They have also been used as immunogens to raise oligomer-selective antibodies in Alzheimer’s disease using the method that we describe here ( 13, 14). They have been increasingly used as therapeutics, often as small molecule drugs to bind targets ( 11, 12). We computationally model some key aspects of the oligomer ensemble using molecular dynamics, in order to predict disease-specific epitopes.Ĭyclic peptides or macrocycles are polymers that have been conjugated to form a ring-like topology ( Fig. Many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE) and amyotropic lateral sclerosis (ALS), spread throughout the brain via a prion-like mechanism involving soluble oligomers ( 1, 4 – 10) Soluble oligomers contain roughly 4-40 chains of protein, which exist in a misfolded conformational ensemble that is conformationally labile and very difficult to experimentally characterize. For example, a peptide consisting of a contiguous fragment of native primary sequence tethered to an immunogen such as keyhole limpet hemocyanin (KLH) will likely exhibit overlap in its presented ensemble with the ensemble of isolated native monomer. Raising an antibody that avoids the majority of diverse conformations of an intrinsically-disordered protein’s ensemble is a challenge. Examples include amyloid- β (A β), tau protein, α-synuclein, and the low-complexity domains in FUS and TDP43. Since the primary sequences of healthy and aberrant protein are generally the same, barring splice variants and perhaps post-translational modifications, the efficacy of an antibody is then due to its selective preference for binding to a misfolded conformation over healthy in-vivo “native” conformations.įor many proteins involved in misfolding disease however, the native conformational ensemble is intrinsically disordered ( 3). For this class of diseases, an effective antibody must be able to spare healthy protein and discriminate misfolded protein species that lead to molecular and cellular pathology ( 1). Nowhere has conformational-selectivity been more important to immunotherapies than in protein-misfolding diseases ( 2). Both primary sequence and conformation of the epitope determine the particular protein morphologies to which the resulting antibodies will be selective. Ensemble comparison and overlap was quantified by the Jensen-Shannon Divergence, and a new measure introduced here-the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble, and which may be a more useful measure in sculpting the conformational-selectivity of an antibody.Ī key step in the development of a therapeutic antibody or active vaccine is the immunization strategy ( 1), namely, the choice of protein epitope and how it will be presented to an animal or human immune system. Different cyclic peptide scaffolds with variable numbers of glycines can have markedly different conformational ensembles. We introduce a method for screening against structured off-pathway targets in the human proteome, by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model, and isolated monomer ensembles. To this end, we scaffolded epitopes in cyclic peptides by varying the number of flanking glycines, to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. Effectively scaffolding epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.
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