Site‐selective cysteine arylation has been achieved by rational 2D reactivity tuning of fluorobenzene probes. In their Communication on page 8022 ff., M. Meldal, F. Diness et al. demonstrate how ...probes may be tailored to selectively target cysteine‐containing proteins by adjusting the numbers of fluoro substituents (Fx) and by selecting substituents based on their electron‐withdrawing capacity represented by their Hammett σp constant.
Monosized beads of polar resins were synthesized for combinatorial chemistry and chemical biology by sustainable microchannel flow synthesis. Regular, biocompatible, and optically encoded beads could ...be efficiently prepared on large scale and in high yield. In a preparative flow polymerization instrument, taking advantage of a designed T-connector for droplet formation, quality beads were synthesized with accurate size control using a minimal amount of recirculating silicon oil as suspension medium. Bead-size was controlled through shear imposed by the silicon oil flow rate. This process provided 86% yield of ∼500 μm macrobeads beads within a 20 μm size range with no deformities or vacuoles, ideally suited for combinatorial chemistry and protein binding studies. The simple flow equipment consisted of a syringe pump for monomer and initiator delivery, a T-connector, a gear pump for oil recirculation, a long, heated coil of Teflon tubing and a collector syringe. The method was used for preparation of PEGA
beads, optically encoded with fluorescent microparticles. The microparticle matrix (MPM) encoded beads were tested in a MPM-decoder showing excellent recognition in bead decoding.
Abstract only
The emergent market of new therapeutic glycoprofiles demands cost‐and‐labor efficient N‐linked glycoengineering, such as rational glycoengineering. However, rational glycoengineering ...remains challenging due to our limited understanding of the complex glycosyltransferase (GT) interactions. Predicting glycosylation outcomes of GT knockins/knockouts requires comprehensive and context‐specific knowledge of GT activities. To combat this issue, we proposed an improved low‐parameter Markov chain model to learn GT activities and used the learnt information to predict glycoprofiles impacted by complex glycoengineering. Specifically, we have quantified the impact of GT isozyme specificities and interactions by fitting the model with a compilation of single‐GT‐knockout erythropoietin (EPO) glycoprofiles for CHO cells. We then showed that the quantified GT impact can be generalized by our model framework to predict complex multiple‐GT‐knockout glycoprofiles of four diverse therapeutic drugs, including EPO, Rituximab, Enbrel, and alpha‐1 antitrypsin. Therefore, the proposed methodology allowed us to systematically unravel the complicated GT interactions beyond simple enzyme rules and with little
a priori
information. Such approach will further promote progress in rational glycoengineering by broadening our scope on the genetic basis of glycosylation, expediting product development for biopharmaceutical research.
Support or Funding Information
This work was conducted with support from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517: A.L., A.W.T.C., A.H.H., B.G.V.) and NIGMS (R35 GM119850: N.E.L.)
Figure 1
Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a ...phage display-based cross-panning strategy, capable of reducing the cytotoxic effects of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to develop more effective, safer, and globally accessible polyvalent antivenoms that can be widely used to treat snakebite envenoming.
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•Improved and more accessible snakebite therapies are needed to reduce the negative effects caused by snakebite envenoming.•Recombinant antivenoms, composed of monoclonal antibodies have been hypothesized as a future snakebite envenoming therapy.•A novel human monoclonal IgG antibody shows broadly-neutralizing capabilities against myotoxicity from viperid snake venoms.•This broad neutralization affects venom from snakes belonging to three genera and originating from three continents.•While cross-reactive, the antibody retains selectivity to bind only a subset of the tested viperid venoms.
The identification of pairs of small peptides that recognize each other in water exclusively through electrostatic interactions is reported. The target peptide and a structure‐biased combinatorial ...ligand library consisting of ≈78 125 compounds were synthesized on different sized beads. Peptide–peptide interactions could conveniently be observed by clustering of the small, fluorescently labeled target beads on the surface of larger ligand‐containing beads. Sequences of isolated hits were determined by MS/MS. The interactions of the complex showing the highest affinity were investigated by a novel single‐bead binding assay and by 2D NMR spectroscopy. Molecular dynamics (MD) studies revealed a putative mode of interaction for this unusual electrostatic binding event. High binding specificity occurred through a combination of topological matching and electrostatic and hydrogen‐bond complementarities. From MD simulations binding also seemed to involve three tightly bound water molecules in the interface between the binding partners. Binding constants in the submicromolar range, useful for biomolecular adhesion and in nanostructure design, were measured.
Beyond recognition: Specific recognition between two small, polar peptides with no lipophilic components is demonstrated (see figure). Pairs of molecules were identified by combinatorial selection and studied in a new on‐bead binding assay. The superior complex was extensively studied by NMR spectroscopy, and a model for the complex was generated by NOE‐restrained molecular dynamics calculations. In the small complex, binding was partially mediated by three tightly bound water molecules.
Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly ...selective acylation of proteins with an N‐terminal Gly‐His6 segment. This tag promoted acylation of the N‐terminal Nα‐amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn‐Lys‐Hism, which we term Lys‐His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ‐amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys‐His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C‐terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys‐His tag acylation method.
A short, designed Lys‐His tag can be inserted into proteins where it autocatalyzes the highly selective acylation of the Lys side‐chain amine with phenyl esters. The reaction conditions of this site‐selective, chemical protein modification method are mild, and no oxidative reagents, reductants nor enzymes are required.
Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. ...Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.
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•Therapeutic antibodies are typically expressed in mammalian cell systems and are inaccessible to resource-poor regions.•Alternative expression systems may be attractive to manufacturers of recombinant antivenoms in resource-poor regions.•Toxin-targeting antibodies were expressed in both tobacco plants and mammalian cells.•Plant and mammalian cell-produced antibodies showed similar antigen binding.•Complete in vitro neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibodies.
The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical ...pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC
values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.
Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific cross-linking, and free of ...any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddition as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatography and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnological applications, in which multiple biocatalysts collaborate at low concentrations, in which diffusion may be limited and/or side-reactions are prone to occur.
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