Four Decades of Hunting the Misfolded Protein: How Prion Research Grew Into a Blueprint for Neurodegenerative Drug Design

When Byron Caughey began his work on prion diseases in the late 1980s, the idea that a protein could replicate, cause disease, and evolve drug resistance without any genetic material seemed almost heretical. Yet over the following four decades, Caughey and his colleagues at the Rocky Mountain Laboratories of the National Institute of Allergy and Infectious Diseases helped transform that heretical notion into one of the most productive frameworks in modern neurodegeneration research. A new review published in Acta Neuropathologica, written by James A. Carroll, Jakub Soukup, Bradley R. Groveman, Christina D. Orrú, Brent Race, and Cathryn L. Haigh, traces this scientific journey and shows how the search for anti-prion therapeutics has repeatedly anticipated, and continues to inform, drug discovery efforts for far more common brain diseases such as Alzheimer’s and Parkinson’s.

The central biological puzzle has never changed. Prion diseases, including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer, are driven by the misfolding of a normal cell-surface protein, the prion protein PrP, into a self-propagating pathogenic conformer known as PrP-Sc. Unlike viruses or bacteria, this infectious entity carries no genome; its biological information is encoded purely in protein conformation. Once a misfolded seed appears, it templates the conversion of the native, protease-sensitive PrP into more of the pathological form, which then aggregates into amyloid fibrils that accumulate in the brain and destroy neurons. Caughey recognized early on that every step of this chain, from the initial conformational conversion to fibril assembly, disassembly, and clearance, represents a potential point of therapeutic attack.

Some of the earliest and most influential work from the Caughey laboratory concerned the structural chemistry of the scrapie-associated protein itself. In 1991, using infrared spectroscopy, Caughey and colleagues demonstrated that the protease-resistant PrP 27-30 core is dominated by beta-sheet secondary structure, a finding that established the conformational difference between the normal and pathological protein and set the stage for decades of structural pharmacology. This was followed by the discovery that sulfated glycosaminoglycans and dyes such as Congo red bind to PrP and block its pathological accumulation, providing the first chemical handles on the conversion process. These studies established a recurring theme: molecules that preferentially interact with amyloid-like surfaces or with the conversion intermediate can inhibit prion propagation, even if their potency in living animals remains limited by permeability and toxicity constraints.

Perhaps the most consequential technical achievement was the development of cell-free conversion assays. In the mid-1990s, Caughey and collaborators showed that aggregates of scrapie-associated PrP could induce the conversion of normal, protease-sensitive PrP to the protease-resistant state in a cell-free system. This single experiment provided powerful biochemical support for the protein-only hypothesis of prion replication and, more practically, created a screening platform. For the first time, researchers could monitor protein misfolding in real time, quantitatively, without infecting animals. The approach matured into real-time quaking-induced conversion, or RT-QuIC, an ultrasensitive amplification assay now used worldwide to detect minute quantities of prion seeding activity in cerebrospinal fluid and other tissues. RT-QuIC has become a clinical diagnostic cornerstone for sporadic and genetic Creutzfeldt-Jakob disease, and it also serves as a critical readout in therapeutic trials, allowing researchers to verify that a candidate treatment genuinely reduces the load of seed-competent misfolded protein.

Armed with these assays, the laboratory systematically screened thousands of drugs and natural products for anti-prion activity. The resulting chemical catalogue is remarkably diverse. Congo red and its analogues, sulfated polyanions, curcumin, synthetic peptides, degenerate phosphorothioate oligonucleotides, and cyclic tetrapyrroles such as porphyrins and phthalocyanines all showed the ability to inhibit protease-resistant PrP formation in cell culture or cell-free systems. Some of these compounds, notably certain porphyrins, extended survival in prion-infected mice after intracerebral challenge, while combination treatments demonstrated that pairing compounds with complementary mechanisms can enhance antiscrapie effects. Equally important was what these experiments taught about failure modes. Work on drug resistance revealed that prions can select for inhibitor-resistant conformations under selective pressure, much as viruses do, meaning that any future therapy must be designed with an eye toward the evolving conformational landscape of the infectious agent.

A second major line of investigation focused on the cell biology of the prion protein. Caughey’s group showed that the scrapie-associated form of PrP derives from a cell surface precursor that is sensitive to both proteases and phospholipases, implicating the plasma membrane as a site of conversion. The glycophosphatidylinositol anchor and the N-linked glycans of PrP were found to influence strain-dependent conformations, linking the molecule’s membrane context to its pathological folding pathway. This cellular perspective suggested therapeutic strategies that go beyond simply blocking conversion: relocating the normal protein substrate away from the compartments where conversion occurs, promoting its removal, or downregulating its expression altogether. Experiments in which neuronal depletion of PrP prevented disease and reversed early spongiform changes in infected mice validated the substrate-targeting logic, and the recent finding that depleting neuronal Ndst1 accelerates prion protein clearance and slows neurodegeneration shows that modifying the protein’s glycan environment remains a viable therapeutic direction.

More recent work from the group has pushed the substrate-targeting strategy into the era of modern nucleic acid therapeutics. Antisense oligonucleotides designed to reduce PrP expression have extended survival in prion-infected mice, and intracerebral infusion studies established proof of concept for delivering these drugs to the central nervous system. Newer reports describe divalent siRNA approaches and the inhibition of the oligosaccharyltransferase complex, which disrupts PrP maturation and effectively treats both rodent and human prions in model systems. Human cerebral organoids have emerged as a clinically relevant screening platform for Creutzfeldt-Jakob disease therapeutics, bridging the gap between cell lines and animal models. Together, these advances suggest that the long-elusive goal of an effective anti-prion treatment may finally be within reach of clinical translation, even though no approved therapy yet exists for these uniformly fatal diseases.

The review also emphasizes how high-resolution structural biology has changed the game. For decades, the infectious prion resisted atomic-level characterization because brain-derived fibrils are heterogeneous and difficult to purify. That barrier has now fallen. Cryo-electron microscopy structures of infectious mammalian prion fibrils, including anchorless RML prions and natural chronic wasting disease fibrils from deer, have revealed parallel in-register intermolecular beta-sheet architectures and defined the conformational motifs that distinguish prion strains. These structures confirm predictions made years earlier by hydrogen-deuterium exchange and other lower-resolution methods, and they open the door to genuine structure-based drug design: small molecules and designed peptides can now be engineered to bind specific pockets or surfaces on the pathogenic fibril rather than discovered by chance. Iterative machine learning approaches, already used to design potent inhibitors of alpha-synuclein and tau aggregation, are being adapted to the prion field, using fibril amplification assays with brain-derived seeds as the feedback loop.

One of the most striking lessons of this four-decade effort is its relevance far beyond the rare diseases that motivated it. Self-propagating misfolded proteins underlie Alzheimer’s disease, Parkinson’s disease, Lewy body dementia, and other common neurodegenerative conditions, and the methodological toolkit built in the prion field, from RT-QuIC and seed amplification assays to structure-guided inhibitor design and substrate-lowering nucleic acid drugs, has been exported almost wholesale to these larger fields. Sensitive detection of misfolded protein seeds in biofluids now enables early diagnosis and objective biomarker monitoring in clinical trials, while drug design pipelines that once screened blindly can target specific fibril polymorphs. The prion concept has also reshaped thinking about biosafety, with rigorous work on disinfectants such as sodium hypochlorite and Wex-cide defining how to inactivate these exceptionally resistant agents in clinical and laboratory settings.

The Caughey story is ultimately a case study in how persistent, mechanistically grounded basic research can convert a scientific pariah into a therapeutic roadmap. By elucidating disease biochemistry, insisting on the need to target conformational change, building assays that measure the pathogenic process directly, and integrating those assays with structural characterization and cell physiology, one laboratory’s quest for prion inhibitors has given neurodegeneration research its most rigorous experimental standards. As antisense oligonucleotides, siRNA platforms, prion vaccines, and structure-based small molecule design converge, the patients and families affected by these devastating diseases have, for the first time in the history of the field, multiple credible paths toward an effective treatment, each one built on foundations laid over forty years of work at the interface of chemistry, structure, and cell physiology.

Subject of Research: Development of therapeutic inhibitors targeting prion protein misfolding through biochemistry, structural biology, and cell physiology

Article Title: Life at the interface: Byron Caughey’s search for prion disease inhibitors through chemistry, structure, and cell physiology

Article References: Life at the interface: Byron Caughey’s search for prion disease inhibitors through chemistry, structure, and cell physiology. (n.d.). https://doi.org/10.1007/s00401-026-03080-9

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03080-9

Keywords: prion diseases, prion protein, PrP-Sc, Congo red, RT-QuIC, antisense oligonucleotides, cryo-EM, drug discovery, neurodegeneration, protein misfolding, Creutzfeldt-Jakob disease, Byron Caughey