Rapid Binding Kinetics Research Articles

Impact of Nonnative Interactions on the Binding Kinetics of Intrinsically Disordered p53 with MDM2: Insights from All-Atom Simulation and Markov State Model Analysis.

Intrinsically disordered proteins (IDPs) lack a well-defined tertiary structure but are essential players in various biological processes. Their ability to undergo a disorder-to-order transition upon binding to their partners, known as the folding-upon-binding process, is crucial for their function. One classical example is the intrinsically disordered transactivation domain (TAD) of the tumor suppressor protein p53, which quickly forms a structured α-helix after binding to its partner MDM2, with clinical significance for cancer treatment. However, the contribution of nonnative interactions between the IDP and its partner to the rapid binding kinetics, as well as their interplay with native interactions, is not well understood at the atomic level. Here, we used molecular dynamics simulation and Markov state model (MSM) analysis to study the folding-upon-binding mechanism between p53-TAD and MDM2. Our results suggest that the system progresses from the nascent encounter complex to the well-structured encounter complex and finally reaches the native complex, following an induced-fit mechanism. We found that nonnative hydrophobic and hydrogen bond interactions, combined with native interactions, effectively stabilize the nascent and well-structured encounter complexes. Among the nonnative interactions, Leu25p53-Leu54MDM2 and Leu25p53-Phe55MDM2 are particularly noteworthy, as their interaction strength is close to the optimum. Evidently, strengthening or weakening these interactions could both adversely affect the binding kinetics. Overall, our findings suggest that nonnative interactions are evolutionarily optimized to accelerate the binding kinetics of IDPs in conjunction with native interactions.

Influence of expression and purification protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles.

Heterotrimeric G proteins are a class of signal transduction complexes with broad roles in human health and agriculturally important plant traits. In the classic paradigm, guanine nucleotide binding to the Gα subunit regulates the activation status of the complex. Using the Arabidopsis thaliana Gα subunit, GPA1, we developed a rapid StrepII-tag mediated purification method that facilitates isolation of protein with increased enzymatic activities as compared to conventional methods, and is demonstrably also applicable to mammalian Gα subunits. We subsequently utilized domain swaps of GPA1 and human GNAO1 to demonstrate the instability of recombinant GPA1 is a function of the interaction between the Ras and helical domains, and can be partially uncoupled from the rapid nucleotide binding kinetics displayed by GPA1.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this β-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based β-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

Natural selection of carotenoid binding in Gloeobacter rhodopsin

Energy transfer from the 4-ketocarotenoid antenna (salinixanthin) and echinenone to xanthorhodopsin has been confirmed in Salinibacter ruber and Gloeobacter violaceus, respectively. Hundreds of carotenoids and numerous rhodopsin homologs are spreading across the life domain. Here, we found that ketocarotenoid binds to Gloeobacter rhodopsin (GR), suggesting an increased likelihood of the existence of the carotenoid-rhodopsin complex. Additionally, the recruitment of the secondary chromophore antenna is precisely controlled by rhodopsin. Therefore, comparing the binding of multiple carotenoids is of interest to GR. Particular attention was paid to the structural interaction of ligands within the protein binding pocket. While different degrees of carotenoid association are evident in the GR-carotenoid experimental data and molecular docking results, the retinal isomerization dynamics defined the relative orientation of the β-ionone ring, and structural changes in the protein acted as modest barriers for the best carotenoid for GR. Notably, the selection of carotenoid binding can be summarized in three points: (1) structural matching of a ligand molecule with the binding pocket; (2) strong binding affinity and rapid binding kinetics; (3) dynamic flexibility that is versatile to rhodopsin activation. The selected carotenoids showed significant enhancement in light-driven proton pumping as extra light-harvesting antennae and provided intrinsic protection from stress.

Silicone-Based Lubricant-Infused Slippery Coating Covalently Bound to Aluminum Substrates for Underwater Applications.

Wetting of solid surfaces is crucial for biological and industrial processes but is also associated with several harmful phenomena such as biofouling and corrosion that limit the effectiveness of various technologies in aquatic environments. Despite extensive research, these challenges remain critical today. Recently, we have developed a facile UV-grafting technique to covalently attach silicone-based coatings to solid substrates. In this study, the grafting process was evaluated as a function of UV exposure time on aluminum substrates. While short-time exposure to UV light results in the formation of lubricant-infused slippery surfaces (LISS), a flat, nonporous variant of slippery liquid-infused porous surfaces, longer exposure leads to the formation of semi-rigid cross-linked polydimethylsiloxane (PDMS) coatings, both covalently bound to the substrate. These coatings were exposed to aquatic media to evaluate their resistance to corrosion and biofouling. While the UV-grafted cross-linked PDMS coating effectively inhibits aluminum corrosion in aquatic environments and allows organisms to grow on the surface, the LISS coating demonstrates improved corrosion resistance but inhibits biofilm adhesion. The synergy between facile and low-cost fabrication, rapid binding kinetics, eco-friendliness, and nontoxicity of the applied materials to aquatic life combined with excellent wetting-repellent characteristics make this technology applicable for implementation in aquatic environments.

Magnetic hydrothermal carbonaceous nanospheres bonded cell membranes as a stable and reusable platform for discovering natural bioactive components

The covalent bionic modifications of hydrothermal carbonaceous nanomaterials (HCNs) are of great potential in extraction and separation engineering because they can avoid present disadvantages of chemical functionalization and achieve selective recognition capacity with stability and sustainability. This article developed novel dual functionalized HCNs with sealed magnetic nanospheres (MNs) and cell membranes (CMs) of osteoblast, via the hydrothermal carbonization of glucose on MN surface and the aqueous amidation between terminal amino groups on CMs and surface carboxyl groups on HCNs. Owing to the specific combinations between drugs and membrane receptors, CMs bonded magnetic HCNs (CM-MHCNs) were applied as a drug discovery platform to screen potential anti-osteoporosis components from traditional herb medicine. The introduction of hydrothermal carbonaceous layer on MNs was proved to sharply improve their water dispersibility, resistance to oxidation and acids and CM grafting capacity with good magnetic property. These combined with the covalent bonding between prepared MHCNs and CMs provided the enhanced stability, where the loss amount (7.0 mg g−1) was dramatically reduced in comparison with that of CMs adsorbed MHCNs (17.9 mg g−1) and MNs (18.7 mg g−1). The resultant CM-MHCNs showed high adsorption ability, rapid binding kinetics, good selectivity and reusability. Consequently, two novel potential anti-osteoporosis compounds: isopimpinellin and xanthotoxol were successfully discovered from Common Cnidium Fruit, which were confirmed by pharmacodynamic experiments. Their possible membrane receptors were further investigated by molecular docking and western blot analysis. These researches on CM-MHCNs offer a deep insight into green design of stable biomaterials and show their promising prospect in the discovery of drug leads, thus promoting the development of traditional herb medicines.

Highly Efficient Radiosynthesis and Biological Evaluation of [18 F]Safinamide, a Radiolabelled Anti-Parkinsonian Drug for Positron Emission Tomography Imaging.

As an add-on drug approved for Parkinson's disease treatment, safinamide has multiple functions, such as selective and reversible monoamine oxidase-B inhibition, voltage-sensitive sodium/potassium channel blockage, and glutamate release inhibition. Meanwhile, safinamide shows tremendous therapeutic potential in the context of other central nervous system diseases (e. g. ischaemic stroke, amyotrophic lateral sclerosis, depression, etc.). In this work, [18 F]safinamide, which is safinamide labelled by the positron-emitting radionuclide [18 F]fluorine, was synthesized automatically based on iodonium ylide precursors with high radiochemical yield and high molar activity. Density functional theory was applied to calculate the Gibbs free energy change during iodonium ylide-mediated fluorination and to interpret the effect of tetraethylammonium (TEA+ ) as the counter cation in these reactions to improve the nucleophilicity of [18 F/19 F]fluoride. In addition, positron emission tomography studies on Sprague Dawley rats were carried out to determine the imaging characteristics, pharmacokinetics, and metabolism of the [18 F]safinamide radiotracer. The results displayed the complete biodistribution of the radiotracer, especially in rat brains, and revealed that [18 F]safinamide has moderate brain uptake, rapid and reversible binding kinetics, and good stability.

Characterization of the physicochemical interactions between exenatide and two intestinal permeation enhancers: Sodium caprate (C10) and salcaprozate sodium (SNAC)

A common approach to tackle the poor intestinal membrane permeability of peptides after oral administration is to formulate them with a permeation enhancer (PE). Increased oral bioavailability for oral peptide candidates has been reported from clinical trials when either salcaprozate sodium (SNAC) or sodium caprate (C10) is incorporated in the formulation. However, little is known about how they physically interact with peptides in solution. Our objective was to compare the biophysical interactions between the GLP-1 analogue exenatide (Byetta®, Lilly), and C10 or SNAC using a variety of advanced analytical techniques. First, critical micelle concentration was measured in different buffers for both PEs. Dynamic light scattering (DLS) measurements revealed specific supramolecular structures arising from exenatide-PE association. Surface plasmon resonance (SPR) indicated the formation of exenatide-PE complexes with a high contribution from non-specific interactions and rapid binding kinetics, resulting in overall low affinities. DLS and isothermal titration calorimetry (ITC) were used to examine the supramolecular organization of the PEs, and revealed thermodynamic signatures characterized by unfavourable enthalpic contributions compensated by favourable entropic ones, but with low-affinity estimates in water (KD in the 10–100 µM range). With affinity capillary electrophoresis (ACE), weak interactions between exenatide and SNAC or C10 were confirmed in saline, with a dissociation constant around 10 µM and 30 µM respectively. In biorelevant intestinal media, the bile salts in FaSSIF and FeSSIF further reduced the binding of both agents to exenatide (KD ≈ 100 µM), indicating that the interaction between the PEs and exenatide might be inhibited by bile salts in the GI lumen. This study suggests that the interactions of both PEs with exenatide follow a similar non-covalent mechanism and are of low affinity.

Nontoxic Liquid-Infused Slippery Coating Prepared on Steel Substrates Inhibits Corrosion and Biofouling Adhesion.

Wetting of surfaces plays a vital role in many biological and industrial processes. There are several phenomena closely related to wetting such as biofouling and corrosion that cause the deterioration of materials, while the efforts to prevent the degradation of surface functionality have spread over several millennia. Antifouling coatings have been developed to prevent/delay both corrosion and biofouling, but the problems remain unsolved, influencing the everyday life of the modern society in terms of safety and expenses. In this study, liquid-infused slippery surfaces (LISSs), a recently developed nontoxic repellent technology, that is, a flat variation of omniphobic slippery liquid-infused porous surfaces (SLIPSs), were studied for their anti-corrosion and marine anti-biofouling characteristics on metallic substrates under damaged and plain undamaged conditions. Austenitic stainless steel was chosen as a model due to its wide application in aquatic environments. Our LISS coating effectively prevents biofouling adhesion and decays corrosion of metallic surfaces even if they are severely damaged. The mechanically robust LISS reported in this study significantly extends the SLIPS technology, prompting their application in the marine environment due to the synergy between the facile fabrication process, rapid binding kinetics, nontoxic, ecofriendly, and low-cost applied materials together with excellent repellent characteristics.

Rational construction of fluorescent molecular imprinted polymers for highly efficient glycoprotein detection

The enrichment of glycoproteins by fluorescence molecular imprinting has shown an attractive application prospect, however, its specificity and sensitivity in actual sample analysis remain a significant challenge. In this work, a novel fluorescent molecular imprinted nanoplatform (MMIPs@QDs) was constructed for the efficient recognition and determination of glycoproteins by combining the glycopeptide template and the post-imprinting modification of nitrogen-doped graphene quantum dots. The synthetic MMIPs@QDs exhibited high adsorption capacity and rapid binding kinetics. Satisfying selectivity and sensitivity were obtained using MMIPs@QDs, with an imprinting factor of 7.71 and a limit of detection of 0.20 μg mL−1, respectively. The efficiency and applicability of this proposed method were validated by the detection of ovalbumin in egg white samples, showed high recoveries (94.3%−100.2%) and acceptable relative standard deviation (1.79%−7.36%). This fluorescent molecular imprinted nanoplatform provides a new approach to address the limitations of existing protein detection techniques.

Characterisation of a novel KRAS G12C inhibitor ASP2453 that shows potent anti-tumour activity in KRAS G12C-mutated preclinical models

KRAS is one of the most frequently mutated oncogenes in various cancers, and several novel KRAS G12C direct inhibitors are now in clinical trials. Here, we characterised the anti-tumour efficacy of ASP2453, a novel KRAS G12C inhibitor, in preclinical models of KRAS G12C-mutated cancer. We evaluated the in vitro and in vivo activity of ASP2453, alone or in combination with targeted agents and immune checkpoint inhibitors, in KRAS G12C-mutated cancer cells and xenograft models. We also assessed pharmacological differences between ASP2453 and AMG 510, another KRAS G12C inhibitor, using an SPR assay, washout experiments and an AMG 510-resistant xenograft model. ASP2453 potently and selectively inhibited KRAS G12C-mediated growth, KRAS activation and downstream signalling in vitro and in vivo, and improved the anti-tumour effects of targeted agents and immune checkpoint inhibitors. Further, ASP2453 had more rapid binding kinetics to KRAS G12C protein and showed more potent inhibitory effects on KRAS activation and cell proliferation after washout than AMG 510. ASP2453 also induced tumour regression in an AMG 510-resistant xenograft model. ASP2453 is a potential therapeutic agent for KRAS G12C-mutated cancer. ASP2453 showed efficacy in AMG 510-resistant tumours, even among compounds with the same mode of action.

Conformational equilibria in allosteric control of Hsp70 chaperones

Heat-shock proteins of 70kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

The Problems of Applying Classical Pharmacology Analysis to Modern In Vitro Drug Discovery Assays: Slow Binding Kinetics and High Target Concentration.

The analysis framework used to quantify drug potency in vitro (e.g., Kd or Ki) was initially developed for classical pharmacology bioassays, for example, organ bath experiments testing moderate-affinity natural products. Modern drug discovery can infringe the assumptions of the classical pharmacology analysis equations, owing to the reduction of assay volume in miniaturization, target overexpression, and the increase of compound-target affinity in medicinal chemistry. These assumptions are that (1) the compound concentration greatly exceeds the target concentration (i.e., minimal ligand depletion), and (2) the compound is at equilibrium with the receptor (i.e., rapid ligand binding kinetics). Unappreciated infringement of these assumptions can lead to substantial underestimation of compound affinity, which negatively impacts the drug discovery process, from early-stage lead optimization to prediction of human dosing. This study evaluates the real-world impact of these factors on the target interaction assays used in drug discovery using literature examples, database searches, and simulations. The ranges of compound affinity and the assay types that are prone to depletion and equilibration artifacts are identified. Importantly, the highest-affinity compounds, usually the highest value chemical matter in drug discovery, are the most affected. Methods and simulation tools are provided to enable investigators to evaluate, manage, and minimize depletion or equilibration artifacts. This study enables the correct application of pharmacological data analysis to accurately quantify affinity using modern drug discovery assay technology.

GS-967 and Eleclazine Block Sodium Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

GS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (I NaL). Here, we took advantage of a high throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (I NaP) recorded from human induced pluripotent stem cell-derived cardiomyocytes. We compared the effects of GS-967 and eleclazine with the antiarrhythmic drug lidocaine, the prototype I NaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of I NaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of I NaP (IC50 = 0.07 and 0.6 µM, respectively) than ranolazine (7.8 µM), lidocaine (133.5 µM), and lacosamide (158.5 µM). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared with the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the higher association rates and moderate unbinding rate of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine. SIGNIFICANCE STATEMENT: We investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell-derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique effects, including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.

MnO2 cacti-like nanostructured platform powers the enhanced electrochemical immunobiosensing of cortisol

Herein, we describe a label-free transition metal oxide based biosensor platform to detect both saliva and sweat cortisol levels at a point-of-care testing (POCT) perspective. The immobilization of ACAs on GCE/β-MnO2 CNs electrode is conducted by electrostatic interaction approach via isoelectric-point (IEP) of β-MnO2 (7.30) and cortisol antibodies (4.50), which establish a suitable atmosphere for effective immobilization. Bovine serum albumin (BSA) was served as blocking-agent for non-specific adsorption on the surface of MnO2 nanostructured electrode. Under optimized conditions, ACAs/beta-MnO2 cacti-like nanostructured (β-MnO2 CNs) electrode specifically interacted with cortisol molecules, which enabled rapid and sensitive electrochemical impedance detection of cortisol via 3D analyte diffusion profile at the electrode interface and also enhanced cortisol capture achieved by large specific surface of MnO2 nanostructures. This ACAs/β-MnO2 nanostructured electrode shows wide-linear EIS signals from 0.1 pM to 1500 pM with a limit of detection (LOD) of 0.023 pM (S/N = 3) and displays rapid binding kinetics with cortisol versus other glucocorticoids in terms of selectivity. Finally, the cortisol detection in human sweat, and saliva samples has been demonstrated for practical applicability and obtained results were compared with ELISA method.

Theory of Allosteric Regulation in Hsp70 Molecular Chaperones.

Heat-shock proteins of 70 kDa (Hsp70s) are ubiquitous molecular chaperones that function in protein folding as well as other vital cellular processes. They bind and hydrolyze ATP in a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides in a substrate-binding domain (SBD). However, the molecular mechanism for this allosteric action has remained unclear. Here, we develop and experimentally quantify a theoretical model for Hsp70 allostery based on equilibria among Hsp70 conformational states. We postulate that, when bound to ATP, Hsp70 is in equilibrium between a restraining state (R) that restricts ATP hydrolysis and binds peptides poorly, if at all, and a stimulating state (S) that hydrolyzes ATP relatively rapidly and has high intrinsic substrate affinity but rapid binding kinetics; after the hydrolysis to ADP, NBD and SBD disengage into an uncoupled state (U) that binds peptide substrates tightly, but now with slow kinetics of exchange.

Detection of cell-surface phosphatidylserine using the fluorogenic probe P-IID.

The fluorogenic probe P-IID enables the detection of cell-surface phosphatidylserine (PS) using both fluorescence imaging and flow cytometry. Here we provide a detailed protocol for the use of P-IID for the qualitative detection of externalized PS in apoptotic cells using confocal microscopy, including the real-time imaging of apoptosis upon drug treatment. We also provide a detailed method for the quantitative analysis of cell death by flow cytometry, using P-IID in conjunction with the nuclear stain propidium iodide. P-IID is superior to commonly used Annexin-V fluorophore conjugates for PS detection as it provides a "turn-on" fluorescence response, displays rapid binding kinetics and can be used at low temperature (4°C), without washing and in the absence of Ca2+ ions.

Selective and Rapid Cell-Permeable Inhibitor of Human Caspase-3.

Individual roles and overlapping functionalities of 12 human caspases during apoptosis and other cellular processes remain poorly resolved primarily due to a lack of chemical tools. Here we present a new selective caspase-3 inhibitor, termed Ac-ATS010-KE, with rapid and irreversible binding kinetics. Relative to previously designed caspase-3-selective molecules that have tremendously abated inhibitory rates and thus limited use in biological settings, the improved kinetics of Ac-ATS010-KE permits its use in a cell-based capacity. We demonstrate that Ac-ATS010-KE prevents apoptosis with comparable efficacy to the general caspase inhibitor Ac-DEVD-KE and surprisingly does so without side-chain methylation. This observation is in contrast to the well-established peptide modification strategy typically employed for improving cellular permeability. Ac-ATS010-KE protects against extrinsic apoptosis, which demonstrates the utility of a thiophene carboxylate leaving group in biological settings, challenges the requisite neutralization of free carboxylic acids to improve cell permeability, and provides a tool-like compound to interrogate the role of caspase-3 in a variety of cellular processes.

Molecularly Imprinted Nanoparticles for Biomedical Applications.

Molecularly imprinted polymers (MIPs) are synthetic receptors with tailor-made recognition sites for target molecules. Their high affinity and selectivity, excellent stability, easy preparation, and low cost make them promising substitutes to biological receptors in many applications where molecular recognition is important. In particular, spherical MIP nanoparticles (or nanoMIPs) with diameters typically below 200 nm have drawn great attention because of their high surface-area-to-volume ratio, easy removal of templates, rapid binding kinetics, good dispersion and handling ability, undemanding functionalization and surface modification, and their high compatibility with various nanodevices and in vivo biomedical applications. Recent years have witnessed significant progress made in the preparation of advanced functional nanoMIPs, which has eventually led to the rapid expansion of the MIP applications from the traditional separation and catalysis fields to the burgeoning biomedical areas. Here, a comprehensive overview of key recent advances made in the preparation of nanoMIPs and their important biomedical applications (including immunoassays, drug delivery, bioimaging, and biomimetic nanomedicine) is presented. The pros and cons of each synthetic strategy for nanoMIPs and their biomedical applications are discussed and the present challenges and future perspectives of the biomedical applications of nanoMIPs are also highlighted.

Controlled preparation of molecularly imprinted polymer microspheres with surface uncrosslinked glycoprotein binding cavities

Molecularly imprinted polymers (MIPs) are synthetic receptors with tailor-made recognition sites for target molecules. Their high affinity and selectivity, excellent stability, easy preparation, and low cost make them promising substitutes to biological receptors (e.g., antibody and enzyme) in many applications where molecular recognition is important. Despite significant progress made in the imprinting of small templates, the imprinting of biomacromolecules (e.g., proteins) remains a big challenge because of their large sizes, complexed structures, and conformational variability. These inherent characteristics of biomacromolecules lead to many significant problems for the resulting macromolecularly imprinted polymers (mMIPs) such as laborious removal of large templates and their slow access to the binding sites. Recent years have witnessed much efforts being devoted to the development of mMIPs due to their great potential in proteome analysis, clinical diagnostics, and biomedicine. So far, some useful strategies have been developed for the imprinting of proteins, mainly including the bulk polymerization method, epitope imprinting strategy, and surface imprinting approach. Among them, the surface imprinting approach has been most widely used because it can readily lead to mMIPs with higher efficiency for removing large templates and more rapid template binding kinetics. Nevertheless, the presently developed mMIPs normally have crosslinked template binding sites, whose rigid structures might have negative influence on the removal of large templates and template binding kinetics. In this sense, the development of mMIPs with more flexible biomacromolecular binding cavities (e.g., uncrosslinked ones) should be useful for solving the above problems. To our knowledge, however, only rather limited numbers of publications relating to mMIPs with uncrosslinked binding sites have been disclosed, which are all based on the use of self-assembled monolayer strategy. The development of versatile new approaches for preparing mMIPs with uncrosslinked binding sites and good molecular imprinting effect is still highly desirable. We demonstrate a facile and efficient new approach for the controlled preparation of MIP microspheres with surface uncrosslinked glycoprotein binding sites. It involves the first one-pot synthesis of uniform “living” polymer microspheres with both surface-bound epoxy groups and alkyl halide groups (i.e., atom transfer radical polymerization (ATRP)-initiating groups) via atom transfer radical precipitation polymerization, their surface modification with 3-aminophenylboronic acid (APBA) for introducing surface phenylboronic acid moieties and surface immobilization of a glycoprotein (ovalbumin (OVA)), subsequent use of the “living” polymer microspheres with surface-immobilized OVA as the ATRP initiator for the controlled grafting of poly( N -isopropylacrylamide) (PNIPAAm) brushes, and final removal of OVA. A series of MIP microspheres with surface uncrosslinked OVA binding sites were readily obtained following the above procedure by just changing the polymerization time for grafting PNIPAAm brushes, and their morphologies, chemical structures, surface hydrophilicity, water dispersion stability, and template binding properties were characterized in detail. The experimental results demonstrated that the above approach could effectively provide MIPs with excellent recognition ability toward OVA in the aqueous medium. The surface hydrophilicity and water dispersion stability of MIP microspheres were largely improved due to their surface-grafting of hydrophilic polymer brushes. Moreover, the chain length of PNIPAAm brushes showed significant influence on the template binding properties of MIP microspheres, and the best template binding capacity and specific binding were achieved for MIPs only when the thickness of PNIPAAm layers was close to the total length of the diameter of OVA plus the length of the surface-attached phenylboronic acid unit. Furthermore, the optimal MIP also exhibited good selectivity toward OVA over other proteins. The strategy presented here paves a new way for controlled and efficient preparation of glycolprotein-imprinted polymer microspheres with good molecular recognition capability.