Covalent hits and where to find them.

Covalent hits for drug discovery campaigns are neither fantastic beasts nor mythical creatures, they can be routinely identified through electrophile-first screening campaigns using a suite of different techniques. These include biophysical and biochemical methods, cellular approaches, and DNA-encoded libraries. Employing best practice, however, is critical to success. The purpose of this review is to look at state of the art covalent hit identification, how to identify hits from a covalent library and how to select compounds for medicinal chemistry programmes.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

Transmembrane Transport of Bicarbonate Unravelled.

Invited for the cover of this issue are Dr. Stephen Butler, Dr. Hennie Valkenier and co-workers at Université Libre de Bruxelles, Loughborough University, Masaryk University, and the University of Bristol. The image depicts the transport of bicarbonate anions versus the spontaneous diffusion of CO2 across the lipid bilayer of a liposome. Read the full text of the article at 10.1002/chem.202100491.

Transmembrane Transport of Bicarbonate Unravelled*.

Anion receptors can be used to transport ions across lipid bilayers, which has potential for therapeutic applications. Synthetic bicarbonate transporters are of particular interest, as defects in transmembrane transport of bicarbonate are associated with various diseases. However, no convenient method exists to directly observe bicarbonate transport and study the mechanisms involved. Here, an assay is presented that allows the kinetics of bicarbonate transport into liposomes to be monitored directly and with great sensitivity. The assay utilises an encapsulated europium(III) complex, which exhibits a large increase in emission intensity upon binding bicarbonate. Mechanisms involving CO2 diffusion and the dissipation of a pH gradient are shown to be able to lead to an increase in bicarbonate concentration within liposomes, without transport of the anion occurring at all. By distinguishing these alternative mechanisms from actual bicarbonate transport, this assay will inform the future development of bicarbonate transporters.

Tuning the anion binding properties of lanthanide receptors to discriminate nucleoside phosphates in a sensing array.

The development of synthetic receptors for the selective binding and discrimination of anions in water requires an understanding of how anions interact with these synthetic receptors. Molecules designed to differentiate nucleoside phosphate anions (e.g. ATP, ADP, GTP, GDP, UDP) under physiological conditions could underpin exciting new sensing tools for biomedical research and drug discovery, but it is very challenging due to the similarities in anion structure, size and charge. We present a series of lanthanide-based anion receptors and establish key structural elements that impact on nucleoside phosphate anion binding and sensing. Structural evidence of anion binding using X-ray crystallographic and NMR data, supported by DFT calculations indicate the binding modes between the lanthanide complexes and certain phosphoanions, revealing a bidentate (α-, γ-) binding mode to ATP. We further use four of the receptors to allow discrimination of eight nucleoside phosphate anions in the first array-based assay using lanthanide complexes, taking advantage of the multiple emission bands and long emission lifetimes associated with luminescent lanthanide complexes.

A simple, robust, universal assay for real-time enzyme monitoring by signalling changes in nucleoside phosphate anion concentration using a europium(iii)-based anion receptor.

Enzymes that consume and produce nucleoside polyphosphate (NPP) anions represent major targets in drug discovery. For example, protein kinases are one of the largest classes of drug targets in the fight against cancer. The accurate determination of enzyme kinetics and mechanisms is a critical aspect of drug discovery research. To increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical researchers have robust, affordable assays to measure enzyme activity accurately. We present a simple, sensitive microplate assay for real-time monitoring of a range of pharmaceutically important enzyme reactions that generate NPP anions, including kinases and glycosyltransferases. Our assay utilises a single, stable europium(iii) complex that binds reversibly to NPP anions, signalling the dynamic changes in NPP product/substrate ratio during an enzyme reaction using time-resolved luminescence. This supramolecular approach to enzyme monitoring overcomes significant limitations in existing assays, obviating the need for expensive antibodies or equipment, chemically labelled substrates or products and isolation or purification steps. Our label and antibody-free method enables rapid and quantitative analysis of enzyme activities and inhibition, offering a potentially powerful tool for use in drug discovery, suitable for high-throughput screening of inhibitors and accurate measurements of enzyme kinetic parameters.

Generation of Dynamic Combinatorial Libraries Using Hydrazone-Functionalized Surface Mimetics.

Dynamic combinatorial chemistry (DCC) represents an approach, whereby traditional supramolecular scaffolds used for protein surface recognition might be exploited to achieve selective high affinity target recognition. Synthesis, in situ screening and amplification under selection pressure allows the generation of ligands, which bear different moieties capable of making multivalent non-covalent interactions with target proteins. Generic tetracarboxyphenyl porphyrin scaffolds bearing four hydrazide moieties have been used to form dynamic combinatorial libraries (DCLs) using aniline-catalyzed reversible hydrazone exchange reactions, in 10 % DMSO, 5 mm NH4OAc, at pH 6.75. High resolution mass spectrometry (HRMS) was used to monitor library composition and establish conditions under which equilibria were established.

Application of lanthanide luminescence in probing enzyme activity.

Enzymes play critical roles in the regulation of cellular function and are implicated in numerous disease conditions. Reliable and practicable assays are required to study enzyme activity, to facilitate the discovery of inhibitors and activators of enzymes related to disease. In recent years, a variety of enzyme assays have been devised that utilise luminescent lanthanide(iii) complexes, taking advantage of their high detection sensitivities, long luminescence lifetimes, and line-like emission spectra that permit ratiometric and time-resolved analyses. In this Feature article, we focus on recent progress in the development of enzyme activity assays based on lanthanide(iii) luminescence, covering a variety of strategies including Ln(iii)-labelled antibodies and proteins, Ln(iii) ion encapsulation within defined peptide sequences, reactivity-based Ln(iii) probes, and discrete Ln(iii) complexes. Emerging approaches for monitoring enzyme activity are discussed, including the use of anion responsive lanthanide(iii) complexes, capable of molecular recognition and luminescence signalling of polyphosphate anions.

Recognition of proximally phosphorylated tyrosine residues and continuous analysis of phosphatase activity using a stable europium complex.

The recognition of proteins and their post-translational modifications using synthetic molecules is an active area of research. A common post-translational modification is the phosphorylation of serine, threonine or tyrosine residues. The phosphorylation of proximal tyrosine residues occurs in over 1000 proteins in the human proteome, including in disease-related proteins, so the recognition of this motif is of particular interest. We have developed a luminescent europium(III) complex, [Eu.1]+ , capable of the discrimination of proximally phosphorylated tyrosine residues, from analogous mono- and non-phosphorylated tyrosine residues, more distantly-related phosphotyrosine residues and over proximally phosphorylated serine and threonine residues. [Eu.1]+ was used to continuously monitor the phosphatase catalysed dephosphorylation of a peptide containing proximally phosphorylated tyrosine residues.

Protein sensing and discrimination using highly functionalised ruthenium(ii) tris(bipyridyl) protein surface mimetics in an array format.

Ruthenium(ii) tris(bipyridyl) protein surface mimetics are used in an array format to sense and discriminate proteins including therapeutically relevant targets, hDM2 and MCL-1, using linear discriminant analysis (LDA).

A continuous luminescence assay for monitoring kinase activity: signalling the ADP/ATP ratio using a discrete europium complex.

We report the application of a stable cationic europium complex [Eu.1]+ in a continuous-read luminescence assay for kinase activity. [Eu.1]+ binds reversibly to ATP and ADP in water, at neutral pH, in the presence of Mg2+ ions, providing distinctive luminescence responses that permits the kinase-catalysed conversion of ATP to ADP to be monitored in real-time.

Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H,15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.

Correction: Metal complexes as "protein surface mimetics".

Correction for 'Metal complexes as "protein surface mimetics"' by Sarah H. Hewitt and Andrew J. Wilson, Chem. Commun., 2016, 52, 9745-9756.

Metal complexes as "protein surface mimetics".

A key challenge in chemical biology is to identify small molecule regulators for every single protein. However, protein surfaces are notoriously difficult to recognise with synthetic molecules, often having large flat surfaces that are poorly matched to traditional small molecules. In the surface mimetic approach, a supramolecular scaffold is used to project recognition groups in such a manner as to make multivalent non-covalent contacts over a large area of protein surface. Metal based supramolecular scaffolds offer unique advantages over conventional organic molecules for protein binding, including greater stereochemical and geometrical diversity conferred through the metal centre and the potential for direct assessment of binding properties and even visualisation in cells without recourse to further functionalisation. This feature article will highlight the current state of the art in protein surface recognition using metal complexes as surface mimetics.