Thermodynamic and Kinetic Study of Diels-Alder Reaction between Furfuryl Alcohol and N-Hydroxymaleimides-An Assessment for Materials Application.

The study of Diels-Alder reactions in materials science is of increasing interest. The main reason for that is the potential thermoreversibility of the reaction. Aiming to predict the behavior of a material modified with maleimido and furyl moieties, 1H NMR and UV-Vis solution studies of the Diels-Alder reaction between furfuryl alcohol and two N-hydroxymaleimides are explored in the present study. Rate constants, activation energy, entropy, and enthalpy of formation were determined from each technique for both reacting systems. Endo and exo isomers were distinguished in 1H NMR, and the transition from a kinetic, controlled Diels-Alder reaction to a thermodynamic one could be observed in the temperature range studied. A discussion on the effect of that on the application in a material was performed. The approach selected considers a simplified equilibrium of the Diels-Alder reaction as the kinetic model, allowing materials scientists to evaluate the suitability of using the reacting molecules for the creation of thermoresponsive materials. The proposed approach determines the kinetic constants without the direct influence of the equilibrium constant value, thereby allowing a more objective data analysis. The effects of the selection of kinetic model, analytical method, and data treatment are discussed.

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.

Single Pair Förster Resonance Energy Transfer: A Versatile Tool To Investigate Protein Conformational Dynamics.

Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer (spFRET) offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence (TIRF) microscopy offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review.

Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts.

Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures that have poor pharmacokinetic properties and decreased efficacy relative to homogenous ADCs. Furthermore, ADCs that are maleimide-based often have inadequate circulatory stability, which can result in premature drug release with consequent off-target toxicities. Selenocysteine-modified antibodies have been developed that allow site-specific antibody conjugation, yielding homogeneous ADCs. Herein, we survey several electrophilic functional groups that react with selenocystine with high efficiency. Several of these result in conjugates with stabilities that are superior to maleimide conjugates. Among these, the allenamide functional group reacts with notably high efficiency, leads to conjugates with remarkable stability, and shows exquisite selectivity for selenocysteine conjugation.

Synthesis and assessment of a maleimide functionalized BF2 azadipyrromethene near-infrared fluorochrome.

The first water soluble maleimide bearing NIR BF2-azadipyrromethene (NIR-AZA) fluorochrome has been synthesised which is capable of rapid thiol conjugations in water with peptides such as glutathione, the cell penetrating peptide (CPP) C(ß-A)SKKKKTKV-NH2 and a thiol substituted cRGD. NIR fluorescence imaging showed rapid cellular delivery of the CPP conjugate and effective in vivo tumour localization for the cRGD conjugate.

Monte Carlo method based QSAR modeling of maleimide derivatives as glycogen synthase kinase-3ß inhibitors.

The Monte Carlo method was used for QSAR modeling of maleimide derivatives as glycogen synthase kinase-3ß inhibitors. The first QSAR model was developed for a series of 74 3-anilino-4-arylmaleimide derivatives. The second QSAR model was developed for a series of 177 maleimide derivatives. QSAR models were calculated with the representation of the molecular structure by the simplified molecular input-line entry system. Two splits have been examined: one split into the training and test set for the first QSAR model, and one split into the training, test and validation set for the second. The statistical quality of the developed model is very good. The calculated model for 3-anilino-4-arylmaleimide derivatives had following statistical parameters: r(2)=0.8617 for the training set; r(2)=0.8659, and r(m)(2)=0.7361 for the test set. The calculated model for maleimide derivatives had following statistical parameters: r(2)=0.9435, for the training, r(2)=0.9262 and r(m)(2)=0.8199 for the test and r(2)=0.8418, r(av)(m)(2)=0.7469 and ∆r(m)(2)=0.1476 for the validation set. Structural indicators considered as molecular fragments responsible for the increase and decrease in the inhibition activity have been defined. The computer-aided design of new potential glycogen synthase kinase-3ß inhibitors has been presented by using defined structural alerts.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Assessment of different functionalization methods for grafting a protein to an alumina-covered biosensor.

The specificity of biosensors is typically obtained by surface biofunctionalization, which enables the selective binding of biomolecules. This critical step is sensitive to the nature of materials and to the overall experimental conditions. Here, we provide a comprehensive study of several biofunctionalization methods, including the layer-by-layer technique and both the gas-phase and liquid-phase silanizations, and we propose a new maleimide-based protocol for grafting a protein to a sensor covered by alumina. This method was then validated by making a respiratory syncitial virus-specific biosensor.

Assessment of physical stability of an antibody drug conjugate by higher order structure analysis: impact of thiol- maleimide chemistry.

PURPOSE: To provide a systematic biophysical approach towards a better understanding of impact of conjugation chemistry on higher order structure and physical stability of an antibody drug conjugate (ADC). METHODS: ADC was prepared using thiol-maleimide chemistry. Physical stabilities of ADC and its parent IgG1 mAb were compared using calorimetric, spectroscopic and molecular modeling techniques. RESULTS: ADC and mAb respond differently to thermal stress. Both the melting temperatures and heat capacities are substantially lower for the ADC. Spectroscopic experiments show that ADC and mAb have similar secondary and tertiary structures, but these are more easily destabilized by thermal stress on the ADC indicating reduced conformational stability. Molecular modeling calculations suggest a substantial decrease in the conformational energy of the mAb upon conjugation. The local surface around the conjugation sites also becomes more hydrophobic in the ADC, explaining the lower colloidal stability and greater tendency of the ADC to aggregate. CONCLUSIONS: Computational and biophysical analyses of an ADC and its parent mAb have provided insights into impact of conjugation on physical stability and pinpointed reasons behind lower structural stability and increased aggregation propensity of the ADC. This knowledge can be used to design appropriate formulations to stabilize the ADC.

Thioredoxin redox status assessment during embryonic development: the redox Western.

Oxidative stress can contribute significantly to the mechanisms that cause teratogenesis and embryotoxicity, but cellular antioxidant defense systems have evolved to counteract the detrimental consequences of oxidative insult. Numerous antioxidant systems exist in developing organisms but have been shown to be dynamically expressed and may only become functional during specific periods of development. Thioredoxins (Trxs) are a class of low-molecular-weight oxidoreductase antioxidants that play an important role in reactive oxygen species detoxification and transcription factor regulation, both of which are critical to normal development. In this chapter, we describe a method by which cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) redox status can be measured. In brief, derivatization of reduced Trx at the active site cysteine thiols using a large maleimide derivative, targeting the redox-sensitive residues making the protein larger. Nonreducing SDS-PAGE is performed to separate reduced (labeled) and oxidized (unlabeled) Trx based on their size. Band densitometries can be measured and used in the Nernst equation to calculate redox potentials. These approaches provide a means to evaluate this unique redox couple during development and allow for the measurement of compartmentalized (mitochondrial vs. cytosolic) oxidation during chemical insult.

Tumor targeting of functionalized lipid nanoparticles: assessment by in vivo fluorescence imaging.

Lipid nanoparticles (LNP) coated by a poly(oxyethylene) polymer have been manufactured from low cost and human use-approved materials, by an easy, robust, and up-scalable process. The incorporation in the formulation of maleimide-grafted surfactants allows the functionalization of the lipid cargos by targeting ligands such as the cRGD peptide binding to alpha(v)beta(3) integrin, a well-known angiogenesis biomarker. LNP are able to encapsulate efficiently lipophilic molecules such as a fluorescent dye, allowing their in vivo tracking using fluorescence imaging. In vitro study on HEK293(beta3) cells over-expressing the alpha(v)beta(3) integrins demonstrates the functionalization, specific targeting, and internalization of cRGD-functionalized LNP in comparison with LNP-cRAD or LNP-OH used as negative controls. Following their intravenous injection in Nude mice, LNP-cRGD can accumulate actively in slow-growing HEK293(beta3) cancer xenografts, leading to tumor over skin fluorescence ratio of 1.53+/-0.07 (n=3) 24h after injection. In another fast-growing tumor model (TS/A-pc), tumor over skin fluorescence ratio is improved (2.60+/-0.48, n=3), but specificity between the different LNP functionalizations is no more observed. The different results obtained for the two tumor models are discussed in terms of active cRGD targeting and/or passive nanoparticle accumulation due to the Enhanced Permeability and Retention effect.

Conformational analysis of trimeric maleimide substituted 1,5,9-triazacyclododecane HIV fusion scaffolds.

An analysis of the conformational preferences of three trimeric maleimide substituted 1,5,9-triazacyclododecane derivatives, proposed as cross linking reagents for HIV-1 fusion inhibitors, is presented. Exhaustive sampling was performed using the mixed Low Mode Monte Carlo conformational searching technique on the corresponding OPLS2005/GBSA(water) potential energy surface. Geometric structure, molecular length, and hydrogen bonding patterns of the compounds are analyzed. Global minimum energy structures were verified as minima using B3LYP/6-31G * geometry optimization. All structures adopt a crown-like 12-membered ring conformation; however, the system with the shortest maleimide arms (1a) can also adopt alternative ring orientations. Overall, derivatives with longer maleimide arms were more flexible and resulted in ensembles with a larger number of low energy structures. Comparison with biological inhibition data indicates that there is very little relationship between molecular size and the ability of the scaffold to orient CD4M9 miniproteins for optimal inhibition; however hydrophobicity may play a role.

LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.