De novo designed protein inhibitors of amyloid aggregation and seeding

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer’s disease (AD) and alpha-synuclein (αSyn) in Parkinson’s disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, “capping” them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

The aberrant aggregation of proteins into amyloid fibrils is a hallmark of many neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) (1). In AD, amyloid-β (Aβ) and tau amyloid fibrils comprise the extracellular amyloid plaques and intracellular neurofibrillary tangles, respectively, characteristic of disease progression (2). Likewise, intracellular Lewy bodies found in the neurons of patients with PD and dementia with Lewy bodies (DLB) are primarily composed of αSyn fibrils (3). There are currently no therapies capable of significantly slowing or stopping the progression of any of these diseases, and inhibition of fibril formation has become a major target for therapeutic development (4, 5). Amyloid fibrils are composed of repeating layers of β-strand–rich protein monomers stacked upon each other, forming β-sheets. The β-sheets interdigitate to form a stable fibril core through interactions known as steric zippers (6). Antiamyloid therapies have typically focused on small molecules that prevent aggregation or dissociate preexisting aggregates and antibodies that promote fibril clearance (7–9). An alternative approach is the design of molecules that bind to the ends of the growing fibrils, capping their growth and preventing the further addition of more protein monomers. This approach has been successfully used to design peptide-based inhibitors of tau, Aβ, and αSyn aggregation (10–14). This design strategy considers the atomic structures of fibrils, employing rational and computational design techniques to derive a peptide sequence complementary to the growing fibril surface.Since the initial designs of structure-based capping inhibitor peptides, many advances have been made in both the determination of amyloid protein structure, as well as in methods of protein design. The first atomic-resolution structures of amyloid fibrils determined by X-ray crystallography were restricted to small peptide segments ∼6 to 11 amino acids in length (15). The recent advent of cryoelectron microscopy (cryo-EM), microelectron diffraction (MicroED), and solid-state NMR (ssNMR) spectroscopy have enabled the determination of amyloid protein structures that were previously unsolvable (16–18). These techniques have been used to solve an ever-growing list of structures of both recombinantly derived fibrils (19–21) as well as fibrils directly extracted from patient tissue (22–29). These structures have provided key insights into fibril architecture and polymorphism in relation to disease.Like the structural knowledge of amyloid fibrils, the toolbox of protein structure prediction and design has been rapidly expanding in recent years (30, 31). Significant advances in algorithms and computing power have facilitated the de novo design of proteins with a variety of properties and functions, ranging from stability, pH sensitivity, to even logic operations, with vast potential for use in therapeutics, diagnostics, etc (32–36). While the underlying design principles of de novo generated proteins are becoming well established, examples of their direct application into biological systems are still limited. In this work, we use de novo protein design to create 35 to 50 residue miniproteins that bind to the growing ends of tau, αSyn, and Aβ fibrils. We target recently determined full-length atomic structures of each amyloid protein in our designs to generate miniproteins capable of inhibiting aggregation, seeding, and toxicity both in vitro and in vivo.