Iterative In Situ Click Chemistry Creates Antibody‐like Protein‐Capture Agents

Special agents for protein capture : Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one‐bead–one‐compound method for the creation of a peptide library enable the fragment‐based assembly of selective high‐affinity protein‐capture agents. The resulting ligands are water‐soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper(I)‐catalyzed cycloaddition.

     

Exploiting Protein Symmetry To Design Light‐Controllable Enzyme Inhibitors

The activity of the metabolic branch‐point enzyme PriA from Mycobacterium tuberculosis (mtPriA) can be controlled reversibly by light. Two‐pronged inhibitors based on the dithienylethene scaffold were designed utilizing mtPriA’s natural rotational symmetry. Switching from the flexible, ring‐open to the rigid, ring‐closed isomer reduces inhibition activity by one order of magnitude.     

In‐Bead Screening of Hydroxamic Acids for the Identification of HDAC Inhibitors

A one bead–one compound screening format is presented. Following solid‐phase synthesis on a photolabile linker, library compounds were readily released and screened inside polymer beads. The release of screening compounds was readily controlled by varying photolysis time and light intensity. Dose‐response experiments were carried out to effectively distinguish high‐ and low‐affinity ligands. A library containing 55 800 compounds was synthesized and screened in a fluorometric assay, thereby identifying potent HDAC inhibitors with IC₅₀ values in the nanomolar range.     

In Situ AFM Investigation of Electrochemically Induced Surface‐Initiated Atom‐Transfer Radical Polymerization

Electrochemically induced surface‐initiated atom‐transfer radical polymerization is traced by in situ AFM technology for the first time, which allows visualization of the polymer growth process. It affords a fundamental insight into the surface morphology and growth mechanism simultaneously. Using this technique, the polymerization kinetics of two model monomers were studied, namely the anionic 3‐sulfopropyl methacrylate potassium salt (SPMA) and the cationic 2‐(metharyloyloxy)ethyltrimethylammonium chloride (METAC). The growth of METAC is significantly improved by screening the ammonium cations by the addition of ionic liquid electrolyte in aqueous solution.     

Substrate Activity Screening with Kinases: Discovery of Small‐Molecule Substrate‐Competitive c‐Src Inhibitors

Substrate‐competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small‐molecule substrates for any protein kinase were discovered, as well as the first substrate‐competitive inhibitors of c‐Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate‐competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP‐competitive inhibitors.     

Structure‐Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure‐based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein‐bound library member(s) by saturation‐transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.     

Analysis of Density‐Dependent Binding of Glycans by Lectins Using Carbohydrate Microarrays

To investigate the density‐dependent binding of glycans by lectins using carbohydrate microarrays, a number of C‐terminal hydrazide‐conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne‐linked peptides possessing C‐terminal hydrazide on a solid support, (2) coupling of azide‐linked, unprotected sugars to the alkyne‐linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on epoxide‐derivatized glass slides and were used to analyze the density‐dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density.     

Fast Epitope Mapping for the Anti‐MUC1 Monoclonal Antibody by Combining a One‐Bead‐One‐Glycopeptide Library and a Microarray Platform

Anti‐MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer‐related MUC1 molecules, the O‐glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) “one‐bead‐one‐compound”‐based preparation of bilayer resins carrying glycopeptides on the shell and mass‐tag tripeptides coding O‐glycan patterns in the core, ii) on‐resin screening with an anti‐MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass‐tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O‐glycosylations against anti‐MUC1 mAb clone VU‐3C6. Qualitative mass‐tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray‐based assay. Our screening provides valuable information on O‐glycosylations of epitopes leading to high affinity with mAb.     

Surface‐Assisted Self‐Assembly Strategies Leading to Supramolecular Hydrogels

Localized molecular self‐assembly processes leading to the growth of nanostructures exclusively from the surface of a material is one of the great challenges in surface chemistry. In the last decade, several works have been reported on the ability of modified or unmodified surfaces to manage the self‐assembly of low‐molecular‐weight hydrogelators (LMWH) resulting in localized supramolecular hydrogel coatings mainly based on nanofiber architectures. This Minireview highlights all strategies that have emerged recently to initiate and localize LMWH supramolecular hydrogel formation, their related fundamental issues and applications.     

Surface‐Assisted Self‐Assembly of a Hydrogel by Proton Diffusion

Controlling supramolecular growth at solid surfaces is of great importance to expand the scope of supramolecular materials. A dendritic benzene‐1,3,5‐tricarboxamide peptide conjugate is described in which assembly can be triggered by a pH jump. Stopped‐flow kinetics and mathematical modeling provide a quantitative understanding of the nucleation, elongation, and fragmentation behavior in solution. To assemble the molecule at a solid–liquid interface, we use proton diffusion from the bulk. The latter needs to be slower than the lag phase of nucleation to progressively grow a hydrogel outwards from the surface. Our method of surface‐assisted self‐assembly is generally applicable to other gelators, and can be used to create structured supramolecular materials.     

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily‐Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self‐assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic “thermal tag” with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily‐selective probes against two clinically important epigenetic enzymes: FTO ( 7 ; IC₅₀=2.6 μm ) and ALKBH3 ( 8 ; IC₅₀=3.7 μm ). To date, this is the first report of a subfamily‐selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.     

毛细管电泳法在酶抑制剂筛选中的应用

毛细管电泳法以其分离效率高、耗时短、样品用量少、灵敏度高以及易于实现自动化等诸多特点,在筛选酶抑制剂研究中有着不可替代的优势.本文综述了毛细管电泳法在酶抑制剂筛选中的应用.主要介绍了应用最广泛的离线分析(Off-line assay)和在线分析(In-line assay),即柱前酶反应(Pre-capillary enzyme reaction)和在柱酶分析(In-capillary enzyme reaction).并对该领域的未来发展进行了展望.