Bioinformatics toolbox for exploring target mutation-induced drug resistance.

Drug resistance is increasingly among the main issues affecting human health and threatening agriculture and food security. In particular, developing approaches to overcome target mutation-induced drug resistance has long been an essential part of biological research. During the past decade, many bioinformatics tools have been developed to explore this type of drug resistance, and they have become popular for elucidating drug resistance mechanisms in a low cost, fast and effective way. However, these resources are scattered and underutilized, and their strengths and limitations have not been systematically analyzed and compared. Here, we systematically surveyed 59 freely available bioinformatics tools for exploring target mutation-induced drug resistance. We analyzed and summarized these resources based on their functionality, data volume, data source, operating principle, performance, etc. And we concisely discussed the strengths, limitations and application examples of these tools. Specifically, we tested some predictive tools and offered some thoughts from the clinician's perspective. Hopefully, this work will provide a useful toolbox for researchers working in the biomedical, pesticide, bioinformatics and pharmaceutical engineering fields, and a good platform for non-specialists to quickly understand drug resistance prediction.

DMSO-Related Effects on Ligand-Binding Properties of Lysine Methyltransferases G9a and SETD8.

Being the standard solvent for preparing stock solutions of compounds for drug discovery, DMSO is always present in assay buffers in concentrations ranging from 0.1 % to 5 % (v/v). Even at the lowest concentrations, DMSO-containing solutions can have significant effects on individual proteins and possible pitfalls cannot be eliminated. Herein, we used two protein systems, the lysine methyltransferases G9a/KMT1 C and SETD8/KMT5 A, to study the effects of DMSO on protein stability and on the binding of the corresponding inhibitors, using different biophysical methods such as nano Differential Scanning Fluorimetry (nanoDSF), Differential Scanning Fluorimetry (DSF), microscale thermophoresis (MST), and surface plasmon resonance (SPR), all widely used in drug discovery screening campaigns. We demonstrated that the effects of DMSO are protein- and technique-dependent and cannot be predicted or extrapolated on the basis of previous studies using different proteins and/or different assays. Moreover, we showed that the application of orthogonal biophysical methods can lead to different binding affinity data, thus confirming the importance of using at least two different orthogonal assays in screening campaigns. This variability should be taken into account in the selection and characterization of hit compounds, in order to avoid data misinterpretation.

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb.

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000-10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

Gold Teflates Revisited: From the Lewis Superacid [Au(OTeF5 )3 ] to the Anion [Au(OTeF5 )4 ].

A new synthetic access to the Lewis acid [Au(OTeF5 )3 ] and the preparation of the related, unprecedented anion [Au(OTeF5 )4 ]- with inorganic or organic cations starting from commercially available and easy-to-handle gold chlorides are presented. In this first extensive study of the Lewis acidity of a transition-metal teflate complex by using different experimental and quantum chemical methods, [Au(OTeF5 )3 ] was classified as a Lewis superacid. The solid-state structure of the triphenylphosphine oxide adduct [Au(OPPh3 )(OTeF5 )3 ] was determined, representing the first structural characterization of an adduct of this highly reactive [Au(OTeF5 )3 ]. Therein, the coordination environment around the gold center slightly deviates from the typical square planar geometry. The [Au(OTeF5 )4 ]- anion shows a similar coordination motif.

Determination of dissociation constants of protein ligands by thermal shift assay.

The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an extrinsic fluorescent dye, the TSA tracks protein unfolding. This method precisely determines the midpoint of the unfolding transition (Tm), which can shift upon the addition of a ligand. Though experimental protocols have been well developed, the thermal shift assay data traditionally yielded qualitative results. Quantitative methods for Kd determination relied either on empirical and inaccurate usage of Tm or on isothermal approaches, which do not take full advantage of the melting point precision provided by the TSA. We present a new analysis method based on a model that relies on the equilibrium system between the native and molten globule state of the protein using the van't Hoff equation. We propose the Kd can be determined by plotting Tm values versus the logarithm of ligand concentrations and fitting the data to an equation we derived. After testing this procedure with the monomeric maltose-binding protein and an allosterically regulated homotetrameric enzyme (ADP-glucose pyrophosphorylase), we observed that binding results correlated very well with previously established parameters. We demonstrate how this method could potentially offer a broad applicability to a wide range of protein classes and the ability to detect both active and allosteric site binding compounds.

Chitosan-based matrix solid-phase dispersion extraction assisted cell membrane magnetic bead ligand-affinity assay for screening active compounds from Fructus Cnidii.

Cell membrane ligand-affinity assay is a useful tool for screening the active compounds from natural products. However, in traditional cell membrane ligand-affinity assays, natural products need to be refluxed, before being analyzed. This process consumes considerable time and energy and cannot be used for screening natural products that contain thermally unstable compounds. Therefore, an efficient analytical method is required. In this study, chitosan-based matrix solid-phase dispersion extraction was combined with cell membrane magnetic bead ligand-affinity assay to form a novel method for identifying the active compounds in Fructus Cnidii such as osthole and imperatorin. When compared with traditional cell membrane ligand-affinity assays, this assay requires less energy, extraction time (7 min), solvent volume (1.2 mL), and fewer natural products (40 mg). This indicates that the chitosan-based matrix solid-phase dispersion extraction assisted cell membrane magnetic beads ligand-affinity assay is an alternative analytical method for studying natural products.

Identifying coupled clusters of allostery participants through chemical shift perturbations.

Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.

Small Heterocyclic Ligands as Anticancer Agents: QSAR with a Model G-Quadruplex.

G-quadruplexes (GQs) have become valid targets for anticancer studies in recent decades due to their multifaceted biological function. Herewith, we aim to quantify interactions of potential heterocyclic ligands (Ls) with model GQs. For seven 4-aminoquinazolines and three 2-heteroaryl perimidines, seven of this ten-membered group so far unknown, we use routine quantum chemical modeling. As shown in the literature, a preferred mode of interaction of heterocycles with cellular structures is stacking to exposable faces of G-quadruplexes. To exploit the energy of this interaction as a molecular descriptor and achieve the necessary chemical precision, we use state of the art large-scale density functional theory (DFT) calculations of stacked heterocycles to a GQ. Actually, the GQ has been simplified for the computation by stripping it off all pentose phosphate residues into a naked model of stacked guanine quartets. The described model thus becomes computable. The obtained heterocyclic ligand GQ.L stacking energies, that is, their GQ affinities, are the necessary ligand descriptors. Using the ligand biological inhibitory activities (IC50) on a human malignant melanoma A375 cell line, we obtain a good linear relationship between computed ligand stacking affinities to GQ, and experimental log (IC50) values. Based on the latter relationship, we discuss a putative mechanism of anticancer activity of heterocyclic ligands via stacking interactions with GQs and thereby controlling cell regulatory activity. This mechanism may tentatively be applied to other condensed five- and six-membered small heterocycles as well.

Method for efficient soluble expression and purification of recombinant human interleukin-15.

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.

Predicting protein-ligand affinity with a random matrix framework.

Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, we show that the similarity of an unknown ligand to the remaining, cleaned chemical features is a robust predictor of ligand-target affinity, performing as well or better than any algorithm in the published literature. We interpret our algorithm as deriving a model for the binding energy between a target receptor and the set of known ligands, where the underlying binding energy model is related to the classic Ising model in statistical physics.

Affinity monolith chromatography: A review of general principles and applications.

Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological-binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose, and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined.

(CF3 )3 Au as a Highly Acidic Organogold(III) Fragment.

The Lewis acidity of perfluorinated trimethylgold (CF3 )3 Au was assessed by theoretical and experimental methods. It was found that the (CF3 )3 Au unit is much more acidic than its nonfluorinated analogue (CH3 )3 Au, and probably sets the upper limit of the acidity scale for neutral organogold(III) species R3 Au. The significant acidity increase on fluorination is in line with the CF3 group being more electron-withdrawing than CH3 . The solvate (CF3 )3 Au⋅OEt2 (1) is presented as a convenient synthon of the unsaturated, 14-electron species (CF3 )3 Au. Thus, the weakly coordinated ether molecule in 1 is readily replaced by a variety of neutral ligands (L) to afford a wide range of (CF3 )3 Au⋅L compounds, which were isolated and characterized. Most of these mononuclear compounds exhibit marked thermal stability. This enhanced stabilization can be rationalized in terms of substantially stronger [Au]-L interactions with the (CF3 )3 Au unit. An affinity scale of this single-site, highly acidic organogold(III) fragment was calculated by DFT methods and experimentally mapped for various neutral monodentate ligands. The high-energy profile calculated for the fluorotropic [Au]-CF3 ⇌F-[Au]←CF2 process makes this potential decomposition path unfavorable and adds to the general stabilization of the fragment.

A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli.

G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

How exogenous ligand enhances the efficiency of cadmium phytoextraction from soils?

Many experiments showed that exogenous ligands could enhance cadmium (Cd) phytoextraction efficiency in soils. Previous studies suggested that the dissociation and the apoplastic uptake of Cd complex could not fully explain the increase of root Cd uptake. Two hypotheses are evaluated to explain enhanced Cd uptake in the presence of ligand: i) enhanced apoplastic uptake of complex due to reduced apoplastic resistance and ii) complex internalization by membrane transporters. RESULTS: show that the ligand affinity for Cd is a key characteristic determining the potential mechanism for enhanced Cd uptake. When low molecular weight organic acids are applied, the complex dissociation could generally be fast (> 10-3.3 s-1) and result in the increased Cd uptake. When hydrophilic aminopolycarboxylic acids (APCAs) are applied in experiments without water or temperature stresses to the plant, the root water uptake flux could very likely be high (> 10-7.8 dm s-1), and the strong apoplastic complex uptake could enhance the root Cd uptake. When lipophilic APCAs are applied, the strong internalization of the complex by membrane transporters could result in the increased Cd uptake if the maximum internalization rate is high (> 10-12 mol dm-2 s-1). However, the complex internalization by membrane transporters must be experimentally confirmed.

Structure-Activity Relationship and Functional Evaluation of Cannabinoid Type-1 Receptor.

The type-1 cannabinoid receptor (CB1R) is a potential therapeutic target in several pathological conditions, including neuropsychological disorders and neurodegenerative diseases. Owing to their structural diversity, it is not easy to derive general structure-activity relationships (SARs) for CB1R ligands. In this study, CB1R ligands were classified into six structural families, and the corresponding SAR was determined for their affinities for CB1R. In addition, we determined their functional activities for the activation of extracellular signal-regulated kinases (ERKs). Among derivatives of indol-3-yl-methanone, the highest ligand affinity was observed when a pentyl and a naphthalenyl group were attached to the N1 position of the indole ring and the carbon site of the methanone moiety, respectively. In the case of adamantane indazole-3-carboxamide derivatives, the presence of fluorine in the pentyl group, the substituent at the N1 position of the indazole ring, strongly increased the affinity for CB1R. For (naphthalen-1-yl) methanone derivatives, the presence of 4-alkoxynaphthalene in the methanone moiety was more beneficial for the affinity to CB1R than that of a heterocyclic ring. The functional activities of the tested compounds, evaluated through ERK assay, were correlated with their affinity for CB1R, suggesting their agonistic nature. In conclusion, this study provides valuable insight for designing novel ligands for CB1R, which can be used to control psychiatric disorders and drug abuse.

Biological and physico-chemical characterization of human norovirus-like particles under various environmental conditions.

Human noroviruses (HuNoVs) are the predominant etiological agent of viral gastroenteritis in all age groups worldwide. Mutations over the years have affected noroviruses' responses to environmental conditions due to the arrangement of amino acid residues exposed on the VP1 capsid surface of each strain. The GII.4 HuNoV genotype has been the predominant variant for decades, while the GII.17 genotype has often been detected in East Asia since 2014. Here, GII.17 and GII.4 baculovirus-expressed VLPs (virus-like particles) were used to study the biological (binding to HuNoV ligand, namely the ABO and Lewis antigens) and physicochemical properties (size, morphology, and charge) of the HuNoV capsid under different conditions (temperature, pH, and ionic strength). GII.17 showed stability at low and high ionic strength, while GII.4 aggregated at an ionic strength of 10 mM. The nature of the buffers influences the morphology and stability of the VLPs. Here, both VLPs were highly stable from pH 7-8.5 at 25 °C. VLPs retained HBGA binding capability for the pH, ionic strength and temperature encountered in the stomach (fed state) and the small intestine. Increasing the temperature to above 65 °C altered the morphology of VLPs, causing aggregation, and decreased their affinity to HBGAs. Comparing both isolates, GII.17 showed a better stability profile and higher affinity to HBGAs than GII.4, making them interesting candidate particles for a future norovirus vaccine. Biological and physicochemical studies of VLPs are as pertinent as ever in view of the future arrival of VLP-based HuNoV vaccines.

Human serum albumin depletion based on dye ligand affinity chromatography via magnetic microcryogels.

One of the primary purposes of proteomic studies is to analyze the proteins in the blood to be considered as biomarkers. Albumin, which constitutes the majority of total serum proteins, complicates the discovery of low-density proteins that are important for the diagnosis of diseases. Based on this, an alternative approach for albumin depletion was developed in this study by covalently attached Cibacron Blue 3GA (CB) to magnetic microcryogels. After detailed characterization of CB attached magnetic microcryogels synthesized via a microstencil array chip, albumin adsorption studies were performed to examine the optimum depletion conditions. In the presented study, the maximum albumin adsorption capacity (Qmax) was calculated as 149.25 mg/mL in pH 5.0 acetate buffer solution, which is the optimum pH value for albumin. Experimental studies have demonstrated that CB-attached magnetic microcryogels can be reused without loss of performance for albumin depletion after 10 adsorption-desorption cycles.

Prediction of protein-ligand binding affinity with deep learning.

The prediction of binding affinities between target proteins and small molecule drugs is essential for speeding up the drug research and design process. To attain precise and effective affinity prediction, computer-aided methods are employed in the drug discovery pipeline. In the last decade, a variety of computational methods has been developed, with deep learning being the most commonly used approach. We have gathered several deep learning methods and classified them into convolutional neural networks (CNNs), graph neural networks (GNNs), and Transformers for analysis and discussion. Initially, we conducted an analysis of the different deep learning methods, focusing on their feature construction and model architecture. We discussed the advantages and disadvantages of each model. Subsequently, we conducted experiments using four deep learning methods on the PDBbind v.2016 core set. We evaluated their prediction capabilities in various affinity intervals and statistically and visually analyzed the samples of correct and incorrect predictions for each model. Through visual analysis, we attempted to combine the strengths of the four models to improve the Root Mean Square Error (RMSE) of predicted affinities by 1.6% (reducing the absolute value to 1.101) and the Pearson Correlation Coefficient (R) by 2.9% (increasing the absolute value to 0.894) compared to the current state-of-the-art method. Lastly, we discussed the challenges faced by current deep learning methods in affinity prediction and proposed potential solutions to address these issues.

A natural goldmine of binding proteins and soluble receptors simplified their translation to blockbuster drugs, all in one decade.

Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the "triumph". My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNß proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn's disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.

Biophysical and biochemical insights in the design of immunoassays.

BACKGROUND: Rapid antigen assays have been attractive for decentralized, point of care diagnostics because of their low cost, robustness, and ease of use. The development of a diagnostic assay for a newly emerging infectious disease needs to take into account the progression of a disease, whether there is human to human transmission, and patient biomarker levels with time, and these all impact the choice of antigen targets and affinity agents. SCOPE OF REVIEW: The factors involved in the biophysical design of rapid antigen immunoassays are discussed, focusing on antigen selection and designing for cross-reactivity. State of the art in the biophysical characterization of protein-ligand or antigen-antibody interactions, the different types of affinity agents used in immunoassays, and biochemical conjugation strategies are described. MAJOR CONCLUSIONS: Antigen choice is a critical factor in immunoassay diagnostic development, and should account for the properties of the virion, virus, and disease progression. Biophysical and biochemical aspects of immunoassays are critical for performance. GENERAL SIGNIFICANCE: This review can serve as an instructive guide to aid in diagnostic development for future emerging diseases.