Molecularly Imprinted Polymer Nanogels for Protein Recognition: Direct Proof of Specific Binding Sites by Solution STD and WaterLOGSY NMR Spectroscopies

Abstract

Molecularly imprinted polymers (MIPs) are tailor‐made synthetic antibodies possessing specific binding cavities designed for a target molecule. Currently, MIPs for protein targets are synthesized by imprinting a short surface‐exposed fragment of the protein, called epitope or antigenic determinant. However, finding the epitope par excellence that will yield a peptide “synthetic antibody” cross‐reacting exclusively with the protein from which it is derived, is not easy. We propose a computer‐based rational approach to unambiguously identify the “best” epitope candidate. Then, using Saturation Transfer Difference (STD) and WaterLOGSY NMR spectroscopies, we prove the existence of specific binding sites created by the imprinting of this peptide epitope in the MIP nanogel. The optimized MIP nanogel could bind the epitope and cognate protein with a high affinity and selectivity. The study was performed on Hepatitis A Virus Cell Receptor‐1 protein, also known as KIM‐1 and TIM‐1, for its ubiquitous implication in numerous pathologies.

Computationally driven discovery of SARS-CoV-2 Mproinhibitors: from design to experimental validation

“…All the NMR screening experiments were performed with a Bruker Neo 600 MHz spectrometer, equipped with a nitrogen cooled 5 mm Prodigy CryoProbe at 298 K. The ligand binding was monitored by WaterLOGSY (wLogsy) 59 and Saturation Transfer Difference (STD) 60 experiments in the presence and in the absence of the protein. Samples contained 10 μM M pro and 100 μM to 2 mM ligand dissolved in 150 mM NaCl, 20 mM phosphate, 5% D 2 O, and 4% DMSO-d6 (pH = 7.3).…”

Molecularly Imprinted Polymer Hydrogel Nanoparticles: Synthetic Antibodies for Cancer Diagnosis and Therapy

“…Click chemistry is advantageous because it enables the immobilization of the template in such a way to leave the functional groups essential for recognition, accessible during the imprinting process. Once the template is covalently immobilized, different protocols are practiced by different laboratories concerning the choice of the pre‐polymerization composition, type of initiation, and the way to elute the high affinity MIP‐NGs [4,5a,32,42–44] . The protocol developed in our group which has given reproducible results is detailed below.…”

“…The highly diluted pre‐polymerization mixture (total monomer concentration of 0.5 %), typically consists of the functional monomers NIPAM as the major component (>80 % mol percentage) which provides hydrogen bonding and confers thermoresponsiveness, TBAM to target hydrophobic groups, 4‐acrylamidophenyl(amino)‐methaniminium acetate (AB, benzamidine‐based monomer) to target acidic moieties or 2‐(trifluoromethyl)acrylic acid, to target basic moieties and N ‐phenylacrylamide (PAA) to form Π–Π interactions. The monomers are added at varying molar amounts, depending on the amino acids present, for a peptide template for instance [4,15,32] . Low amount (5 % mol percentage) of BIS is used as crosslinker.…”

“…The monomers are added at varying molar amounts, depending on the amino acids present, for a peptide template for instance. [4,15,32] Low amount (5 % mol percentage) of BIS is used as crosslinker. The polymer is synthesized in the collapsed state, at 37 °C, a temperature above the lower critical solution temperature (LCST) of poly(NIPAM), i. e. 32 °C.…”

“…This strategy, inspired by nature, consists of imprinting a short surface-exposed fragment called epitope (antigenic determinant of a protein that binds to an antibody). [27][28][29][30] The resulting MIP is able to recognize the epitope, as well as the protein or glycan, [31][32][33][34] from which it is derived. When the MIPs are loaded with drugs, the latter can be released at the tumor site.…”