China JLM-Lifetech latest company blog about FCS Interaction Analyzer Helps Fujian Medical University Team Discover Small-Molecule AD Drug

Journal: Pharmacological Research

Research Team: Xiaochun Chen / Feisheng Zhong / Zucheng Ye Team, Fujian Medical University

DOI: https://doi.org/10.1016/j.phrs.2025.107834

01 Research Overview

Galectin-3 (Gal-3) is a key protein regulating neuroinflammatory processes and plays a crucial role in the pathological progression of Alzheimer's disease (AD), making it a highly promising therapeutic target. However, developing small-molecule inhibitors that can efficiently inhibit Gal-3 activity while possessing strong blood-brain barrier (BBB) permeability remains a major challenge in the field.

The research team led by Xiaochun Chen, Feisheng Zhong, and Zucheng Ye at Fujian Medical University published a study titled "AI-driven discovery of brain-penetrant Galectin-3 inhibitors for Alzheimer's disease therapy" in the authoritative pharmacology journal Pharmacological Research (IF = 10.5). Using an AI-assisted drug screening platform, the study successfully identified FJMU1887, a small-molecule compound with potential therapeutic value, and verified its direct binding interaction with Gal-3 through subsequent experiments. Mechanistic studies showed that FJMU1887 reversibly binds to Gal-3, interfering with the interaction between Gal-3 and TREM2 (Triggering Receptor Expressed on Myeloid Cells 2). Further in vitro anti-inflammatory assays, pharmacokinetic analysis, and in vivo efficacy evaluations collectively demonstrated that FJMU1887 is a BBB-permeable small-molecule Gal-3 inhibitor with dual effects: inhibiting neuroinflammation and improving cognitive function. Overall, FJMU1887 represents the first oral Gal-3 inhibitor with favorable brain permeability that exhibits both anti-neuroinflammatory and cognitive-enhancing effects, providing a promising lead compound for AD therapy while validating the feasibility of AI-assisted drug discovery strategies.

02 Investigating Small-Molecule Effects on Protein Complex Dynamics via FCS

TREM2 is a microglia-specific receptor involved in modulating neuroinflammatory responses and microglial phagocytosis in AD. Recent studies have shown that Gal-3 interacts with TREM2, thereby influencing microglial activation states and associated inflammatory signaling pathways.

In this study, the authors first confirmed the binding activity of FJMU1887 to Gal-3 using conventional interaction assay methods, including Bio-Layer Interferometry (BLI) and MicroScale Thermophoresis (MST), obtaining a dissociation constant ( $K_d$ ) of $1.55\ \mu\text{M}$ . To further explore the specific impact of FJMU1887 on Gal-3-mediated protein interactions, the team employed Fluorescence Correlation Spectroscopy (FCS) to monitor the dynamic formation and dissociation of the Gal-3/TREM2 complex in situ within living cells.

In the experimental setup, Gal-3 and TREM2 tagged with fluorescent proteins moxCerulean3 and mGold, respectively, were co-transfected into HEK293 cells. Following confocal imaging, FCS was performed in regions of fluorescence colocalization to measure the diffusion coefficient ( $D$ ) of TREM2-mGold, continuously monitoring dynamic changes during compound treatment and washout (Figure A). Results showed that FJMU1887 disrupted the interaction between Gal-3 and soluble TREM2 (sTREM2), leading to a significant increase in the intracellular diffusion coefficient of sTREM2. Upon washing out the compound, Gal-3 re-bound to sTREM2, and the diffusion coefficient returned to baseline. This indicated that the reversible binding of FJMU1887 to Gal-3 dynamically modulates the Gal-3/TREM2 interaction. This conclusion was further validated by Fluorescence Resonance Energy Transfer (FRET) experiments, providing complementary evidence that FJMU1887 affects the formation or stability of the Gal-3/TREM2 complex. The dynamic equilibrium of this complex plays an important role in regulating AD-associated neuroinflammation.

Figure. Disruption of Gal-3/TREM2 complex formation by FJMU1887 evaluated via FCS. The diffusion coefficient ( $D$ ) of the Gal-3–TREM2 complex was measured under different conditions. Treatment with $10\ \mu\text{M}$ FJMU1887 significantly increased the diffusion coefficient compared to the untreated complex (*** $P < 0.001$ ), indicating complex disruption. Notably, the effect of FJMU1887 was reversible—after washout, the diffusion coefficient largely recovered to baseline levels, demonstrating complex re-formation. Individual data points represent single measurements; bars indicate mean ±SEM. Statistical significance was determined using one-way ANOVA followed by Tukey's post-hoc test.

03 Key Technological Advantages of FCS

FCS not only successfully captured the disruption of protein complex dynamics by the drug, but also clearly revealed the full mechanism chain: reversible binding $\rightarrow$ complex dissociation → functional inhibition. This highlights the unique value of FCS in studying live-cell protein-protein interactions and small-molecule regulatory mechanisms in situ:

Real-Time Dynamic Monitoring: Monitors processes in native cellular microenvironments without requiring fixed intermolecular distance thresholds. Any change in hydrodynamic radius or conformation resulting from binding can be detected through shifts in the diffusion coefficient.
In-Situ Equilibrium Detection: Measures directly under binding-dissociation equilibrium conditions in solution or living cells without wash steps, reaching single-molecule sensitivity.
Quantitative Kinetic Analysis: Outputs diffusion coefficients as continuous quantitative metrics, unlike conventional endpoint or qualitative assays. Combined with washout experiments, it directly proves the reversibility of compound inhibition, supplying quantitative data for efficacy and mechanistic assessments.

Through these advantages, FCS achieved real-time dynamic observation of FJMU1887's reversible inhibition of the Gal-3/TREM2 interaction—a dimension difficult to access via traditional biochemical techniques.

04 Research Significance and Future Outlook for FCS

This study successfully leveraged AI-assisted drug screening to identify the lead compound FJMU1887 from a large chemical library. By combining multiple orthogonal techniques—affinity analysis (BLI/MST), intermolecular distance measurement (FRET), and protein interaction dynamic monitoring (FCS)—the team systematically decoded the mechanism of action from multiple perspectives. Extensive in vitro and in vivo evaluations confirmed that FJMU1887 possesses dual anti-neuroinflammatory and cognitive-enhancing activities, establishing its potential as an AD therapeutic candidate.

In this integrated platform, FCS provided critical live-cell dynamic diffusion data unattainable by other methods, proving that FJMU1887 reversibly disrupts Gal-3/TREM2 binding. This offered direct evidence for elucidating the drug mechanism and provided additional validation of the direct interaction between Gal-3 and TREM2. The study showcases the core advantages of FCS: real-time monitoring of complex dynamics in solution, maskless non-tethered measurement, and direct quantitative output of diffusion coefficients.

About FCS Technology

Fluorescence Correlation Spectroscopy (FCS) quantitatively measures parameters such as molar concentration, fluorescence brightness/aggregation state, diffusion coefficient/hydrodynamic radius, and binding affinity ( $K_D$ ) with single-molecule resolution in micro-volume (a few microliters) solution samples or individual living cells. Compatible with physiological samples (e.g., cell lysates, blood), it is an in situ, homogeneous, high-content analytical tool. FCS and its derivative methods have been widely applied in signal transduction, liquid-liquid phase separation, biomolecular aggregation, structural-functional mechanisms, nanomedicine development, exosome characterization, fluorescent probe development, antibody/drug screening, and microfluidics, with over 15,000 research articles indexed in PubMed.

About JLM-Lifetech FCS Interaction Analyzer

FCS Interaction Analyzer Helps Fujian Medical University Team Discover Small-Molecule AD Drug

The MIA series FCS Molecular Interaction Analyzer for Single-molecule Kinetics is a high-sensitivity analysis platform based on FCS technology. By detecting fluorescence fluctuation signals in extremely small observation volumes, it enables single-molecule biomolecular interaction and characterization analysis. Using correlation algorithms to analyze time-domain signals, the system operates without separation or purification steps (homogeneous assay). With only tens of microliters of sample, it delivers high-throughput quantitative analysis of concentration, particle size, diffusion coefficient, and binding affinity ( $K_d$ ) in seconds.