YIAD-0501 directly dissociates aggregates of full-length and N-terminal pyroglutamate-modified forms of Aβ

Abstract

Background

Recent approvals of amyloid-β (Aβ) antibody drugs have established amyloid clearance as a viable therapeutic approach in Alzheimer’s disease (AD). However, despite substantial amyloid reduction, their cognitive benefits remain modest, potentially reflecting incomplete targeting of the structurally diverse pathogenic Aβ assemblies that drive AD progression. Given this molecular heterogeneity, a therapeutic strategy capable of targeting multiple toxic Aβ forms is required to achieve broader efficacy. To address this need, we investigated YIAD-0501, a small-molecule candidate designed to simultaneously engage multiple pathogenic Aβ species, including oligomeric and fibrillar forms of Aβ (1–42) and pyroglutamate Aβ(pE3–42).

Methods

A series of 6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine derivatives was synthesized and screened by Thioflavin T fluorescence and A11 dot blot assays to identify compounds active against diverse pathogenic Aβ assemblies. The lead compound, YIAD-0501, was further characterized by transmission electron microscopy, circular dichroism, microscale thermophoresis, molecular docking, and amyloid plate mapping to define its Aβ interaction and structural effects. For in vivo evaluation, YIAD-0501 (10 mg/kg, daily for 4 weeks) was administered to 6-month-old male 5XFAD mice, followed by Y-maze testing for spatial working memory and contextual fear conditioning for hippocampal-dependent memory. Biochemical analyses, including immunoblotting, immunohistochemistry, and ELISA, were subsequently conducted to quantify Aβ plaque burden, soluble Aβ levels, and gliosis.

Results

YIAD-0501 effectively reduced both oligomeric and fibrillar assemblies of Aβ (1–42) and Aβ(pE3–42) in vitro. Molecular docking and amyloid mapping analyses indicated interactions between YIAD-0501 and both the C-terminal hydrophobic region and the KLVFFA aggregation core of Aβ, consistent with the observed reduction in β-sheet content and direct binding. In 5XFAD mice, YIAD-0501 treatment decreased amyloid plaque burden, soluble Aβ levels, and neuroinflammation in the hippocampus, accompanied by improvements in spatial working and hippocampal-dependent memory.

Conclusions

Collectively, our findings identify YIAD-0501 as a small-molecule candidate that reduces multiple pathogenic Aβ assemblies and ameliorates hippocampal pathology and memory deficits in the 5XFAD mouse model. These findings highlight a chemically driven, multi-target mode of Aβ clearance, representing a strategy for broader intervention across the heterogeneous pathogenic landscape of AD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13195-026-01999-5.

Background

Amyloid-β (Aβ) deposits are a key pathological hallmark of Alzheimer’s disease (AD) [1]. Following aberrant proteolytic cleavage, Aβ peptides assemble into toxic soluble oligomers and insoluble fibrils that accumulate as extracellular plaques, trigger chronic neuroinflammation, and ultimately lead to cognitive decline [2–4]. Among the Aβ species abundant in plaques, Aβ (1–42) and its pyroglutamate-modified variant Aβ(pE3–42) are particularly prone to aggregation and closely associated with heightened neurotoxicity [5–9].

Recent clinical trials with lecanemab, which preferentially binds Aβ (1–42) protofibrils, and donanemab, which targets Aβ(pE3–42) aggregates, have further highlighted the pathological significance of these distinct Aβ forms [10–12]. Although these immunotherapies markedly reduced amyloid burden on positron-emission tomography imaging, their cognitive benefits remain modest, underscoring the complexity of AD pathogenesis and the need for more comprehensive therapeutic strategies [13–15]. Given the structural heterogeneity of pathogenic Aβ aggregates, a small-molecule strategy capable of simultaneously targeting multiple toxic species represents a promising approach to achieve broader efficacy, with advantages including ease of administration and cost-effectiveness [16–25].

Building on this rationale, we explored a 6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine scaffold, whose π-electron-rich and conformationally rigid tricyclic core was anticipated to facilitate interactions with multiple Aβ assemblies [26–30]. To translate this concept into a chemical platform, we synthesized a focused library of derivatives (YIAD-0500 to YIAD-0505) [31].

The primary objective of this study was to evaluate the anti-Aβ efficacy of diazepine derivatives, with particular emphasis on both Aβ (1–42) and Aβ(pE3–42). To this end, Thioflavin T (ThT) fluorescence and dot blot assays were performed to assess effects on oligomer and fibril formation, complemented by aggregation kinetics and transmission electron microscopy (TEM) analyses. Structural and binding interactions were further examined using circular dichroism (CD), microscale thermophoresis (MST), mapping amyloid plate (MAP) assays, and molecular docking. The lead compound, YIAD-0501, was subsequently evaluated in 5XFAD transgenic (TG) mice for cognitive performance, amyloid pathology, neuroinflammation, and safety profiles.

Methods

Study design

This study combined in vitro and in vivo experiments to evaluate the anti-amyloid activity of YIAD-0501. In vitro screening included ThT fluorescence assays to quantify Aβ fibrils and A11 dot blot assays to detect oligomers, using both Aβ (1–42) and Aβ(pE3–42), complemented by aggregation kinetics and TEM analyses. Structural and binding interactions were further examined using CD, MST, MAP assays, and molecular docking. In vivo efficacy was assessed in 5XFAD mice following daily intraperitoneal administration of YIAD-0501 (10 mg/kg) for 4 weeks. Cognitive function was evaluated using the Y-maze and contextual fear conditioning tests. Aβ burden was analyzed by immunohistochemistry (IHC), 6E10 dot blot, and enzyme-linked immunosorbent assay (ELISA), while neuroinflammation was examined by western blot and IHC. Safety was evaluated by short-term administration in Institute of Cancer Research (ICR) mice with monitoring of body weight, food intake, and gross organ morphology, along with NeuN IHC and in vitro cell viability assays.

Synthesis of YIAD compounds

YIAD-0500 (2-(3-Methoxyphenyl)-5-phenyl-6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile), YIAD-0501 (2-([1,1′-Biphenyl]-4-yl)-5-phenyl-6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile), YIAD-0502 (2-(5-Bromothiophen-2-yl)-5-(4-methoxyphenyl)-6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile), YIAD-0503 (5-(4-Chlorophenyl)-2-(4-methoxyphenyl)-6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile), YIAD-0504 (5-(Furan-2-yl)-2-(4-methoxyphenyl)-6H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile) and YIAD-0505 (2-(4-Methoxyphenyl)-5-oxo-5,6-dihydro-4H-furo[3,2-f]pyrrolo[1,2-d][1,4]diazepine-1-carbonitrile) compounds were synthesized by a one-pot four-component coupling reaction, as previously reported [31].

Preparation of Aβ peptides

Aβ (1–42) (DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) and Aβ(pE3–42) (pyroEFRHDSGYEV HHQKLVFFAE DVGSNKGAII LMVGGVVIA) peptides were synthesized by Fmoc solid-phase peptide synthesis as previously described [32]. Peptide-bound resins were washed with dichloromethane and cleaved using trifluoroacetic acid (TFA). After TFA removal, peptides were lyophilized to yield monomeric Aβ (1–42) and Aβ(pE3–42).

ThT fluorescence assays

For the inhibition assay, YIAD compounds (5 or 50 µM) were co-incubated with monomeric Aβ (1–42) in 10% DMSO at 37 °C for 1 day. For the dissociation assay, monomeric Aβ (1–42) was first pre-incubated in 10% DMSO at 37 °C for 1 day to form pre-aggregates. YIAD compounds were then added, followed by an additional day of incubation under the same conditions.

For the dose-dependent inhibition and dissociation assay of YIAD-0501, Aβ (1–42) and Aβ(pE3–42) peptides were used. Incubation protocols were identical to those described above. YIAD-0501 was tested from 50 to 0.1 µM (2-fold serial dilutions). Final peptide concentration was 12.5 µM.

For aggregation and dissociation kinetics of Aβ (1–42) and Aβ(pE3–42), each peptide (10 µM) was co-incubated with YIAD-0501 (50 µM, 10% DMSO) at 37 °C under inhibition conditions, and ThT fluorescence was measured at 0, 2, 6, 9, 18, 24, and 48 h. For dissociation kinetics, Aβ peptides were first incubated for 24 h to generate pre-aggregated species, followed by treatment with YIAD-0501 (50 µM) or vehicle (DMSO). ThT fluorescence was then recorded at 0.5, 2, 6, 9, 18, 24, and 48 h after treatment.

After incubation, 25 µL of each sample was loaded into a black 96-well half-area plate (Corning, 3694) in triplicate. Then, 75 µL of ThT solution (5 µM in 50 mM glycine buffer, pH 8.5) was added to each well. Fluorescence intensity was measured at Ex 450/Em 485 nm using a TECAN Infinite 200 PRO microplate reader. Values obtained from 0-day incubated Aβ (1–42) and Aβ(pE3–42) samples were subtracted from the corresponding experimental measurements prior to normalization.

TEM

Aβ aggregates prepared under inhibition conditions (1 day of incubation) or dissociation conditions (1 day to generate pre-aggregates followed by an additional 1 day of incubation) in the absence or presence of YIAD-0501 (50 µM) were applied to carbon-coated copper grids (CF200-CU, YMS, Korea). Aβ (1–42) or Aβ(pE3–42) was used at a final concentration of 10 µM. Aliquots (10 µL) were loaded onto the grids for 1 min, after which excess solution was removed using filter paper. The grids were negatively stained with 5 µL of 2% (w/v) uranyl acetate for 10 s and allowed to air-dry completely. Grids were glow-discharged using a PELCO easiGlow system (Ted Pella, USA) prior to imaging. Samples were imaged using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, USA).

MAP assays

Full-length Aβ (1–42) and its hexamer fragments were immobilized on a maleimide-activated microplate and incubated with 10 µM of Flamma 552–conjugated Aβ (1–42) at 37 ℃ for 6 h to form pre-formed oligomers, as previously described [33]. Then, YIAD-0501 (50 µM in 10% DMSO) was added and incubated with pre-formed oligomers at 37 ℃ for 1 day. After incubation, the wells were washed three times with 200 µL of wash buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 0.05% Tween-20; pH 7.2). Each well was then filled with 100 µL of binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM ethylenediaminetetraacetic acid (EDTA); pH 7.2). Fluorescence intensity was measured at Ex 550/Em 564 nm using a TECAN Infinite 200 PRO microplate reader.

Docking simulations

To obtain the three-dimensional structure of YIAD-0501, Avogadro 2, a chemical editor and visualization application was utilized for geometry optimization, including energy minimization [34]. The structural model of Aβ (1–42) was derived from the Protein Data Bank (PDB) structure (PDB ID: 2NAO) [35]. The docking simulation between YIAD-0501 and Aβ (1–42) was performed through GNINA (version 1.3) [36]. The result of the docking simulation was visualized and analyzed using PyMOL software (version 2.1), a molecular visualization system. The interaction between Aβ (1–42) and YIAD-0501 was investigated by utilizing the Protein-Ligand Interaction Profiler server [37].

CD spectroscopy

Secondary structure changes of Aβ peptides were analyzed by CD spectroscopy using a Jasco J-815 spectropolarimeter. Aβ (1–42) or Aβ(pE3–42) (25 µM) was incubated in deionized water containing 10% acetonitrile in the absence or presence of YIAD-0501 (50 µM, 10% acetonitrile) for 12 h. CD spectra were recorded at 25 °C using a quartz cuvette with a 1-mm path length (Hellma) over a wavelength range of 200–250 nm, with a data pitch of 1 nm, a bandwidth of 1 nm, and a scan speed of 50 nm/min. Raw ellipticity data (mdeg) were converted to molar ellipticity (Δε) using the BeStSel web-based analysis server.

MST

Binding interactions between YIAD-0501 and Aβ (1–42) were assessed by MST using a Monolith X instrument (NanoTemper Technologies, Germany). FITC-labeled Aβ (1–42) (8 µM in 10% DMSO, dissolved in 1× PBS) was mixed with YIAD-0501 prepared as a 2-fold serial dilution ranging from 40 µM to 0.313 µM (eight concentration points; 10% DMSO in 1× PBS). Following mixing, the final concentrations were adjusted to 4 µM for FITC-labeled Aβ (1–42), with YIAD-0501 concentrations ranging from 20 µM to 0.156 µM. Samples were briefly vortexed and loaded into premium MST capillaries. MST measurements were performed with LED excitation settings optimized to keep fluorescence signals within the recommended detection range. Thermophoretic traces were analyzed using MO.Affinity Analysis software (NanoTemper Technologies), and binding curves were fitted using a one-site binding model to determine the dissociation constant (K_d).

Dot blot assays

To analyze oligomeric Aβ levels in vitro, samples obtained from inhibition and dissociation assay of YIAD-0501 were blotted onto nitrocellulose membranes, dried (30 min), and blocked with 5% skim milk in TBS-T for 1 h at room temperature. Membranes were then incubated overnight at 4 ℃ with anti-oligomer A11 antibody (ThermoFisher, AHB0052, 1:1,000), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, 111-035-144, 1:10,000) for 1 h at room temperature.

To analyze amyloid levels in brain tissue, the hippocampus from the left hemisphere was homogenized in radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, R0278) containing protease inhibitor cocktail (Roche, 11836170001). After centrifugation (14,000 rpm, 4 ℃, 30 min), protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225). A total of 10 µg of protein in a 3 µL volume was blotted on the nitrocellulose membrane, dried (30 min), and blocked with 5% skim milk in TBS-T for 1 h at room temperature. Membranes were then incubated overnight at 4 ℃ with anti-Aβ 6E10 antibody (BioLegend, 803003, 1:2,000), followed by HRP-conjugated anti-mouse IgG antibody (BETHYL, A90-116P, 1:20,000) for 1 h at room temperature. In both assays, protein signals were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher, 34580). Between each step, membranes were washed three times for 5 min with TBS-T (tris-buffered saline with 0.1% Tween-20).

Administration of YIAD-0501 to 5XFAD mice

To test the efficacy of YIAD-0501 in vivo, 5XFAD (B6SJL-Tg(APPSwFILon, PSEN1*M146L*L286V)6799Vas/Mmjax) transgenic mouse model was used. YIAD-0501 (10 mg/kg dissolved in PBS with 15% DMSO and 15% Tween-80) was administered daily via intraperitoneal injection to six-month-old 5XFAD mice (male, n = 8) for 4 weeks. The dose was selected based on reported in vivo efficacy of amyloid-modulating small molecules and the maximum concentration achievable in this formulation [38–40]. As controls, 15% DMSO and 15% Tween-80 dissolved in PBS were intraperitoneally injected into age-matched wild-type (WT) (male, n = 10) and 5XFAD (male, n = 10) littermates. An additional group of 5XFAD mice sacrificed prior to administration (male, n = 8) was used as a baseline reference. The administration was completed prior to the initiation of behavioral testing. After the administration period, mice underwent deep anesthesia induced by 4% avertin solution administered via intraperitoneal injection. Plasma was collected from the inferior vena cava into EDTA-coated tubes and centrifuged at 2,000 × g for 15 min at 4 ℃ to isolate plasma. Subsequently, transcardiac perfusion was performed with 0.9% saline to remove the blood and preserve brain tissue for further analysis. After brain excision, the left hemisphere was homogenized for immunoassay, and the right hemisphere was preserved for IHC analysis. All animal experiments were conducted under the guide for care and use of laboratory animals of the National Institutes of Health. All mice were maintained in the animal facility of Yonsei University, under a 12-hour light-dark cycle, with controlled temperature and humidity, and ad libitum access to food and water. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-A-202107-1300-01).

Y-maze tests

All mice were placed in a black opaque Y-shaped maze to evaluate short-term working memory and exploratory activity by calculating spontaneous alternation and the total number of arm entries. No explicit spatial cues were provided in this test. Each arm was 10 cm in width, 40 cm in length, and 12 cm in height, angled at 120° from each other. Every mouse began exploration from the same arm and was allowed to move freely during a 10-minute session. An entry was marked once a mouse passed the midpoint of an arm. Behavioral performance was scored manually based on recorded exploration behavior. The spontaneous alternation was calculated by recording the number of arm entries and triads.

Contextual fear conditioning tests

Mice were placed in the same conditioning chamber for 5 min per day across three consecutive days. On day 1, mice were placed in the chamber for 5 min for habituation. On day 2, mice received foot shocks twice (0.3 mA, 2 s each) at 3 and 4 min after entering the chamber. On day 3, freezing behavior was recorded using the Packwin software. A breathing filter was applied to improve motion detection accuracy. To minimize potential confounding by body weight, a subset analysis including only mice within ± 1 standard deviation of the overall mean was performed [41].

IHC staining

Right hemispheres of mouse brains were fixed overnight at 4 ℃ in 4% paraformaldehyde and cryoprotected in 30% sucrose until fully submerged. Brains were embedded in optimal cutting temperature compound (Tissue-Tek® O.C.T. Compound, Sakura, 4583) and sectioned at 25 μm using a cryostat (Leica, CM1860). Sections were mounted on glass slides and treated with 1% sodium dodecyl solution (SDS) in PBS for antigen retrieval. To block nonspecific binding, slides were incubated with 5% horse serum in PBS for 1 h at room temperature. Sections were then incubated overnight at 4 ℃ with primary antibodies against Aβ (6E10, BioLegend, 803003, 1:200) and GFAP (Millipore, AB5541, 1:200), followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) (Invitrogen, A11001, 1:200) and Alexa Fluor 568-conjugated goat anti-chicken IgY (H + L) (A11041, Invitrogen, 1:200) for 1 h at room temperature.

For assessment of potential neuronal toxicity, sections were incubated overnight at 4 °C with primary antibodies against NeuN (Millipore, MAB377, 1:500), followed by incubation with Alexa Fluor 488–conjugated goat anti-mouse IgG (H + L) (Invitrogen, A11001, 1:1,000) for 1 h at room temperature.

For microglial staining, sections were incubated overnight at 4 °C with primary antibodies against Iba1 (Wako, 019-19741, 1:200), followed by incubation with Alexa Fluor 555–conjugated goat anti-rabbit IgG (H + L) (abcam, ab150078, 1:200) for 1 h at room temperature. Fluorescence images were acquired using a fluorescence microscope (DM2500, Leica) with LAS X software and analyzed using ImageJ software.

ELISA

To detect and quantify human Aβ (1–42) levels in hippocampal brain lysates, a human Aβ42 ELISA kit (Invitrogen, KHB3441) was used according to the manufacturer’s instructions. Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225). Aβ (1–42) standards were prepared by serial dilution to final concentrations of 1,000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg/mL. A total of 50 µL of each standard and 5 µg of hippocampal protein were loaded into each well. Then, 50 µL of detection antibody was added and the plate was incubated overnight at 4 °C. The following day, the plate was incubated with HRP-conjugated anti-rabbit IgG antibody for 30 min at room temperature. Between each step, the plate was washed three times with wash buffer. A stabilized chromogen solution was then added and incubated in the dark for 30 min. After addition of a stop solution to terminate the reaction, absorbance was measured at 450 nm using a SpectraMax M2 microplate reader. To detect and quantify human Aβ (1–42) levels in plasma, a human Aβ42 ultrasensitive ELISA kit (Invitrogen, KHB3544) was used according to the manufacturer’s instructions. Plasma samples were diluted 1:2 in the provided dilution buffer. Aβ (1–42) standards were prepared by serial dilution to final concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0 pg/mL. All subsequent steps were performed as described above.

Western blots

Hippocampal protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher, 23225), and 15 µg of protein per sample was loaded per lane. Proteins were separated by SDS-PAGE on 12% gradient polyacrylamide gel (Gradi-Gel II, ELPIS) at 100 V and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk in TBS-T for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against GFAP (Millipore, AB5541, 1:2,000), Iba1 (Cell Signaling, 17198 S, 1:1,000), and β-actin (Millipore, MAB1501R, 1:10,000). The next day, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies: anti-rabbit IgG (Jackson ImmunoResearch, 111-035-144, 1:10,000), anti-chicken IgY (Jackson ImmunoResearch, 703-035-155, 1:50,000), and anti-mouse IgG (Bethyl, A90-116P, 1:50,000). Between each step, membranes were washed three times for 5 min with TBS-T (Tris-buffered saline with 0.1% Tween-20). Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher, 34580).

Biosafety assessment

Biosafety assessment was performed to evaluate potential systemic and neuronal toxicity associated with YIAD-0501 treatment. ICR mice were administered YIAD-0501 (10 mg/kg dissolved in PBS containing 15% DMSO and 15% Tween-80) via daily intraperitoneal injection for 2 weeks. Baseline body weight and food intake were recorded on Day 0 prior to drug administration. Body weight was monitored daily, and food intake was measured every two days throughout the treatment period. Gross examination of major organs was conducted at the end of the study, and hair loss was visually inspected before and after the administration period.

Cell viability assay

Cell viability was evaluated using the murine hippocampal cell line HT22. Cells were seeded into 96-well plates (CLS3596, Corning, USA) at a density of 1 × 10³ cells per well and subjected to a 4-hour serum-free starvation period. Cells were then treated with YIAD-0501 diluted in Dulbecco’s Modified Eagle Medium (DMEM; LM001-05, WELGENE, Korea) supplemented with 1% penicillin–streptomycin (15140122, Gibco, USA) and 1% DMSO, or with vehicle control (1% DMSO), for 1 day. Cell viability was assessed using the D-Plus™ CCK cell viability assay kit (Eubiogene, Korea) according to the manufacturer’s instructions.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software). For in vitro biochemical assays, including Thioflavin T (ThT) fluorescence assays and dot-blot quantification, statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc comparison test. For in vivo behavioral experiments, including contextual fear conditioning, group comparisons were conducted using one-way ANOVA followed by Bonferroni’s post-hoc test. For histological and biochemical analyses of brain tissues, including immunohistochemistry (6E10 and GFAP), in vivo dot blot assays, and western blot densitometry, statistical analyses were performed using one-way ANOVA with Bonferroni’s post-hoc test. For NeuN immunohistochemistry, one-way ANOVA followed by Bonferroni’s post-hoc test was used for multiple comparisons. For ELISA measurements, Y-maze spontaneous alternation, Iba1 immunohistochemistry, and cell viability assays, comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. All quantitative data are presented as mean ± standard error of the mean (SEM). In graphical representations, each data point represents an individual biological replicate (one animal for in vivo experiments or one independent sample for in vitro assays). Statistical significance thresholds and exact P values are indicated in the corresponding figure legends.

Results

YIAD-0501 inhibits formation and promotes dissociation of Aβ(1–42) fibrils and oligomers

To identify chemical compounds capable of inhibiting further formation of Aβ aggregates and dissociating pre-existing aggregates, we screened six YIAD compounds for their ability to reduce fibrillar and oligomeric Aβ (1–42) species. Aβ (1–42) is the predominant plaque-forming isoform and is therefore widely used in aggregation screens [5, 6]. The chemical structures of the six compounds are shown in Fig. 1A. Fibril formation was quantified by ThT fluorescence, which reflects β-sheet–rich amyloid structures [42].

Fig. 1.

Screening of YIAD compounds identifies YIAD-0501 as a potent inhibitor and dissociator of Aβ (1–42) fibrils and oligomers. A Chemical structures of six YIAD compounds. B Inhibition of Aβ (1–42) fibril formation by YIAD compounds (5 and 50 µM). YIAD compounds were co-incubated with Aβ (1–42) monomers for 1 day. C Dissociation of fibrils in Aβ (1–42) pre-aggregates (pre-agg.) by YIAD compounds (5 and 50 µM). Aβ (1–42) monomers were incubated for 1 day to form pre-aggregates, followed by compound treatment and an additional day of incubation to form post-aggregates (post-agg.). D Dose-dependent inhibitory effect of YIAD-0501 on Aβ (1–42) fibril formation. E Dose-dependent dissociative effect of YIAD-0501 on fibrils in Aβ (1–42) pre-aggregates. All fluorescence intensity values were normalized to the Aβ (1–42)-only or Aβ (1–42)-only pre-aggregates control, set as 100%. F Dot blot analysis of Aβ (1–42) oligomer formation inhibition and dissociation by YIAD-0501. Oligomers were detected using the anti-oligomer A11 antibody. For inhibition assay, YIAD-0501 was co-incubated with Aβ (1–42) monomers for 1 day. For dissociation assay, Aβ (1–42) monomers were incubated for 1 day to form pre-aggregates, followed by compound treatment and an additional day of incubation to form post-aggregates. A11 signal intensities were normalized to the Aβ (1–42)-only control (for inhibition) or Aβ (1–42)-only pre-aggregates control (for dissociation), set as 100%. For statistical analysis, one-way ANOVA followed by Bonferroni’s post-hoc comparison test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Aβ (1–42)-only or Aβ (1–42)-only pre-aggregates control. Data are presented as mean ± SEM. Aβ (1–42)-only control is denoted as “–”. pre-agg., pre-aggregates; post-agg., post-aggregates

In the fibril-inhibition assays, monomeric Aβ (1–42) (12.5 µM) was co-incubated with each compound (5 or 50 µM) for 1 day at 37 °C. All compounds reduced ThT fluorescence to varying degrees, indicating inhibition of fibril formation. YIAD-0501 exhibited the strongest inhibitory effect among the six compounds (Fig. 1B). To evaluate dissociative activity, Aβ (1–42) monomers were incubated for 1 day to generate pre-aggregates, followed by compound treatment for an additional 1 day. At 50 µM, YIAD-0501 reduced ThT fluorescence by 52.5% relative to the pre-aggregate control (P < 0.0001), the most pronounced effect among the YIAD compounds (Fig. 1C).

Dose-response analysis of YIAD-0501 (0.1–50 µM) revealed clear, concentration-dependent inhibition of fibril formation and dissociation of pre-formed aggregates (Fig. 1D, E), consistent with a specific interaction with amyloid aggregates. The corresponding IC₅₀ and EC₅₀ values are shown in Fig. S1A and B. Supporting these dose-dependent effects, aggregation kinetics analysis demonstrated that co-incubation of Aβ (1–42) with YIAD-0501 markedly suppressed fibril formation throughout the incubation period (Fig. S1C). Time-course analysis showed that treatment of pre-aggregated Aβ (1–42) with YIAD-0501 resulted in a rapid and sustained reduction in ThT fluorescence beginning at early time points (0.5 h) and persisting for up to 48 h (Fig. S1D).

Aβ oligomers are widely recognized as the most neurotoxic conformers in the aggregation cascade. Oligomer levels were quantified by dot blot using the conformation-specific A11 antibody. Dot blot analysis confirmed that YIAD-0501 significantly reduced oligomer formation and promoted dissociation of pre-formed oligomers at both tested concentrations (Fig. 1F, Fig. S1E, F). Morphological changes in fibrillar assemblies following YIAD-0501 treatment were further visualized by TEM, which revealed a reduction in mature fibrillar structures compared with control conditions (Fig. S1G). Collectively, these data show that YIAD-0501 reduces both Aβ (1–42) fibrils and oligomers.

YIAD-0501 inhibits formation and promotes dissociation of Aβ(pE3–42) fibrils and oligomers

Pyroglutamate-modified Aβ, Aβ(pE3–42), is less abundant than Aβ (1–42) yet is exceptionally stable and neurotoxic, making it a key pathological species in AD [7–9]. We therefore evaluated whether YIAD-0501 can reduce fibrillar and oligomeric Aβ(pE3–42) species.

When monomeric Aβ(pE3–42) was co-incubated with serial dilutions of YIAD-0501 (0.1–50 µM), YIAD-0501 reduced ThT fluorescence signals in a dose-dependent manner, indicating inhibition of Aβ(pE3–42) fibril formation (Fig. 2A). In a subsequent dissociation assay, pre-aggregated fibrils exposed to the same concentration range showed a comparable, dose-responsive decline in ThT fluorescence, indicating that YIAD-0501 effectively dissociates existing Aβ(pE3–42) fibrils (Fig. 2B). The corresponding IC₅₀ and EC₅₀ values are presented in Fig. S2A, B and closely align with the values obtained for Aβ (1–42), illustrating YIAD-0501’s comparable potency toward canonical Aβ (1–42) and its pyroglutamate-modified form. Supporting these dose-dependent effects, aggregation kinetics analysis demonstrated that co-incubation of Aβ(pE3–42) with YIAD-0501 suppressed fibril formation throughout the incubation period (Fig. S2C). Time-course analysis showed that treatment of pre-aggregated Aβ(pE3–42) with YIAD-0501 resulted in a rapid and sustained reduction in ThT fluorescence beginning at early time points (0.5 h) and persisting for up to 48 h (Fig. S2D).

Fig. 2.

YIAD-0501 reduces Aβ(pE3–42) fibrils and oligomers. A Dose-dependent inhibitory effect of YIAD-0501 on Aβ(pE3–42) fibril formation. YIAD-0501 was co-incubated with Aβ(pE3–42) monomers for 1 day. B Dose-dependent dissociative effect of YIAD-0501 on fibrils in Aβ(pE3–42) pre-aggregates (pre-agg.) samples. Aβ(pE3–42) monomers were incubated for 1 day to form pre-aggregates, followed by compound treatment and an additional day of incubation to form post-aggregates (post-agg.). All fluorescence intensity values were normalized to the Aβ(pE3–42)-only or Aβ(pE3–42)-only pre-aggregates control, set as 100%. C Dot blot analysis of Aβ(pE3–42) oligomer formation inhibition and dissociation by YIAD-0501. Oligomers were detected using the anti-oligomer A11 antibody. For inhibition assay, YIAD-0501 was co-incubated with Aβ(pE3–42) monomers for 1 day. For dissociation assay, Aβ(pE3–42) monomers were incubated for 1 day to form pre-aggregates, followed by compound treatment and an additional day of incubation to form post-aggregates. A11 signal intensities were normalized to the Aβ(pE3–42)-only control (for inhibition) or Aβ(pE3–42)-only pre-aggregates control (for dissociation), set as 100%. For statistical analysis, one-way ANOVA followed by Bonferroni’s post-hoc comparison test was performed. ***P < 0.001, ****P < 0.0001 vs. Aβ(pE3–42)-only or Aβ(pE3–42)-only pre-aggregates control. Data are presented as mean ± SEM. Aβ(pE3–42)-only control is denoted as “–”. pre-agg., pre-aggregates; post-agg., post-aggregates

To determine whether these effects extend to oligomers, we quantified dot blot signals using the conformation-specific A11 antibody. YIAD-0501 showed a trend toward reduced oligomer formation and dissociation of pre-formed Aβ(pE3–42) oligomers (Fig. 2C, Fig. S2E, F). Consistent with these biochemical findings, TEM revealed reduced fibrillar assemblies of Aβ(pE3–42) following treatment with YIAD-0501 (Fig. S2G). Taken together, YIAD-0501 inhibited formation of Aβ(pE3–42) fibrils and oligomers and dissociated pre-existing aggregates of both conformers, thereby extending its anti-amyloid activity to the highly pathogenic pyroglutamate-modified peptide.

YIAD-0501 targets hydrophobic C-terminal and aggregation-core motifs of Aβ

Characterizing the binding interface between small-molecule modulators and Aβ assemblies is essential for understanding and ultimately optimizing anti-amyloid mechanisms [43]. To investigate how the small-molecule modulator YIAD-0501 engages Aβ, we performed a MAP assay and molecular docking simulation, supported by CD spectroscopy and MST analyses. We selected Aβ (1–42) as the reference isoform because its aggregation pathway and high-resolution structures are extensively documented [44–46].

To identify the residues targeted by YIAD-0501 on Aβ (1–42) oligomers, we carried out a MAP assay [33]. A plate immobilized with full-length Aβ (1–42) and 37 hexamer fragments was pre-incubated with Flamma 552–conjugated Aβ (1–42) (10 µM, 6 h, 37 °C) to allow oligomer formation, followed by YIAD-0501 treatment for 24 h (Fig. 3A). YIAD-0501 dissociated 57.0% of full-length oligomers. Among the fragments, dissociation was strongest for the Aβ [25–41] region, particularly the GSNKGAIIGLMVGGVVI sequence (Fig. 3B, C), a hydrophobic C-terminal domain critical for β-sheet stabilization [4].

Fig. 3.

YIAD-0501 targets hydrophobic and aggregation-core motifs of Aβ (1–42) assemblies. A Schematic overview of the Aβ (1–42) oligomer dissociation site-mapping assay. B Dissociation rates of full-length Aβ (1–42) and each of its 37 hexamer fragments after YIAD-0501 treatment. C Heatmap of the dissociation rates (white to red, 0–100%). D Top two molecular-docking poses of YIAD-0501 on Aβ (1–42) fibrils. Data are presented as mean ± SEM. Norm., normalized

To corroborate the MAP findings and visualize plausible binding modes within a β-sheet context, we performed molecular docking of YIAD-0501 to the amyloid-fibril structure of Aβ (1–42) (PDB ID: 2NAO) using GNINA (Fig. 3D) [36]. The two highest-scoring poses yielded binding affinities of − 8.52 kcal/mol (Pose 1) and − 7.42 kcal/mol (Pose 2). Pose 1 features hydrophobic contacts with Gln15, Phe20, Val24 and Ile32, along with π–π stacking with Phe19, whereas Pose 2 forms hydrophobic interactions with Ile31, Val36, Val39 and Ile41.

CD spectroscopy showed that co-incubation with YIAD-0501 reduced β-sheet secondary structure content in both Aβ (1–42) and Aβ(pE3–42) assemblies (Fig. S3A, B), consistent with destabilization of β-sheet-rich amyloid structures. In line with these structural effects, MST analysis demonstrated direct binding of YIAD-0501 to Aβ (1–42), with a dissociation constant (K_d) of 1.56 µM (Fig. S3C).

Taken together, these data indicate that YIAD-0501 engages hydrophobic C-terminal and aggregation-core motifs of Aβ and is associated with reduced β-sheet secondary structure and inhibition of Aβ self-assembly.

YIAD-0501 improves spatial working and contextual memory in 5XFAD mice

After establishing robust anti-amyloid activity in vitro, we investigated whether YIAD-0501 could enhance cognition in an AD mouse model. We used the 5XFAD transgenic mouse model, which develops robust amyloid pathology, to test whether the compound’s anti-amyloid actions translate in vivo [47]. Six-month-old male 5XFAD mice received daily intraperitoneal injections of YIAD-0501 (10 mg/kg, TG 0501, n = 8) for 4 weeks. Age-matched vehicle-treated wild-type (WT vehicle, n = 10) and 5XFAD littermates (TG vehicle, n = 10) served as controls. A separate TG group (n = 8) was sacrificed prior to drug administration to provide a pre-treatment baseline (Fig. 4A).

Fig. 4.

YIAD-0501 improves cognitive performance in 5XFAD mice. A In vivo experimental design. Six-month-old male 5XFAD transgenic (TG) mice received daily intraperitoneal injections of YIAD-0501 (TG 0501, 10 mg/kg/day, n = 8) for 4 weeks. Age-matched vehicle-treated wild-type mice (WT Veh, n = 10) and 5XFAD littermates (TG Veh, n = 10) served as controls. A separate group of TG mice (n = 8) was sacrificed prior to drug administration to serve as a pre-treatment baseline. Behavioral assessments were performed after the treatment period, and brain hemispheres were collected for further analysis. B Schematic representation of the Y-maze test. C Representative Y-maze heatmaps. D Spontaneous alternation percentage. E Schematic timeline of the contextual fear conditioning test. F Freezing time percentage on day 3. Data are presented as mean ± SEM. An unpaired t-test was used for the Y-maze test, and contextual freezing was analyzed with one-way ANOVA followed by Bonferroni’s post-hoc comparison test. *P < 0.05, **P < 0.01 vs. TG Veh group. IP, intraperitoneal; Pre-admin., pre-administration; Post-admin., post-administration; IHC, immunohistochemistry; WT, wild-type; TG, 5XFAD transgenic; Veh, vehicle; 0501, YIAD-0501; Min., minimum; Max., maximum

Spatial working memory was assessed with the Y-maze spontaneous alternation test (Fig. 4B). TG 0501 mice showed a higher alternation percentage compared to TG vehicle (Fig. 4C–D), indicating improved spatial working memory after YIAD-0501 treatment. Total arm entries did not differ significantly between the TG 0501 group and the TG vehicle groups (Fig. S4A), excluding altered locomotor activity as a confounding factor.

Encouraged by the rescue of short-term spatial memory, we next examined another cognitive domain, contextual associative memory, using a fear conditioning test. The test consisted of habituation (day 1), two 0.3 mA foot shocks of two seconds each (day 2) and freezing measurement (day 3) (Fig. 4E). TG 0501 mice showed a significantly higher freezing response compared to the TG vehicle group, indicating rescued hippocampus-dependent memory (Fig. 4F, Fig. S4B). To assess potential confounding by body weight, a subset analysis including only mice within ± 1 standard deviation of the overall mean body weight was conducted and yielded comparable results (Fig. S4B) [41]. Spatial working and contextual associative memory both rely heavily on hippocampal circuitry. Taken together, these findings indicate that YIAD-0501 mitigates cognitive deficits in 5XFAD mice, possibly via enhancement of hippocampus-dependent function.

YIAD-0501 reduces plaque density and soluble Aβ levels in the hippocampus of 5XFAD mice

Given that the Y-maze and contextual fear conditioning tests predominantly rely on hippocampal circuits mediating spatial and contextual memory [48, 49], we next examined the effect of YIAD-0501 on Aβ pathology in this region. Plaque number and area were first quantified across the total brain hemisphere and then specifically within the hippocampus using 6E10 IHC (Fig. 5A-C, Fig. S5, 6). In the total brain hemisphere, plaque number did not differ significantly among the pre-administration, post-administration TG vehicle, and TG 0501 mice. Plaque area was significantly smaller in the pre-administration group than in post-administration TG vehicle group, suggesting progressive plaque growth during the four-week interval (Fig. 5D). In the hippocampus, YIAD-0501 exerted a robust effect. There was a significant reduction in both Aβ plaque number and area in the hippocampus of TG 0501 mice compared to TG vehicle, restoring plaque burden to baseline levels (Fig. 5E).

Fig. 5.

YIAD-0501 reduces plaque burden and soluble Aβ in the hippocampus of 5XFAD mice. A Schematic of brain regions analyzed for plaque quantification. B, C Representative images from post-administration (post-admin.) (B), and pre-administration (pre-admin.) (C) groups, stained with the anti-Aβ 6E10 antibody. Scale bars: 2 mm (whole brain hemisphere), 500 μm (hippocampus). D, E Quantification of Aβ plaque number and area in the total brain hemisphere (D) and hippocampus (E), normalized to the post-admin. 5XFAD transgenic vehicle (TG Veh) group (set to 100%). F Dot blot analysis of soluble hippocampal Aβ species using the anti-Aβ 6E10 antibody, and ELISA quantification of soluble Aβ (1–42) levels in the hippocampus. ELISA data were analyzed by unpaired t-test. All other comparisons used one-way ANOVA followed by Bonferroni’s post-hoc comparison test. *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant vs. post-admin. TG vehicle. Data are presented as mean ± SEM. Pre-admin., pre-administration; Post-admin., post-administration; WT, wild-type; TG, 5XFAD transgenic; 0501, YIAD-0501

To assess soluble Aβ levels in the hippocampus, we conducted dot blot assay and ELISA using RIPA-soluble hippocampal lysates. Dot blot analysis with anti-Aβ 6E10 antibody showed significantly lower Aβ signal in TG 0501 mice compared to TG vehicle (Fig. 5F, Fig. S7), indicating a reduction in total soluble Aβ species. In addition, we performed ELISA to specifically quantify soluble Aβ (1–42). Consistent with the dot blot result, soluble Aβ (1–42) concentrations were significantly lower in TG 0501 than TG vehicle mice (Fig. 5F). Collectively, these results suggest that YIAD-0501 reduces both plaque burden and soluble Aβ, including Aβ (1–42) in the hippocampus, providing a pathological correlate to its cognitive benefit.

YIAD-0501 alleviates hippocampal neuroinflammation in 5XFAD mice

In line with the observed reductions in aggregated and soluble Aβ species following YIAD-0501 treatment, we next examined whether downstream neuroinflammatory pathology in the hippocampus was attenuated. Neuroinflammation driven by reactive astrocytes and microglia is a hallmark of AD and frequently observed in proximity to Aβ plaques [50, 51]. To evaluate the effect of YIAD-0501 on hippocampal neuroinflammation, we first performed IHC staining for GFAP, a marker of reactive astrocytes. Consistent with the Aβ findings, TG 0501 mice showed a significant reduction in both number and area of GFAP-positive astrocytes in the hippocampus relative to TG vehicle, returning neuroinflammatory burden to baseline levels (Fig. 6A–C, Fig. S8). In addition to astrocytic responses, we assessed microglial activation in the hippocampus by IHC staining for Iba1. Although the total number of Iba1-positive microglia did not differ significantly between TG vehicle and TG 0501 mice, quantitative analysis revealed a significant reduction in the Iba1-positive area in the hippocampus following YIAD-0501 treatment (Fig. S9).

Fig. 6.

YIAD-0501 attenuates astrocytic and microglial activation in the hippocampus of 5XFAD mice. A Representative images from pre-administration (pre-admin.) and post-administration (post-admin.) groups, stained with anti-glial fibrillary acidic protein (GFAP) antibody. Scale bars: 500 μm. B Schematic of the hippocampal area for GFAP-positive astrocyte quantification. C Quantification of GFAP-positive astrocyte number and area in the hippocampus, normalized to the post-admin. 5XFAD transgenic vehicle (TG Veh) group (set to 100%). D Western blot analysis of hippocampal lysates for GFAP and ionized calcium-binding adaptor molecule 1 (Iba1). β-actin was used as a loading control. E Quantification of GFAP and Iba1 protein levels by densitometry. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc comparison test. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns = not significant vs. post-admin. TG vehicle. Data are presented as mean ± SEM. GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adaptor molecule 1; Pre-admin., pre-administration; Post-admin., post-administration; WT, wild-type; TG, 5XFAD transgenic; 0501, YIAD-0501

We next analyzed hippocampal lysates by western blot. In line with IHC findings, YIAD-0501 treatment significantly decreased the protein levels of both GFAP and Iba1 (Fig. 6D–E, Fig. S10). Collectively, these results indicate that YIAD-0501 alleviates hippocampal neuroinflammation, as reflected by reduced astrocytic reactivity and decreased microglial immunoreactive area.

Discussion

In this study, targeting multiple pathogenic Aβ forms, including early oligomeric species, was associated with improved cognitive performance in 5XFAD mice. This association was supported by in vitro evidence showing that YIAD-0501 reduced the formation of Aβ (1–42) and Aβ(pE3–42) aggregates, as well as by in vivo reductions in hippocampal amyloid pathology, soluble Aβ levels, and neuroinflammation.

A key finding of this study is that YIAD-0501 interacts with both Aβ (1–42) and Aβ(pE3–42) aggregates. Molecular docking and MAP analyses indicate that YIAD-0501 primarily engages the KLVFFA self-recognition core and the C-terminal hydrophobic region, which contribute to fibril stability [4]. These sites are conserved across the two peptides, which may explain why YIAD-0501 retains activity despite N-terminal variation.

Our in vitro assays showed that YIAD-0501 reduces both β-sheet fibrillar structures and A11-positive oligomers. In 5XFAD mice, reductions in Aβ plaques were accompanied by a decrease in total soluble Aβ levels in both 6E10 dot blot and Aβ (1–42) ELISA analyses, with no evidence of an increase in soluble intermediates. Although these assays do not differentiate individual conformers, these findings indicate that YIAD-0501 does not shift Aβ aggregates toward a more soluble and potentially toxic species in vivo. Given the established association between soluble Aβ species and synaptic dysfunction [21, 22], the cognitive improvements observed in YIAD-0501–treated 5XFAD mice may therefore reflect, at least in part, a reduction in soluble Aβ species, in addition to the observed decrease in plaque burden.

YIAD-0501 did not induce overt systemic or neuronal toxicity under the experimental conditions used. In ICR mice treated with YIAD-0501 (10 mg/kg), no significant changes in body weight or food intake were observed, and gross examination revealed no noticeable abnormalities in major organs (Fig. S11A-C). In addition, NeuN immunostaining with quantitative analysis did not reveal significant differences among experimental groups, indicating no apparent neurotoxic effects associated with YIAD-0501 treatment (Fig. S11D). Consistent with these findings, no detectable cytotoxicity was observed in HT22 murine hippocampal cells following exposure to YIAD-0501 (25 µM) (Fig. S11E).

From a translational perspective, small-molecule approaches such as YIAD-0501 may offer practical advantages, including ease of administration and cost-efficiency, compared with antibody-based therapies [16, 17]. While Aβ-targeting immunotherapies can effectively reduce aggregated Aβ, their clinical implementation is often constrained by the risk of amyloid-related imaging abnormalities, which limits patient eligibility [13, 52, 53]. In this context, a small-molecule dissociation strategy represents a non-immunogenic alternative that may circumvent antibody-mediated inflammatory responses and their associated safety concerns.

Limitations

This study evaluated a single fixed dose over a 4-week regimen and did not include pharmacokinetic (PK) or blood-brain barrier penetration data, limiting the interpretation of brain exposure and dose–response relationships. In addition, a positive control group was not included in the in vivo behavioral experiments, and future studies incorporating appropriate positive controls will be required to further validate the behavioral effects observed in this study.

The analyses focused primarily on Aβ-related endpoints. Plasma Aβ (1–42), which may reflect central Aβ dynamics, did not differ between TG vehicle and TG 0501 mice (Fig. S12), possibly due to the 2-week drug-free interval required for behavioral testing that may have allowed peripheral clearance of dissociated peptides. Furthermore, reliable in vivo validation of pyroglutamate-modified Aβ species could not be achieved, and conclusions regarding Aβ(pE3–42) are therefore limited to in vitro analyses.

The reduction in Aβ pathology was most prominent in the hippocampus. Such region-specific differences in therapeutic responsiveness have been described in amyloid mouse models and may reflect underlying differences in local pathology or compound exposure [54], although these factors remain to be fully elucidated. Structure–activity relationship analysis was also limited, as only a subset of compounds from the previously reported library could be evaluated due to solubility constraints.

Future studies incorporating pharmacokinetic profiling, multiple dosing paradigms, metabolic rate, and additional pathological markers—including tau pathology, synaptic integrity, neuronal damage, and direct comparative evaluation with established Aβ-targeting therapies—will be necessary to more comprehensively define the pharmacological profile and therapeutic potential of YIAD-0501.

Conclusion

YIAD-0501 is a multiple-targeting small-molecule candidate that reduces both Aβ (1–42) and Aβ(pE3–42) aggregates, including oligomers and fibrils. While clinical experience with Aβ-directed immunotherapies underscores the relevance of reducing pathogenic Aβ species, their restricted target range may limit cognitive benefits. In this study, YIAD-0501 reduced amyloid pathology and improved cognitive performance, supporting its potential as a small-molecule strategy for targeting heterogeneous Aβ pathology. Together with practical advantages such as ease of administration and cost-efficiency, YIAD-0501 represents a promising candidate for addressing the complexity of amyloid pathology in AD.

Abbreviations

Aβ

Amyloid-β

AD

Alzheimer’s disease

ThT

Thioflavin T

TEM

Transmission electron microscopy

CD

Circular dichroism

MST

Microscale thermophoresis

MAP

Mapping amyloid plate

IP

Intraperitoneal

IHC

Immunohistochemistry

RIPA

Radio-immunoprecipitation assay

TFA

Trifluoroacetic acid

EDTA

Ethylenediaminetetraacetic acid

PDB

Protein Data Bank

HRP

Horseradish peroxidase

ELISA

Enzyme-linked immunosorbent assay

GFAP

Glial fibrillary acidic protein

Iba1

Ionized calcium-binding adaptor molecule 1

ICR

Institute of Cancer Research

SEM

Standard error of the mean

WT

Wild-type

TG

5XFAD transgenic

PK

Pharmacokinetic

Authors’ contributions

H.S. and S.H.L. contributed equally to this work. H.S. performed all ThT, dot blot, TEM, CD, MST, and in vivo studies along with subsequent biochemical analysis. S.H.L. synthesized and purified YIAD compounds, including YIAD-0501 for in vivo studies. W.S conducted molecular docking simulations and wrote the corresponding section of the Results. S.H.Y. synthesized and purified YIAD compounds. I.C. synthesized Aβ(1–42) hexamer and performed the MAP assay. S.Y., I.W.P., S.J.Y., and S.K. synthesized and purified Aβ peptides. M.S.P synthesized and purified FITC labelled Aβ(1–42). S.M.L. prepared the animals. H.S., H.Y.K., I.K., and Y.S.K. contributed to the manuscript preparation. H.Y.K., I.K., and Y.S.K. designed and supervised the study.

Funding

This research was supported by a grant of the Korea Dementia Research Project through the Korea Dementia Research Center (KDRC), funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (Grant Number : RS-2024-00349158), and Mid-Career Researcher Program (Grant Number: RS-2025-00523607, I.K.; RS-2021-NR059653, Y.S.K.), and Basic Science Research Program (Grant Number: RS-2018-NR031048, H.Y.K., I.K., and Y.S.K.) through the National Research Foundation of Korea (NRF), funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea. This research was also supported by Korea Institute for Advancement of Technology (KIAT) funded by the Ministry of Trade, Industry and Energy in 2024 (Grant Number: RS-2024-00418203) and Amyloid Solution Inc.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The protocols of animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-A-202107-1300-01).

Consent for publication

Not applicable.

Competing interests

Y.K. is an employee of Amyloid Solution and received equity or equity options. The rest of the authors do not have any conflict of interest.