Search

Design of the next-generation of therapeutics, biosensors, and molecular tools for basic research requires that we bring protein activity under control. Each protein has unique properties, and therefore, it is critical to tailor the current techniques to develop new regulatory methods and regulate new proteins of interest (POIs). This perspective gives an overview of the widely used stimuli and synthetic and natural methods for conditional regulation of proteins.

Many bioconjugation strategies for DNA oligonucleotides and antibodies suffer limitations, such as site���specificity, stoichiometry and hydrolytic instability of the conjugates, which makes them unsuitable for biological applications. Here, we report a new platform for the preparation of DNA���antibody bioconjugates with a simple benzoylacrylic acid pentafluorophenyl ester reagent. Benzoylacrylic���labelled oligonucleotides prepared with this reagent can be site���specifically conjugated to a range of proteins and antibodies through accessible cysteine residues. The homogeneity of the prepared DNA���antibody bioconjugates was confirmed by a new LC���MS protocol and the bioconjugate probes were used in fluorescence or super���resolution microscopy cell imaging experiments. This work demonstrates the versatility and robustness of our bioconjugation protocol that gives site���specific, well���defined and plasma���stable DNA���antibody bioconjugates for biological applications.

© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation., This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement N° 836698 and under grant agreement N° 852985. Funding from the Scripps Research Institute (A.A.), FCT Portugal (PhD scholarship PD/BD/135512/2018 to C.F.A. and FCT Stimulus CEECIND/00453/2018 to G.J.L.B.) and Agencia Estatal Investigación of Spain (AEI; Grant RTI2018-099592-B-C21 to F.C.) is also acknowledged.

Funder: Frances and Augustus Newman Foundation, Funder: Emmanuel College, University of Cambridge, Funder: Biotechnology and Biological Sciences Research Council, Funder: Centre for Misfolding Diseases, University of Cambridge, Funder: Wellcome Trust, Biomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer., We computationally identify and experimentally validate TRPV2 as a target for piperlongumine and establish a link between allosteric target engagement and impaired glioblastoma progression in vivo.

Endogenous long-chain metabolites of vitamin E (LCMs) mediate immune functions by targeting 5-lipoxygenase (5-LOX) and increasing the systemic concentrations of resolvin E3, a specialized proresolving lipid mediator. SAR studies on semisynthesized analogues highlight α-amplexichromanol (27a), which allosterically inhibits 5-LOX, being considerably more potent than endogenous LCMs in human primary immune cells and blood. Other enzymes within lipid mediator biosynthesis were not substantially inhibited, except for microsomal prostaglandin E2 synthase-1. Compound 27a is metabolized by sulfation and β-oxidation in human liver-on-chips and exhibits superior metabolic stability in mice over LCMs. Pharmacokinetic studies show distribution of 27a from plasma to the inflamed peritoneal cavity and lung. In parallel, 5-LOX-derived leukotriene levels decrease, and the inflammatory reaction is suppressed in reconstructed human epidermis, murine peritonitis, and experimental asthma in mice. Our study highlights 27a as an orally active, LCM-inspired drug candidate that limits inflammation with superior potency and metabolic stability to the endogenous lead.

This journal is © The Royal Society of Chemistry 2020. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence., The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines., We acknowledge the Ministerio de Ciencia, Innovación y Universidades and the Agencia Estatal de Investigación (projects RTI2018-099592-B-C21 and RTI2018-097609-B-C21), the Ministerio de Economía y Competitividad (SAF2016-76083-R), European Regional Development Funds (FEDER-UE), the Royal Society (URF\R\180019 to G. J. L. B.) and FCT Portugal (PhD studentship, SFRH/BD/115932/2016 to A. G. and FCT Investigator IF/00624/2015 to G. J. L. B.). M. C. O further thanks CITIUS for technical support. I. A. B. thanks the Asociación Española Contra el Cancer en La Rioja for a grant. E. J.-M. thanks Universidad de La Rioja for a postdoctoral fellowship. R. H.-G. acknowledges Diamond Light Source (Oxford, UK) synchrotron beamline I04 (experiment numbers mx10121-19). He also acknowledges ARAID, MEC (CTQ2013-44367-C2-2-P and BFU2016-75633-P) and Gobierno de Aragón (E34_R17 and LMP58_18) with FEDER (2014–2020) funds for ‘Building Europe from Aragón’ for financial support. The research leading to these results has also received funding from FP7 442 (2007–2013) under BioStruct-X (grant agreement no. 283570 and BIOSTRUCTX_5186). F. G.-M. of Hokkaido University acknowledges grant from the Naito Foundation.