Multi-task bioassay pre-training for protein-ligand binding affinity prediction.

Protein-ligand binding affinity (PLBA) prediction is the fundamental task in drug discovery. Recently, various deep learning-based models predict binding affinity by incorporating the three-dimensional (3D) structure of protein-ligand complexes as input and achieving astounding progress. However, due to the scarcity of high-quality training data, the generalization ability of current models is still limited. Although there is a vast amount of affinity data available in large-scale databases such as ChEMBL, issues such as inconsistent affinity measurement labels (i.e. IC50, Ki, Kd), different experimental conditions, and the lack of available 3D binding structures complicate the development of high-precision affinity prediction models using these data. To address these issues, we (i) propose Multi-task Bioassay Pre-training (MBP), a pre-training framework for structure-based PLBA prediction; (ii) construct a pre-training dataset called ChEMBL-Dock with more than 300k experimentally measured affinity labels and about 2.8M docked 3D structures. By introducing multi-task pre-training to treat the prediction of different affinity labels as different tasks and classifying relative rankings between samples from the same bioassay, MBP learns robust and transferrable structural knowledge from our new ChEMBL-Dock dataset with varied and noisy labels. Experiments substantiate the capability of MBP on the structure-based PLBA prediction task. To the best of our knowledge, MBP is the first affinity pre-training model and shows great potential for future development. MBP web-server is now available for free at: https://huggingface.co/spaces/jiaxianustc/mbp.

Phomoxanthone A Targets ATP Synthase.

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.

Affinity-Guided Site-Selective Labeling of Nanobodies with Aldehyde Handles.

Antigen-binding proteins such as nanobodies are extensively used in biomedicine, diagnostics, and as tools for molecular biology. Often such applications require modification of the nanobodies with fluorophores or drugs. Here, we describe a robust method for introduction of aldehyde handles into His-tagged nanobodies and further derivatization of these proteins with hydroxylamine functionalized compounds of interest. The method allows for isolation of nanobodies containing one or more labels.

Effect of Alkynyl Group on Reactivity in Photoaffinity Labeling with 2-Thienyl-Substituted α-Ketoamide.

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.

Multiplexed Small-Molecule-Ligand Binding Assays by Affinity Labeling and DNA Sequence Analysis.

Small-molecule binding assays to target proteins are a core component of drug discovery and development. While a number of assay formats are available, significant drawbacks still remain in cost, sensitivity, and throughput. To improve assays by capitalizing on the power of DNA sequence analysis, we have developed an assay method that combines DNA encoding with split-and-pool sample handling. The approach involves affinity labeling of DNA-linked ligands to a protein target. Critically, the labeling event assesses ligand binding and enables subsequent pooling of several samples. Application of a purifying selection on the pool for protein-labeled DNAs allows detection of ligand binding by quantification of DNA barcodes. We demonstrate the approach in both ligand displacement and direct binding formats and demonstrate its utility in determination of relative ligand affinity, profiling ligand specificity, and high-throughput small-molecule screening.

Evolution of the Selection Methods of DNA-Encoded Chemical Libraries.

In the past two decades, a DNA-encoded chemical library (DEL or DECL) has emerged and has become a major technology platform for ligand discovery in drug discovery as well as in chemical biology research. Although based on a simple concept, i.e., encoding each compound with a unique DNA tag in a combinatorial chemical library, DEL has been proven to be a powerful tool for interrogating biological targets by accessing vast chemical space at a fraction of the cost of traditional high-throughput screening (HTS). Moreover, the recent technological advances and rapid developments of DEL-compatible reactions have greatly enhanced the chemical diversity of DELs. Today, DELs have been adopted by nearly all major pharmaceutical companies and are also gaining momentum in academia. However, this field is heavily biased toward library encoding and synthesis, and an underexplored aspect of DEL research is the selection methods. Generally, DEL selection is considered to be a massive binding assay conducted over an immobilized protein to identify the physical binders using the typical bind-wash-elute procedure. In recent years, we and other research groups have developed new approaches that can perform DEL selections in the solution phase, which has enabled the selection against complex biological targets beyond purified proteins. On the one hand, these methods have significantly widened the target scope of DELs; on the other hand, they have enabled the functional and potentially phenotypic assays of DELs beyond simple binding. An overview of these methods is provided in this Account.Our laboratory has been using DNA-programmed affinity labeling (DPAL) as the main strategy to develop new DEL selection methods. DPAL is based on DNA-templated synthesis; by using a known ligand to guide the target binding, DPAL is able to specifically establish a stable linkage between the target protein and the ligand. The DNA tag of the target-ligand conjugates serves as a programmable handle for protein characterization or hit compound decoding in the case of DEL selections. DPAL also takes advantage of the fast reaction kinetics of photo-cross-linking to achieve high labeling specificity and fidelity, especially in the selection of DNA-encoded dynamic libraries (DEDLs). DPAL has enabled DEL selections not only in buffer and cell lysates but also with complex biological systems, such as large protein complexes and live cells. Moreover, this strategy has also been employed in other biological applications, such as site-specific protein labeling, protein detection, protein profiling, and target identification. In the Account, we describe these methods, highlight their underlying principles, and conclude with perspectives of the development of the DEL technology.

Janus Bioparticles: Asymmetric Nucleosomes and Their Preparation Using Chemical Biology Approaches.

The fundamental repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around two copies each of four canonical histone proteins. Nucleosomes possess 2-fold pseudo-symmetry that is subject to disruption in cellular contexts. For example, the post-translational modification (PTM) of histones plays an essential role in epigenetic regulation, and the introduction of a PTM on only one of the two "sister" histone copies in a given nucleosome eliminates the inherent symmetry of the complex. Similarly, the removal or swapping of histones for variants or the introduction of a histone mutant may render the two faces of the nucleosome asymmetric, creating, if you will, a type of "Janus" bioparticle. Over the past decade, many groups have detailed the discovery of asymmetric species in chromatin isolated from numerous cell types. However, in vitro biochemical and biophysical investigation of asymmetric nucleosomes has proven synthetically challenging. Whereas symmetric nucleosomes are readily formed via a stochastic combination of their histone and DNA components, asymmetric nucleosome assembly demands the selective incorporation of a single modified/mutant histone copy alongside its wild-type counterpart.Herein we describe the chemical biology tools that we and others have developed in recent years for investigating nucleosome asymmetry. Such approaches, each with its own benefits and shortcomings, fall into five broad categories. First, we discuss affinity tag-based purification methods. These enable the assembly of theoretically any asymmetric nucleosome of interest but are frequently labor-intensive and suffer from low yields. Second, we detail transient cross-linking strategies that are amenable to the preparation of histone H3- or H4-modified/mutant asymmetric species. These yield asymmetric nucleosomes in a traceless fashion, albeit through the use of more complicated synthesis techniques. Third, we describe a synthetic biology technique based on the generation of bump-hole mutant H3 histones that selectively heterodimerize. Although currently developed only for H3 and a related isoform, this method uniquely allows for the interrogation of nucleosome asymmetry in yeast. Fourth, we outline a method for generating H2A- or H2B-modified/mutant asymmetric nucleosomes that relies on the differential DNA-histone contact strength inherent in the Widom 601 DNA sequence. This technique involves the initial formation of hexasomes which are then complemented with distinct H2A/H2B dimers. Finally, we review an approach that utilizes split intein technology to isolate asymmetric H2A- or H2B-modified/mutant nucleosomes. This method shares steps in common with the former but exploits tagged, intein-fused dimers for the facile purification of asymmetric products.Throughout the Account, we highlight various biological questions that drove the development of these methods and ultimately were answered by them. Though each technique has its own shortcomings, collectively these chemical biology tools provide a means to biochemically interrogate a plethora of asymmetric nucleosome species. We conclude with a discussion of remaining challenges, particularly that of endogenous asymmetric nucleosome detection.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption.

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.

Essential requirement for JPT2 in NAADP-evoked Ca2+ signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Direct ligand screening against membrane proteins on live cells enabled by DNA-programmed affinity labelling.

Membrane proteins are important drug targets; however, ligand discovery for membrane proteins is highly challenging due to their hydrophobic nature. We show that membrane proteins may be specifically labelled with a DNA tag by DNA-programmed affinity labelling (DPAL), thereby enabling the screening of chemical compounds against membrane proteins directly on live cells.

2-BFI attenuates ischemic injury by modulating mTOR signaling and neuroinflammation in rats.

Ischemic stroke is one of the major diseases that cause mortality and morbidity of human beings, but there is still lack of effective treatment and prevention. We found that 2-(2-Benzofuranyl)-2-Imidazoline (2-BFI) is potently protective against stroke and acute inflammatory immune disease. Moreover, the mammalian target of rapamycin (mTOR) signaling contributes effectively to the modulation of post-stroke neuroinflammatory response. However, whether the protection of 2-BFI against ischemic injury is through mTOR-mediated neuroinflammatory response remains unestablished. Here, we used 2-BFI to treat ischemic rats induced by distal middle cerebral artery occlusion (dMCAO). We found that 2-BFI administration after dMCAO improved the neurological deficits and decreased the infarct volume. 2-BFI reduced phosphorylation of mTOR and p70S6, increased IL-10 and TGF-ß, and decreased IFN-γ levels in ischemic rats. Our results demonstrated that 2-BFI attenuates ischemic injury by inhibiting the activation of mTOR signaling and modulating neuroinflammation after stroke in rats.

Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag.

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Affinity Tags in Protein Purification and Peptide Enrichment: An Overview.

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific toward particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications in proteins and peptides widen the application of affinity ligand-tag receptors pairs toward universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely the affinity tags and receptors employed on the production of recombinant fusion proteins, and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.

GLOBAL AND TARGETED PROFILING OF GTP-BINDING PROTEINS IN BIOLOGICAL SAMPLES BY MASS SPECTROMETRY.

GTP-binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide-binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP-binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP-binding proteins. We review herein the recent developments in the application of MS-based techniques, together with general and widely used affinity enrichment approaches, for the proteome-wide and targeted capture, identification, and quantification of GTP-binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP-binding proteins are discussed. It can be envisaged that future applications of MS-based proteomics will lead to a better understanding about the roles of GTP-binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes.

In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.

Modular affinity-labeling of the cytosine demethylation base elements in DNA.

5-methylcytosine is the most studied DNA epigenetic modification, having been linked to diverse biological processes and disease states. The elucidation of cytosine demethylation has drawn added attention the three additional intermediate modifications involved in that pathway-5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-each of which may have distinct biological roles. Here, we extend a modular method for labeling base modifications in DNA to recognize all four bases involved in demethylation. We demonstrate both differential insertion of a single affinity tag (biotin) at the precise position of target elements and subsequent repair of the nicked phosphate backbone that remains following the procedure. The approach enables affinity isolation and downstream analyses without inducing widespread damage to the DNA.

Region-specific and dose-specific effects of chronic haloperidol exposure on [3H]-flumazenil and [3H]-Ro15-4513 GABAA receptor binding sites in the rat brain.

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABAAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.

Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells.

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.