Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes.

Covalent inhibitors are emerging as a promising therapeutic means for efficient and sustained targeting of key disease-driving proteins. As for classic non-covalent inhibitors, understanding target engagement and selectivity is essential for determining optimal dosing and limiting potential on- or off-target toxicity. Here, we present a complementary activity-based protein profiling (ABPP) strategy for unbiased proteome-wide profiling of cysteine-reactive inhibitors based on two orthogonal approaches. We illustrate the use of clickable alkyne probes for in-gel fluorescence and mass spectrometry studies using a series of therapeutic XPO1 inhibitors as an example.

Low-cost luminescent scaffolds-based on thiol chitosans by microwave radiation for vertebral disc repair/theragnostic.

This study introduces a new 3D scaffold based on thiolated chitosans with luminescence by microwave radiation using cysteine (Chi_CT_Cys) and 11-mercaptoundecanoic acid (Chi_CT_MUA) for vertebral disc regeneration/theragnostic. These scaffolds were characterized by Raman, PL spectroscopy, swelling, gel-fraction, and morphologies. Cytocompatibility and mechanical behavior were evaluated. Raman showed that disulfide bonds improved the grafting degree (Chi_CT_Cys (1072 ± 136) µmol·g-1 and Chi_CT_MUA (3245 ± 105) µmol·g-1). Morphologies showed interesting characteristics. Swelling behavior showed that Chi_CT_MUA presented a slight minor swelling (2101 ± 251) % compared to Chi_CT_Cys (2589 ± 188) %. Differently, gel-fraction showed that the chemical stability of Chi_CT_Cys was worse (29 ± 4) % than Chi_CT_MUA (15 ± 3) %. PL showed a possibility to use theragnostic evaluation of points of greater compression in a vertebral disc. The mechanical behavior of Chi_CT_MUA presented better results ((70 ± 3) MPa) than Chi_CT_Cys ((37 ± 3) MPa). Cytocompatible showed that the scaffolds presented cell viability >90%. Thusly, these 3D scaffolds presented an incredible potential for tissue engineering applications.

Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation.

Mass spectrometry-based chemoproteomics has enabled functional analysis and small molecule screening at thousands of cysteine residues in parallel. Widely adopted chemoproteomic sample preparation workflows rely on the use of pan cysteine-reactive probes such as iodoacetamide alkyne combined with biotinylation via copper-catalyzed azide-alkyne cycloaddition (CuAAC) or "click chemistry" for cysteine capture. Despite considerable advances in both sample preparation and analytical platforms, current techniques only sample a small fraction of all cysteines encoded in the human proteome. Extending the recently introduced labile mode of the MSFragger search engine, here we report an in-depth analysis of cysteine biotinylation via click chemistry (CBCC) reagent gas-phase fragmentation during MS/MS analysis. We find that CBCC conjugates produce both known and novel diagnostic fragments and peptide remainder ions. Among these species, we identified a candidate signature ion for CBCC peptides, the cyclic oxonium-biotin fragment ion that is generated upon fragmentation of the N(triazole)-C(alkyl) bond. Guided by our empirical comparison of fragmentation patterns of six CBCC reagent combinations, we achieved enhanced coverage of cysteine-labeled peptides. Implementation of labile searches afforded unique PSMs and provides a roadmap for the utility of such searches in enhancing chemoproteomic peptide coverage.

Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Glutenin and Gliadin, a Piece in the Puzzle of their Structural Properties in the Cell Described through Monte Carlo Simulations.

Gluten protein crosslinking is a predetermined process where specific intra- and intermolecular disulfide bonds differ depending on the protein and cysteine motif. In this article, all-atom Monte Carlo simulations were used to understand the formation of disulfide bonds in gliadins and low molecular weight glutenin subunits (LMW-GS). The two intrinsically disordered proteins appeared to contain mostly turns and loops and showed "self-avoiding walk" behavior in water. Cysteine residues involved in intramolecular disulfide bonds were located next to hydrophobic peptide sections in the primary sequence. Hydrophobicity of neighboring peptide sections, synthesis chronology, and amino acid chain flexibility were identified as important factors in securing the specificity of intramolecular disulfide bonds formed directly after synthesis. The two LMW-GS cysteine residues that form intermolecular disulfide bonds were positioned next to peptide sections of lower hydrophobicity, and these cysteine residues are more exposed to the cytosolic conditions, which influence the crosslinking behavior. In addition, coarse-grained Monte Carlo simulations revealed that the protein folding is independent of ionic strength. The potential molecular behavior associated with disulfide bonds, as reported here, increases the biological understanding of seed storage protein function and provides opportunities to tailor their functional properties for different applications.

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies.

The production of antibody-drug conjugates (ADCs) has been in great demand in the field of cancer therapeutics. Although cysteine-based conjugation is the most common and well known process for producing ADCs, multiple analytical methods are required for accurate drug-antibody ratio (DAR) determination due to the heterogeneity of the ADCs. Here we report various analytical methods for DAR analysis of traditional cysteine-based ADCs; additionally, apply a good manufacturing practice (GMP) strategy to produce a four hundred milligram ADC batch for use in good laboratory practice (GLP) studies. The work described herein not only evaluates several analytical performances but also provides guidance for future phase appropriate ADC production while establishing a unique analytical strategy.

A Simple and Cost Effective Turn off Fluorescence Sensor for Biliverdin and Bilirubin Based on L-Cysteine Modulated Copper Nanoclusters.

The present article reports the efficiency of L-cysteine modulated copper nanoclusters (L-cys-CuNCs) as a fluorescent probe for the selective determination of naturally occurring bile pigments biliverdin (BVD) and bilirubin (BLR). These pigments were found to quench the fluorescence of L-cys-CuNCs through static processes. Under optimized conditions, the proposed strategy permitted the quantification of BVD and BLR in the range 4.00 × 10-5 to 5.00 × 10-7M and 1.00×10-5 to 1.00×10-6 M respectively with limits of detection 2.33 × 10-7M and 2.29 × 10-7 M. The practical utility of the developed sensor have been investigated in spiked blood and urine samples.

An efficient, step-economical strategy for the design of functional metalloproteins.

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein-protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres-including a penta-His-coordinated non-haem Fe site-and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.

An Assessment of Computational Methods for Calculating Accurate Structures and Energies of Bio-Relevant Polysulfur/Selenium-Containing Compounds.

The heavier chalcogens sulfur and selenium are important in organic and inorganic chemistry, and the role of such chalcogens in biological systems has recently gained more attention. Sulfur and, to a lesser extent selenium, are involved in diverse reactions from redox signaling to antioxidant activity and are considered essential nutrients. We investigated the ability of the DFT functionals (B3LYP, B3PW91, ωB97XD, M06-2X, and M08-HX) relative to electron correlation methods MP2 and QCISD to produce reliable and accurate structures as well as thermochemical data for sulfur/selenium-containing systems. Bond lengths, proton affinities (PA), gas phase basicities (GPB), chalcogen⁻chalcogen bond dissociation enthalpies (BDE), and the hydrogen affinities (HA) of thiyl/selenyl radicals were evaluated for a range of small polysulfur/selenium compounds and cysteine per/polysulfide. The S⁻S bond length was found to be the most sensitive to basis set choice, while the geometry of selenium-containing compounds was less sensitive to basis set. In mixed chalcogens species of sulfur and selenium, the location of the sulfur atom affects the S⁻Se bond length as it can hold more negative charge. PA, GPB, BDE, and HA of selenium systems were all lower, indicating more acidity and more stability of radicals. Extending the sulfur chain in cysteine results in a decrease of BDE and HA, but these plateau at a certain point (199 kJ mol-1 and 295 kJ mol-1), and PA and GPB are also decreased relative to the thiol, indicating that the polysulfur species exist as thiolates in a biological system. In general, it was found that ωB97XD/6-311G(2d,p) gave the most reasonable structures and thermochemistry relative to benchmark calculations. However, nuances in performance are observed and discussed.

A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.

A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine-Maleimide-Based Antibody Drug Conjugates.

Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol-maleimide conjugation chemistry.

Assessment of Cysteine Reactivity of Human Hemoglobin at Its Residue Level: A Mass Spectrometry-Based Approach.

In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α2ß2) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site-specific reactivities of cysteine residues of Hb were investigated using alkylation kinetics with iodoacetamide (IAM). The observed reactivities corroborated well with the differential surface accessibilities of cysteine residues in the crystal structures of human Hb. The proposed method might be used to investigate cysteine reactivities of all the genetic and post-translational variants of Hb discovered to date. In addition, this method can be extended to explore cysteine reactivities of proteins, irrespective of the presence of chromophores and availability of crystal structures.

Ion Channel Conformation and Oligomerization Assessment by Site-Directed Spin Labeling and Pulsed-EPR.

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail.

Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes.

Protein cysteinyl residues are the mediators of hydrogen peroxide (H2O2)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H2O2 in human cells. We identified >900 H2O2-sensitive cysteines, which are defined as the H2O2-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H2O2-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H2O2-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.

The role of electrostatics in TrxR electron transfer mechanism: A computational approach.

Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a "shuttle," the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson-Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N-terminal (FAD and Cys59/64) and C-terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired "shuttle." Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the "shuttle" mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836-1843. © 2016 Wiley Periodicals, Inc.

Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1.

Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1). Thermogenesis from these adipocytes can combat obesity and diabetes, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.

Characterization and Higher-Order Structure Assessment of an Interchain Cysteine-Based ADC: Impact of Drug Loading and Distribution on the Mechanism of Aggregation.

The impact of drug loading and distribution on higher order structure and physical stability of an interchain cysteine-based antibody drug conjugate (ADC) has been studied. An IgG1 mAb was conjugated with a cytotoxic auristatin payload following the reduction of interchain disulfides. The 2-D LC-MS analysis shows that there is a preference for certain isomers within the various drug to antibody ratios (DARs). The physical stability of the unconjugated monoclonal antibody, the ADC, and isolated conjugated species with specific DAR, were compared using calorimetric, thermal, chemical denaturation and molecular modeling techniques, as well as techniques to assess hydrophobicity. The DAR was determined to have a significant impact on the biophysical properties and stability of the ADC. The CH2 domain was significantly perturbed in the DAR6 species, which was attributable to quaternary structural changes as assessed by molecular modeling. At accelerated storage temperatures, the DAR6 rapidly forms higher molecular mass species, whereas the DAR2 and the unconjugated mAb were largely stable. Chemical denaturation study indicates that DAR6 may form multimers while DAR2 and DAR4 primarily exist in monomeric forms in solution at ambient conditions. The physical state differences were correlated with a dramatic increase in the hydrophobicity and a reduction in the surface tension of the DAR6 compared to lower DAR species. Molecular modeling of the various DAR species and their conformers demonstrates that the auristatin-based linker payload directly contributes to the hydrophobicity of the ADC molecule. Higher order structural characterization provides insight into the impact of conjugation on the conformational and colloidal factors that determine the physical stability of cysteine-based ADCs, with implications for process and formulation development.

Sorting of Single Biomolecules based on Fourier Polar Representation of Surface Enhanced Raman Spectra.

Surface enhanced Raman scattering (SERS) spectroscopy becomes increasingly used in biosensors for its capacity to detect and identify single molecules. In practice, a large number of SERS spectra are acquired and reliable ranking methods are thus essential for analysing all these data. Supervised classification strategies, which are the most effective methods, are usually applied but they require pre-determined models or classes. In this work, we propose to sort SERS spectra in unknown groups with an alternative strategy called Fourier polar representation. This non-fitting method based on simple Fourier sine and cosine transforms produces a fast and graphical representation for sorting SERS spectra with quantitative information. The reliability of this method was first investigated theoretically and numerically. Then, its performances were tested on two concrete biological examples: first with single amino-acid molecule (cysteine) and then with a mixture of three distinct odorous molecules. The benefits of this Fourier polar representation were highlighted and compared to the well-established statistical principal component analysis method.

Organic chloramines in drinking water: An assessment of formation, stability, reactivity and risk.

Although organic chloramines are known to form during the disinfection of drinking water with chlorine, little information is currently available on their occurrence or toxicity. In a recent in vitro study, some organic chloramines (e.g. N-chloroglycine) were found to be cytotoxic and genotoxic even at micromolar concentrations. In this paper, the formation and stability of 21 different organic chloramines, from chlorination of simple amines and amino acids, were studied, and the competition between 20 amino acids during chlorination was also investigated. For comparison, chlorination of two amides was also conducted. The formation and degradation of selected organic chloramines were measured using either direct UV spectroscopic or colorimetric detection. Although cysteine, methionine and tryptophan were the most reactive amino acids towards chlorination, they did not form organic chloramines at the chlorine to precursor molar ratios that were tested. Only 6 out of the 21 organic chloramines formed had a half-life of more than 3 h, although this group included all organic chloramines formed from amines. A health risk assessment relating stability and reactivity data from this study to toxicity and precursor abundance data from the literature indicated that only N-chloroglycine is likely to be of concern due to its stability, toxicity and abundance in water. However, given the stability of organic chloramines formed from amines, more information about the toxicity and precursor abundance for these chloramines is desirable.

In Vitro Assessment of Interaction Between Amino Acids and Copper in Neonatal Parenteral Nutrition.

BACKGROUND: The repeated blackening of in-line filters has been observed during the infusion of parenteral nutrition 2-in-1 mixtures (binary parenteral nutrition [BPN]) delivered in a neonatal intensive care unit. This study aimed to examine the elemental content of precipitates isolated from infused BPN bags and determine the main physicochemical interactions occurring in these bags. MATERIALS AND METHODS: The infusion of BPN mixtures was simulated in vitro following hospital practices. Filter membranes were examined by scanning electron microscopy and energy dispersion spectroscopy (EDS). Amino acid (AA) profiles were obtained from BPN mixtures to determine the concentrations of each AA. RESULTS: Analyzed filter membranes revealed conglomerates of particles on filter surfaces. An EDS analysis generated spectra from isolated particles, identifying copper and sulfur as the major chemical elements. AA mean concentrations were relatively close to the expected value for each AA, except cysteine. Cysteine concentrations were very significantly lower than the expected values. CONCLUSION: A specific interaction was identified between 1 AA (cysteine) and a trace element (copper) in our BPN mixtures.