ABSTRACT
The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human ß-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.
Subject(s)
HumansABSTRACT
Amyloid fibrils are characteristic of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. While different diseases may have fibrils formed of the same protein, the supramolecular morphology of these fibrils is disease-specific. Here, a method is reported to distinguish eight morphologically distinct amyloid fibrils based on differences in ligand binding properties. Eight fibrillar polymorphs of α-synuclein (αSyn) were investigated: five generated de novo using recombinant αSyn and three generated using protein misfolding cyclic amplification (PMCA) of recombinant αSyn seeded with brain homogenates from deceased patients diagnosed with Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). Fluorescence binding assays were carried out for each fibril using a toolkit of six different ligands. The fibril samples were separated into five categories based on a binary classification of whether they bound specific ligands or not. Quantitative binding measurements then allowed every fibrillar polymorph to be uniquely identified, and the PMCA fibrils derived from PD, MSA, and DLB patients could be unambiguously distinguished. This approach constitutes a novel and operationally simple method to differentiate amyloid fibril morphologies and to identify disease states using PMCA fibrils obtained by seeding with patient samples.
Subject(s)
Amyloid , Parkinson Disease , alpha-Synuclein , alpha-Synuclein/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/analysis , Humans , Parkinson Disease/metabolism , Parkinson Disease/diagnosis , Amyloid/metabolism , Amyloid/analysis , Ligands , Multiple System Atrophy/metabolism , Multiple System Atrophy/diagnosis , Lewy Body Disease/metabolism , Lewy Body Disease/diagnosis , Brain/metabolismABSTRACT
The accumulation of amyloid fibrils is characteristic of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. Detecting these fibrils with fluorescent or radiolabelled ligands is one strategy for diagnosing and better understanding these diseases. A vast number of amyloid-binding ligands have been reported in the literature as a result. To obtain a better understanding of how amyloid ligands bind, we have compiled a database of 3457 experimental dissociation constants for 2076 unique amyloid-binding ligands. These ligands target Aß, tau, or αSyn fibrils, as well as relevant biological samples including AD brain homogenates. From this database significant variation in the reported dissociation constants of ligands was found, possibly due to differences in the morphology of the fibrils being studied. Ligands were also found to bind to Aß(1-40) and Aß(1-42) fibrils with similar affinities, whereas a greater difference was found for binding to Aß and tau or αSyn fibrils. Next, the binding of ligands to fibrils was shown to be largely limited by the hydrophobic effect. Some Aß ligands do not fit into this hydrophobicity-limited model, suggesting that polar interactions can play an important role when binding to this target. Finally several binding site models were outlined for amyloid fibrils that describe what ligands target what binding sites. These models provide a foundation for interpreting and designing site-specific binding assays.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Humans , Amyloid beta-Peptides/metabolism , Protein Aggregates , Amyloid/chemistry , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Amyloidogenic ProteinsABSTRACT
The presence of amyloid fibrils is a characteristic feature of many diseases, most famously neurodegenerative disease. The supramolecular structure of these fibrils appears to be disease-specific. Identifying the unique morphologies of amyloid fibrils could, therefore, form the basis of a diagnostic tool. Here we report a method to characterize the morphology of α-synuclein (αSyn) fibrils based on profiling multiple different ligand binding sites that are present on the surfaces of fibrils. By employing various competition binding assays, seven different types of binding sites were identified on four different morphologies of αSyn fibrils. Similar binding sites on different fibrils were shown to bind ligands with significantly different affinities. We combined this information to construct individual profiles for different αSyn fibrils based on the distribution of binding sites and ligand interactions. These results demonstrate that ligand-based profiling can be used as an analytical method to characterize fibril morphologies with operationally simple fluorescence binding assays.
Subject(s)
Neurodegenerative Diseases , alpha-Synuclein , Humans , alpha-Synuclein/chemistry , Ligands , Amyloid/chemistry , Binding SitesABSTRACT
Fibrillar protein aggregates are characteristic of neurodegenerative diseases but represent difficult targets for ligand design, because limited structural information about the binding sites is available. Ligand-based virtual screening has been used to develop a computational method for the selection of new ligands for Aß(1-42) fibrils, and five new ligands have been experimentally confirmed as nanomolar affinity binders. A database of ligands for Aß(1-42) fibrils was assembled from the literature and used to train models for the prediction of dissociation constants based on chemical structure. The virtual screening pipeline consists of three steps: a molecular property filter based on charge, molecular weight, and logP; a machine learning model based on simple chemical descriptors; and machine learning models that use field points as a 3D description of shape and surface properties in the Forge software. The three-step pipeline was used to virtually screen 698 million compounds from the ZINC15 database. From the top 100 compounds with the highest predicted affinities, 46 compounds were experimentally investigated by using a thioflavin T fluorescence displacement assay. Five new Aß(1-42) ligands with dissociation constants in the range 20-600 nM and novel structures were identified, demonstrating the power of this ligand-based approach for discovering new structurally unique, high-affinity amyloid ligands. The experimental hit rate using this virtual screening approach was 10.9%.
Subject(s)
Amyloidogenic Proteins , Software , Ligands , Binding Sites , Protein BindingABSTRACT
The development of an iterative one-pot peptide ligation strategy is described that capitalises on the rapid and efficient nature of the diselenide-selenoester ligation reaction, together with photodeselenisation chemistry. This ligation strategy hinged on the development of a novel photolabile protecting group for the side chain of selenocysteine, namely the 7-diethylamino-3-methyl coumarin (DEAMC) moiety. Deprotection of this DEAMC group can be effected in a mild, reagent-free manner using visible light (λ = 450 nm) without deleterious deselenisation of selenocysteine residues, thus enabling a subsequent ligation reaction without purification. The use of this DEAMC-protected selenocysteine in iterative DSL chemistry is highlighted through the efficient one-pot syntheses of 60- and 80-residue fragments of mucin-1 as well as apolipoprotein CIII in just 2-4 hours.
ABSTRACT
A key process in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases is the aggregation of proteins to produce fibrillary aggregates with a cross ß-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aß, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson's disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.
ABSTRACT
Peptide ligation chemistry has revolutionized protein science by providing access to homogeneously modified peptides and proteins. However, lipidated polypeptides and integral membrane proteins-an important class of biomolecules-remain enormously challenging to access synthetically owing to poor aqueous solubility of one or more of the fragments under typical ligation conditions. Herein we describe the advent of a reductive diselenide-selenoester ligation (rDSL) method that enables efficient ligation of peptide fragments down to low nanomolar concentrations, without resorting to solubility tags or hybridizing templates. The power of rDSL is highlighted in the efficient synthesis of the FDA-approved therapeutic lipopeptide tesamorelin and palmitylated variants of the transmembrane lipoprotein phospholemman (FXYD1). Lipidation of FXYD1 was shown to critically modulate inhibitory activity against the Na+/K+ pump.
Subject(s)
Peptides/chemistry , Selenium Compounds/chemistry , Esters/chemistry , Light , Oxidation-ReductionABSTRACT
Native chemical ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by chemical synthesis. Herein, we outline the use of flow chemistry for the ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL-desulfurization methods. The power of the new ligation-photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.