Abiotic Synthetic Antibodies to Target a Specific Protein Domain and Inhibit Its Function

Abstract

The Bacillus thuringiensis (Bt) Cry proteins are widely used in insect pest control. Despite their economic benefits, remaining concerns over potential ecological and health risks warrant their ongoing surveillance. Affinity reagents, most often antibodies, protein scaffolds, and aptamers, are the traditional tools used for protein binding and detection. We report a synthetic antibody (SA) alternative to traditional biological affinity reagents for binding Bt Cry proteins. Analysis of hotspots of the Bt Cry protein–insect midgut cadherin-like receptor complexes was used for the design of the SA. The SA was selected from a small focused library of hydrogel copolymers containing functional monomers complementary to key exposed hotspots of Bt Cry proteins. A directed chemical evolution identified a SA, APhe-NP23, with affinity and selectivity for Bt Cry1Ab/Ac proteins. The putative intermolecular polymer–protein interfaces were identified by the SA’s uptake of Bt Cry1Ac pepsin hydrolysates, binding epitope mutation studies, and protein–protein inhibition studies of the toxin binding to its native insect receptor binding domains. The SA inhibitor binds to the same protein domains as the insect’s cadherin-like receptors, Bt-R1 and SeCad1b. The SA binds rapidly to Bt Cry1Ab/Ac with high capacity, is pH-responsive, and is synthesized reproducibly. We believe that a hotspot-directed approach is general for creation of abiotic protein affinity reagents that target functional protein domains. Affinity ligands are typically high-information content biologicals. Their structure and function are determined from their amino acid or oligo sequence. In contract, the SA described in this work is a statistical copolymer that lacks sequence specificity. These results are an important contribution to the concept that randomness and biospecificity are not mutually exclusive.

Poly(N-acryloylglycine-acrylamide) Hydrogel Mimics the Cellular Microenvironment and Promotes Neurite Growth with Protection from Oxidative Stress

“…In leading pathophysiological conditions, a change in the intracellular pH in neurons is correlated with neurodegenerative diseases; brain acidosis mediates neuronal death; AD, HD, PD, and cerebral alkalosis lead to ischemic stroke; , and N -acyloylation of amino acids could favor the development of pendant polymers and the charge distribution in a stereoselective configuration along with a higher gelling property. , Due to the presence of carboxylic acid groups (−COOH) and amide groups (−CO-NH) in N -acryloyl-based polymeric chains (Figures S1–S3), pH- and temperature-responsive behaviors in aqueous medium are exhibited. Hence, it could be a good candidate for the loading of various therapeutic molecules (Figure a) and useful in various brain physiopathological conditions and for neurite growth.…”

“…It is observed that amino acid-based polymers are biologically active and helpful for the release of fibroblast growth factors (FGFs) and promote toxin binding activities and protein–protein inhibition activities. , Amino acid-derived acrylic polymers such as poly­(acrylic acid), poly acrylamide, and poly­( N -acryloyl glycinamide) have been studied for their upper critical solution temperature . Poly­( N -acryloyl glycinamide) possesses a gelling property and robust adhesion behavior, promotes wound healing efficacy, and exhibits osteo-regeneration properties .…”

Abiotic Synthetic Antibody Inhibitor with Broad-Spectrum Neutralization and Antiviral Efficacy against Escaping SARS-CoV-2 Variants

“…Synthetic hydrogel polymer nanoparticles (NPs) contain statistical distributions of functional monomers. , Although lacking sequence specificity, hydrogel polymer NPs can be formulated with broad chemical diversity by incorporating various combinations and ratios of functional monomers. , This would enable the acquisition of abiotic synthetic antibodies with peptide/protein affinity and selectivity through a simple selection process referred to as directed chemical evolution. , In addition to specific affinities, synthetic polymer antibodies have many outstanding features, including facile synthesis and functionalization, low production cost, flexible conformation, high stability and robustness, good biocompatibility, stimuli-responsive catch and release, and tunable size and molecular weight, enabling their application in various fields, including toxin sequestration, ,− clinical diagnostics and therapy, , protein purification, ,, affinity precipitation, and heat protection . Earlier polymer NP libraries are tedious, time-consuming, and do not always result in abiotic synthetic antibodies with inherent affinity and specificity.…”

“…This rational approach facilitates the discovery of abiotic synthetic antibodies by the construction and screen of a small focused synthetic polymer NP library that incorporates monomers with functionalities complementary to a critical receptor-binding domain of the target protein. ,,, Polymer candidates selected from this strategy were found to have biomimetic properties of the target protein counterparts. Successful examples include engineered polymer affinity ligands that mimic the heparin, a protein counterpart of the vascular endothelial growth factor, the insect midgut cadherin-like receptors of Bacillus thuringiensis insecticidal crystal proteins, , and the endogenous tissue inhibitors of snake venom metalloproteinases . These limited but successful findings provide a general approach for the discovery of abiotic polymer inhibitors, that is, rational design inspired by molecular details between the target proteins and their receptors, antibodies, or other counterparts.…”

“…Recently, a molecular level understanding of the antigen–receptor/antibody interactions provides insight for the design of therapeutic inhibitors targeting an important protein domain involved in receptor/antibody binding. This rational approach facilitates the discovery of abiotic synthetic antibodies by the construction and screen of a small focused synthetic polymer NP library that incorporates monomers with functionalities complementary to a critical receptor-binding domain of the target protein. ,,, Polymer candidates selected from this strategy were found to have biomimetic properties of the target protein counterparts. Successful examples include engineered polymer affinity ligands that mimic the heparin, a protein counterpart of the vascular endothelial growth factor, the insect midgut cadherin-like receptors of Bacillus thuringiensis insecticidal crystal proteins, , and the endogenous tissue inhibitors of snake venom metalloproteinases .…”