High-throughput discovery of fluoroprobes that recognize amyloid fibril polymorphs

nature chemistry Article https://doi.org/10.1038/s41557-025-01889-7 High-throughput discovery of fluoroprobes that recognize amyloid fibril polymorphs Received: 2 September 2024 Accepted: 30 June 2025 Published online: xx xx xxxx Check for updates Emma C. Carroll 1,2, Hyunjun Yang 2,3,4, Wyatt C. Powell 2, Annemarie F. Charvat 2, Abby Oehler2, Julia G. Jones2, Kelly M. Montgomery2, Anthony Yung2, Zoe Millbern5, Alexander I. P. Taylor 6, Martin Wilkinson 6, Neil A. Ranson 6, Sheena E. Radford 6, Nelson R. Vinueza5, William F. DeGrado3, Daniel A. Mordes2,7, Carlo Condello 2,8 & Jason E. Gestwicki 2,3 Aggregation of microtubule-associated protein tau into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes) such as thioflavin T have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here we leverage advances in protein-adaptive differential scanning fluorimetry to screen the Aurora collection of 300+ fluoroprobes against multiple synthetic fibril polymorphs, including those formed from tau, α-synuclein and islet amyloid polypeptide. This screen—coupled with excitation-multiplexed bright-emission recording (EMBER) imaging and orthogonal secondary assays—revealed pan-fibril-binding chemotypes, as well as fluoroprobes selective for fibril subsets. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer’s disease and rodent models. We propose that these scaffolds represent entry points for developing fibril-selective ligands. Tau is an intrinsically disordered, microtubule-binding protein that assembles into β-sheet rich fibrils within the neurons of patients suffering from a family of devastating neurodegenerative diseases, known as tauopathies1–3. In Alzheimer’s disease—one of the most common tauopathies—these tau fibrils are characterized as either straight filaments or paired helical filaments4, suggesting that the same protein might be able to form fibrils with distinct molecular structures. Indeed, cryo-electron microscopy experiments have revealed that the core structure of patient-derived tau fibrils adopts different molecular conformations or folds5, referred to here as polymorphs. Interestingly, these fibril polymorphs seem to be disease-specific; tau fibril structures differ between some clinically distinct tauopathies but are recapitulated in patients with the same disease6. Together, these observations have driven interest in the development of optical reagents that can rapidly discriminate between tau fibril polymorphs. Organic dyes have for many decades been essential tools for studying amyloid fibrils7,8. The power of these reagents is that their spectral properties change when they are bound to fibrils, making them relatively straightforward to use. For example, the most widely used fluorescent probe (fluoroprobe) is thioflavin T (ThT) and its fluorescence Department of Chemistry, San José State University, San José, CA, USA. 2Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA. 3Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA. 4Department of Biochemistry, Brandeis University, Waltham, MA, USA. 5Department of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC, USA. 6Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK. 7Department of Pathology, University of California San Francisco, San Francisco, CA, USA. 8Department of Neurology, University of California San Francisco, San Francisco, CA, USA. e-mail: 1 Nature Chemistry Article intensity is dramatically increased when bound to amyloids7,9–12. Likewise, fluoroprobes based on Congo Red, curcumin, polythiophenes and other scaffolds13–18 have proven to be convenient tools for studying how fibrils form in vitro and in cells and tissues. Fluoroprobes have also been used as competitors to identify non-fluorescent compounds by displacement19. Although fluoroprobes have played critical roles in studying tauopathies, they typically lack specificity for different fibril polymorphs20,21. Indeed, their generality is often a great strength because a single fluoroprobe such as ThT has the versatility to detect a wide range of fibrils, largely independent of sequence or substructures. Yet the field would also benefit from complementary fluoroprobes that are selective for subsets of tau polymorphs. Most amyloid ligands have been generated by creating close structural analogues of established amyloid-binding scaffolds such as ThT or curcumin14,22,23. Although those efforts are often successful in producing analogues with improved properties such as brightness or permeability, they do not typically involve sampling of a wide range of chemical space. We hypothesized that more diverse starting points might be uncovered by screening larger dye collections containing a greater variety of chemical scaffolds. We saw an opportunity to address this persistent challenge in the recent development of a protein-adaptative differential scanning fluorimetry (paDSF)-based platform that leverages the Aurora collection of 300+ chemically diverse dyes24. To test this idea, we produced tau fibrils formed from either wild type (WT) or the P301S point mutation in MAPT. This mutation is linked to frontotemporal dementia; our data, and the work of others25–28, have shown that tau containing the P301S (or the related P301L) mutation feature distinct fibril structures. To further diversify the structure(s) of these fibrils, we also varied the polyanion used to induce in vitro tau aggregation reactions, as it has recently been shown that the identity of the polyanion inducer also contributes to fibril structure29–31. We then screened each of these fibril samples against the Aurora collection using fluorescence-based paDSF in 384-well plates and validated the resulting dye hits using two orthogonal secondary assays: multidimensional spectral confocal microscopy and kinetic aggregation assays. Using this workflow we found that a subset of the hit molecules bound most of the tau polymorphs (that is, pan-fibril binders), whereas others were relatively specific to subsets of fibril conformers (that is, selective fibril binders). These molecules included compounds with coumarin and polymethine scaffolds, chemotypes that are under-represented in the field of amyloid-binding dyes32,33, as well as chemotypes not previously associated with amyloid recognition. To demonstrate the generality of this screening workflow, we also performed paDSF assays on fibrils composed of α-synuclein and islet amyloid polypeptide (IAPP), again (...truncated)