetts 02215, United States

Thomas M. T. Jensen − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Shawna M. Henry − Department of Biology, Boston

University, Boston, Massachusetts 02215, United States

Anna L. Colliander − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Vita Sereikaite − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Marcella Wenzler − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Palash Jain − Department of Drug Design and Pharmacology,

University of Copenhagen, DK-2100 Copenhagen, Denmark

Hans M. Maric − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Kasper Harpsøe − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark; orcid.org/0000-0002-9326-9644

Søren W. Pedersen − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

(

Invitrogen).

Molecular Dynamics Simulations. The Desmond package in

Schrodinger’s Maestro suite was used to perform molecular dynamics

simulations. Each simulation was run in duplicate (200 ns) under

standard temperature and pressure. The simulation output was parsed

frame by frame using Python, and helical propensity values were

determined.

In vitro Plasma Stability. Peptides were incubated in human

plasma at 37 °C for up to 24 h. The peptides were extracted from the

plasma at various time points and analyzed using UPLC.

In vitro Hepatic Clearance. Peptides were incubated in mouse

hepatic microsomes supplemented with reduced β-nicotinamide

adenine dinucleotide 2′-phosphate (NADPH) and MgCl at 37 °C

for up to 60 min. The peptides were extracted from the microsomes at

various time points and analyzed using UPLC.

Isothermal Titration Calorimetry (ITC). Experiments were

performed using an ITC200 (Malvern). Ligand-to-buﬀer titrations

were performed to subtract the heat produced by injection, mixing,

and dilution. The binding enthalpy was directly measured, and the

dissociation constant (K ) and stoichiometry (N) were obtained by

data analysis using Origin software (OriginLab).

Neuronal Cell Viability. Primary neuron cultures were treated

with peptides or DMSO as indicated. The cell viability and

cytotoxicity were determined using a CellTiter 96 AQ_u_e_o_u_sOne

Solution Cell Proliferation Assay (MTS) Kit (Promega) and a

CyQUANT LDH Cytotoxicity Assay (Thermo Fisher).

Immobilization of Peptides. Peptide KSL-221036 and the

APP_W_Tpeptide comprising a C-terminal Cys and a PEG2 linker were

loaded onto Dynabeads M-270 Epoxy Beads (Thermo Fischer

Scientiﬁc) according to the protocol of the manufacturer.

Protein Isolation from Neuronal Cell Lysate. Primary

neuronal cultures were prepared as described above. Neurons

expressing Mint2 were infected with lentiviral inactive cre

recombinase, while Mint2 knockout neurons were infected with

active cre recombinase. At DIV15, neuronal lysate was collected and

incubated with peptide-loaded Dynabeads. Isolated proteins were

resolved by SDS-PAGE and immunoblotted for Mint2 and GAPDH.

ELISA Aβ Assay Following Peptide Treatment. Primary

neuronal cultures were prepared from newborn transgenic mice

carrying the APPswe/PS1ΔE9 transgene. At 15 DIV, neurons were

incubated with peptides, DAPT (γ-secretase inhibitor), and vehicle

control 0.25% DMSO. The collected conditioned media were handled

according to the protocol of the Human Aβ₄₂Ultrasensitive ELISA

Kit (Invitrogen).

Louise S. Clemmensen − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Linda M. Haugaard-Kedstrom − Department of Drug Design

and Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

David E. Gloriam − Department of Drug Design and

Pharmacology, University of Copenhagen, DK-2100

Copenhagen, Denmark

Angela Ho − Department of Biology, Boston University,

Boston, Massachusetts 02215, United States

Complete contact information is available at:

J. Am. Chem. Soc. 2021, 143, 891−901

Journal of the American Chemical Society

https://pubs.acs.org/10.1021/jacs.0c10696

Article

behavioral, and electrophysiological abnormalities of APP-deficient

mice. J. Neurosci. 2007, 27 (29), 7817−7826.

(17) Ho, A.; Liu, X.; Sudhof, T. C. Deletion of Mint proteins

Author Contributions

decreases amyloid production in transgenic mouse models of

C.R.O.B., T.M.T.J., and S.M.H. contributed equally.

Alzheimer ’s disease. J. Neurosci. 2008, 28 (53), 14392−14400.

Notes

(18) Lau, K. F.; McLoughlin, D. M.; Standen, C.; Miller, C. C. X11

The authors declare no competing ﬁnancial interest.

alpha and x11 beta interact with presenilin-1 via their PDZ domains.

Mol. Cell. Neurosci. 2000, 16 (5), 557−565.

ACKNOWLEDGMENTS

(19) Biederer, T.; Cao, X.; Sudhof, T. C.; Liu, X. Regulation of APP-

dependent transcription complexes by Mint/X11s: differential

functions of Mint isoforms. J. Neurosci. 2002, 22 (17), 7340 −7351.

■

We thank the Lundbeck Foundation for ﬁnancial support

provided to K.S. and NIH R01 AG044499 for ﬁnancial support

provided to A.H. We also thank Christian A. Olsen, Stephan A.

(20) Xie, Z.; Romano, D. M.; Tanzi, R. E. RNA interference-

mediated silencing of X11alpha and X11beta attenuates amyloid beta-

protein levels via differential effects on beta-amyloid precursor protein

processing. J. Biol. Chem. 2005, 280 (15), 15413−15421.

Pless, Mette Ishøy Rosenbaum, and Dennis Ozcelik for their

critical input.

(21) Sullivan, S. E.; Dillon, G. M.; Sullivan, J. M.; Ho, A. Mint

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