Modulating the Folding Landscape of Superoxide Dismutase 1 with Targeted Molecular Binders

Amyotrophic lateral sclerosis, or Lou Gehrig's disease, is characterized by motor neuron death with average survival times of 2 ‐ 5 years. One cause of this disease is the misfolding of superoxide dismutase 1 (SOD1), a protein whose stability and aggregation propensity are affected by point mutations spanning the protein. Here, we use an epitope‐specific, high‐throughput screen to identify peptides that both stabilize the native conformation of SOD1 as well as accelerate its folding by 2.5‐fold. Ligands targeted to the electrostatic loop on the periphery of the protein tightened the non‐metalated structure and accelerated its folding. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies

Modulating SOD1 Folding Landscapes with Targeted Molecular Binders

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the deterioration of motor neurons that abates essential biological functions and exhibits survival times of 3 - 5 years after diagnosis. One driver of this disease derives from inherited mutations to the protein superoxide dismutase 1 (SOD1), which hinder proper folding and result in the accumulation of toxic aggregates. We identified cyclic peptides that target precise epitopes on SOD1 through an emerging screening platform that furnishes high-affinity binders against regions of a protein independent of secondary or tertiary structure. Binding these epitopes both stabilizes the native state and accelerates folding. In this context, these small peptides function as molecular chaperones and mitigate the impact of deleterious mutations to SOD1. They also display the traditional benefits of small molecules, such as straightforward chemical modifications and long-term stability. Overall, this method provides a route to rationally perturb the energy landscape of any protein through noncovalent binding, making it useful in fundamental studies of protein folding as well as designing therapeutics for misfolding diseases

Bulk Synthesis of Exfoliated Two-Dimensional Polymers Using Hydrazone-Linked Covalent Organic Frameworks

Two-dimensional (2D) polymers assemble organic subunits into covalently linked, high-aspect-ratio networks with long-range order. Despite recent advances in 2D polymerization, scalable and general methods to access few- and single-layer materials are limited. Here we exfoliate a hydrazone-linked covalent organic framework (COF) to yield bulk quantities of few-layer two-dimensional (2D) polymers. Immersing the COF powder in several laboratory solvents exfoliates and disperses thin COF-43 samples, which maintain their characteristic periodic hexagonal structure. This phenomenon was characterized using infrared spectroscopy, dynamic light scattering, atomic force microscopy, transmission electron microscopy, and selected area electron diffraction. 2D COFs with reduced interlayer interaction energies offer a new means to access high-aspect-ratio 2D polymers whose structure may be designed using established principles of COF synthesis

Molecular Modulation of Protein Energy Landscapes

Protein catalyzed capture agents are an emerging class of oligopeptides that combine the benefits of small molecules and antibodies to furnish ligands with picomolar binding affinity, serum stability, and cell permeability. Their identification involves screening a synthetic, alkyne-functionalized epitope from a target protein against a library of cyclic peptides bearing terminal azides. We identified ligands that bind regions of superoxide dismutase 1 (SOD1), a protein that misfolds to cause amyotrophic lateral sclerosis (ALS), consistently destabilized upon mutation. Treatment of the disease is challenging because there are over 180 heritable mutations of SOD1 and virtually no well-defined binding sites addressable by traditional ligand identification strategies. These mutations ultimately cause the protein to adopt toxic conformations that aggregate and damage cellular functions within the central nervous system. PCC agents targeting regions consistently destabilized across several mutations bind and stabilize its native conformation. We characterized the impact of binding, both on the ground state stability of several mutants as well as the kinetics of SOD1 folding and denaturation

Molecular Modulation of Protein Energy Landscapes

Protein catalyzed capture agents are an emerging class of oligopeptides that combine the benefits of small molecules and antibodies to furnish ligands with picomolar binding affinity, serum stability, and cell permeability. Their identification involves screening a synthetic, alkyne-functionalized epitope from a target protein against a library of cyclic peptides bearing terminal azides. We identified ligands that bind regions of superoxide dismutase 1 (SOD1), a protein that misfolds to cause amyotrophic lateral sclerosis (ALS), consistently destabilized upon mutation. Treatment of the disease is challenging because there are over 180 heritable mutations of SOD1 and virtually no well-defined binding sites addressable by traditional ligand identification strategies. These mutations ultimately cause the protein to adopt toxic conformations that aggregate and damage cellular functions within the central nervous system. PCC agents targeting regions consistently destabilized across several mutations bind and stabilize its native conformation. We characterized the impact of binding, both on the ground state stability of several mutants as well as the kinetics of SOD1 folding and denaturation

Noncovalent modulation of protein energy landscapes with an emerging class of cyclic-peptide chaperones

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the deterioration of motor neurons that abates essential biol. functions and exhibits survival times of 3 - 5 years after diagnosis. One driver of this disease derives from inherited mutations to the protein superoxide dismutase 1 (SOD1), which hinder proper folding and result in the accumulation of toxic aggregates. We identified cyclic peptides that target precise epitopes on SOD1 through a new high-throughput screening platform that furnishes high-affinity binders against regions of a protein independent of secondary and tertiary structure. Binding these epitopes both stabilizes the native state and accelerates folding. In this context, these small peptides function as mol. chaperones and mitigate the impact of deleterious mutations to SOD1. They also display the traditional benefits of small mols., such as straightforward chem. modifications and long-term stability. Overall, this method provides a route to rationally perturb the energy landscape of any protein through noncovalent binding, making it useful in fundamental studies of protein folding as well as designing therapeutics for misfolding diseases