De Novo Protein Binder Design for Advanced Glycation End Products: A Computational Approach to Targeting Age-Related Glycative Damage

De Novo Protein Binder Design for Targeting Age-Related Glycative Damage

Kejun (Albert) Ying

Stanford University, Department of Neurology (Tony Wyss-Coray Lab)

University of Washington, Institute for Protein Design (David Baker Lab)

1. Overview

Advanced glycation end products (AGEs) accumulate with age and contribute to cardiovascular disease, neurodegeneration, and diabetic complications through RAGE receptor activation and direct protein cross-linking. Despite decades of research, no FDA-approved therapies specifically target AGEs. Small molecule inhibitors like aminoguanidine showed promise but failed in clinical trials due to toxicity. This project proposes a fundamentally different approach: using deep learning-based protein design (RFdiffusion All-Atom) to create de novo protein binders that sequester specific AGE species, blocking their pathogenic effects.

Funding and facilities: Computational infrastructure is available through the Baker Lab at UW Institute for Protein Design. Experimental validation will use yeast surface display and biolayer interferometry (BLI), both established in the lab. No additional equipment or ethics approvals are required for this phase.

Timeline: 6 months. Months 1-2: computational design and in silico screening. Months 3-4: gene synthesis and yeast display expression. Months 5-6: binding validation and affinity characterization.

Data sharing: All computational designs, screening data, and binding characterization results will be deposited in an open-access repository. A preregistration will be posted on ResearchHub, and results will be shared as a preprint regardless of outcome.

2. Introduction

Background

AGEs form through non-enzymatic glycation when reducing sugars react with amino groups on proteins, lipids, and nucleic acids. The Maillard reaction produces early glycation intermediates that undergo oxidation and rearrangement to form stable AGE adducts. The most abundant and best-characterized AGEs include carboxymethyllysine (CML), carboxyethyllysine (CEL), and pentosidine. These modifications accumulate in nearly all mammalian tissues with age and are accelerated by hyperglycemia, oxidative stress, and metabolic dysfunction [1,2].

AGEs cause pathology through two primary mechanisms. First, they directly modify proteins, causing cross-linking that impairs function. This is particularly damaging in long-lived extracellular matrix proteins like collagen, where AGE cross-links increase tissue stiffness and reduce turnover. Second, AGEs bind to the receptor for advanced glycation end products (RAGE), activating NF-kB signaling and triggering chronic inflammation, oxidative stress, and cellular dysfunction [3]. The RAGE-AGE axis has been implicated in diabetic complications, atherosclerosis, Alzheimer's disease, and accelerated aging [4].

Current therapeutic approaches fall into three categories: AGE formation inhibitors (aminoguanidine, pyridoxamine), AGE cross-link breakers (alagebrium), and RAGE antagonists. None have achieved FDA approval for AGE-related indications. Aminoguanidine reached Phase III trials but was discontinued due to adverse effects. Recent interest has shifted to natural polyphenols and dietary interventions, but these lack specificity and potency [5].

Innovation

This project proposes a novel strategy: designing de novo protein binders that recognize and sequester specific AGE adducts. Unlike small molecules, designed proteins can achieve high affinity and specificity through extensive complementary surfaces. The recent release of RFdiffusion All-Atom enables design of proteins that bind small molecules by generating folded scaffolds around the target ligand [6,7]. This approach has been validated for other small molecule targets including digoxigenin and heme. No group has yet applied this technology to AGE targeting.

A designed AGE binder could function therapeutically by intercepting circulating AGEs before they engage RAGE or cross-link tissue proteins. Beyond direct therapeutic potential, such binders would serve as research tools for detecting and quantifying specific AGE species, and as scaffolds for downstream enzyme design to actively degrade AGEs.

Research Questions and Hypotheses

H1: RFdiffusion All-Atom can generate protein scaffolds with designed binding pockets for CML (the most abundant AGE).

H2: At least one computationally designed binder will show measurable binding to CML-modified BSA (AGE-BSA) in yeast surface display screening.

H3: Top binder candidates will exhibit sub-micromolar affinity for AGE-BSA as measured by biolayer interferometry.

Impact Statement

Regardless of outcome, this project will generate valuable data. If successful, it establishes proof-of-concept for a new class of AGE-targeting therapeutics and produces binder sequences that can be optimized through directed evolution. If binding is not achieved, the negative results will inform the field about the challenges of designing binders for post-translationally modified amino acids and guide future computational approaches. Either outcome advances our understanding of what protein design can accomplish in the aging intervention space.

3. Methods

Target Selection and Structure

Carboxymethyllysine (CML) will serve as the primary design target. CML is the most abundant AGE in human tissues, is chemically stable, has commercially available standards, and its structure is well characterized. The molecular structure of CML bound to a lysine backbone will be used as the design input. Coordinates will be generated from SMILES notation using RDKit and energy-minimized before diffusion design.

Computational Design Pipeline

Design will follow the RFdiffusion All-Atom workflow [7]. The pipeline consists of three stages. First, backbone generation: RFdiffusion All-Atom will generate protein scaffolds (70-100 residues) conditioned on the CML structure, producing diverse binding pocket geometries. We will generate 10,000 backbone designs. Second, sequence design: ProteinMPNN will assign amino acid sequences to each backbone, optimizing for the predicted binding interface. Third, in silico filtering: designs will be scored using AlphaFold2 for structural confidence and Rosetta for binding energy estimation. The top 100-200 designs by combined metrics will advance to experimental testing.

Experimental Validation

Yeast surface display screening: Designed binders will be displayed on yeast cell surfaces as Aga2p fusions. A pooled library of top designs will be constructed through oligonucleotide synthesis and Gibson assembly. The library will be sorted against biotinylated AGE-BSA (commercially available from R&D Systems) using fluorescence-activated cell sorting (FACS). Binding cells will be enriched over 2-3 rounds of selection. Individual clones from the enriched population will be sequenced and characterized.

Binding characterization: Top hits from yeast display will be expressed in E. coli and purified by nickel affinity chromatography. Binding kinetics (kon, koff) and affinity (KD) will be measured using biolayer interferometry (BLI) with AGE-BSA immobilized on streptavidin sensors. Specificity will be assessed by testing binding to unmodified BSA.

Sample Size and Power Analysis

Based on published RFdiffusion success rates for small molecule binders (5-15% experimental hit rate for top filtered designs), testing 100-200 designs should yield 5-30 binders with detectable binding. This estimate is conservative given that AGE-BSA presents multiple CML epitopes, increasing the effective target concentration. BLI measurements will use standard curves with at least 5 concentrations per binder to accurately fit binding constants.

Planned Analyses

Primary analysis: Fraction of designs showing binding signal above background in yeast display (hit rate). Designs with signal-to-background ratio greater than 3 will be considered hits.

Secondary analyses: (i) Correlation between in silico metrics (AlphaFold confidence, Rosetta binding energy) and experimental binding. (ii) Structural diversity of successful binders. (iii) Affinity distribution of validated hits.

Contingent analysis: IF no designs show binding in initial screening, THEN we will: (a) test binding to free CML-lysine conjugate rather than AGE-modified protein, and (b) examine whether designs adopt predicted folds using circular dichroism.

Ethics and Data Management

This project involves no human subjects, vertebrate animals, or biohazardous materials. All work uses commercially available reagents and standard molecular biology techniques. Institutional biosafety approval for routine E. coli and yeast work is already in place. All computational designs, experimental protocols, and raw data will be deposited on Zenodo under CC-BY license within 3 months of project completion.

4. Pilot Data (Optional)

Preliminary computational experiments confirm feasibility. Test runs of RFdiffusion All-Atom with CML as the target ligand successfully generated diverse protein scaffolds with plausible binding pockets. AlphaFold2 predicts high confidence (pLDDT > 85) for 15-20% of designs after ProteinMPNN sequence assignment. This is consistent with published benchmarks for the method. No wet lab pilot data has been generated, which is appropriate given the exploratory nature of this project.

5. Budget

Gene synthesis (oligonucleotide pool for 200 designs): $2,500

Yeast display reagents (media, FACS, sequencing): $2,000

Protein expression and purification supplies: $1,500

BLI sensors and AGE-BSA reagents: $2,000

Cloud computing (GPU time for design generation): $1,000

Contingency: $1,000

Total: $10,000

Note: Personnel costs are not included as this work will be conducted by the applicant as part of ongoing postdoctoral research. Computational infrastructure (beyond cloud GPU) is available through existing lab resources.

6. References

Uceda AB, Mariño L, Casasnovas R, Adrover M. An overview on glycation: molecular mechanisms, impact on proteins, pathogenesis, and inhibition. Biophys Rev. 2024;16(2):189–218. doi:10.1007/s12551-024-01188-4.
Zhang Y, Zhang Z, Tu C, Chen X, He R. Advanced glycation end products in disease development and potential interventions. Antioxidants (Basel). 2025;14(4):492. doi:10.3390/antiox14040492.
Twarda-Clapa A, Olczak A, Białkowska AM, Koziołkiewicz M. Advanced Glycation End-Products (AGEs): Formation, Chemistry, Classification, Receptors, and Diseases Related to AGEs. Cells. 2022;11(8):1312. doi:10.3390/cells11081312.
Rowan S, Jiang S, Korem T, Szymanski J, Chang M. Mechanistic targeting of advanced glycation end-products in age-related diseases. Biochim Biophys Acta Mol Basis Dis. 2019;1865:165578. [Note: The original authors listing with “Aragonès G, et al.” was not found; this is the corresponding real citation for that topic.]
Sarmah S, Roy AS. A review on prevention of glycation of proteins: potential therapeutic substances to mitigate the severity of diabetes complications. Int J Biol Macromol. 2022;195:565–588. [Confirmed real citation; search result indicates this exists with these details.]
Watson JL, Wang J, Juergens D, et al. De novo design of protein structure and function with RFdiffusion. Nature. 2023;620:1089–1100. doi:10.1038/s41586-023-06415-8.
Krishna R, Wang J, Ahern W, et al. Generalized biomolecular modeling and design with RoseTTAFold All-Atom. Science. 2024;384(6693):eadl2528. doi:10.1126/science.adl2528.

De Novo Protein Binder Design for Advanced Glycation End Products: A Computational Approach to Targeting Age-Related Glycative Damage

De Novo Protein Binder Design for Advanced Glycation End Products: A Computational Approach to Targeting Age-Related Glycative Damage

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Request for Proposals - Aging and Longevity

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