Interaction of Colchicine-Site Ligands With the Blood Cell-Specific Isotype of β-Tubulin-Notable Affinity for Benzimidazoles.

Tubulin, the main component of microtubules, is an α-β heterodimer that contains one of multiple isotypes of each monomer. Although the isotypes of each monomer are very similar, the beta tubulin isotype found in blood cells is significantly divergent in amino acid sequence compared to other beta tubulins. This isotype, beta class VI, coded by human gene TUBB1, is found in hematologic cells and is recognized as playing a role in platelet biogenesis and function.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton.

A BODIPY-Based Probe Enables Fluorogenicity via Thiol-Dependent Modulation of Fluorophore Aggregation.

Given the popular usage of BODIPY fluorophores in biological research, their propensity to aggregate in aqueous solution and impact their spectroscopic properties arguably warrants more attention. The probe under study herein serves as a case in point. A -maleimide-substituted -phenyl BODIPY (p-MB) had previously been characterized in organic media, where its inherently high fluorescence ruled out its fluorogenic potential.

A Novel Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue.

Drug-induced kidney injury frequently leads to aborted clinical trials and drug withdrawals. Sufficiently sensitive sensors capable of detecting mild signs of chemical insult in cell-based screening assays are critical to identifying and eliminating potential toxins in the preclinical stage. Oxidative stress is a common early manifestation of chemical toxicity, and biomolecule carbonylation is an irreversible repercussion of oxidative stress.

Visualization of oxidative stress-induced carbonylation in live mammalian cells.

Oxidative stress (OS) is associated with a wide variety of diseases and disorders. Detection of oxidative stress in living systems typically relies on fluorescent probes for reactive oxygen species (ROS), which is challenging because of their short life span and high reactivity. Oxidative damage caused by OS produces a more stable signal, but these biomarkers are usually detected using techniques that are not compatible with live cells.

Taurine Is Covalently Incorporated into Alpha-Tubulin.

Taurine is the most abundant free amino acid in the human body. It is found in relatively high concentrations (1-10 mM) in many animal tissues but not in plants. It has been studied since the early 1800s but has not been found to be covalently incorporated into proteins in any animal tissue.

Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues.

Click Chemistry Conjugations.

Click chemistry has found wide application in bioconjugation, enabling control over the site of modification in biomolecules. Demonstrations of this chemistry to construct chemically defined antibody-drug conjugates (ADCs) have increased in recent years, following studies that support benefits of homogeneity and site-specificity of drug placement on the antibody. In this chapter, a brief history of early applications of this chemistry in ADCs is presented.

β-Hydroxy-Stabilized Boron-Nitrogen Heterocycles Enable Rapid and Efficient C-Terminal Protein Modification.

Bioorthogonal chemistry has enabled the development of bioconjugates in physiological environments while averting interference from endogenous biomolecules. Reactions between carbonyl-containing molecules and alkoxyamines or hydrazines have experienced a resurgence in popularity in bioorthogonal chemistry owing to advances that allow the reactions to occur under physiological conditions. In particular, -carbonyl-substituted phenylboronic acids (CO-PBAs) exhibit greatly accelerated rates of hydrazone and oxime formation via intramolecular Lewis acid catalysis.

Site-Specific Bioconjugation and Multi-Bioorthogonal Labeling via Rapid Formation of a Boron-Nitrogen Heterocycle.

Precise control of covalent bond formation in the presence of multiple functional groups is pertinent in the development of many next-generation bioconjugates and materials. Strategies derived from bioorthogonal chemistries are contributing greatly in that regard; however, the gain of chemoselectivity is often compromised by the slow rates of many of these existing chemistries. Recent work on a variation of the classical aldehyde/ketone condensation based on ortho-carbonylphenylboronic acids has uncovered markedly accelerated rates compared to those of the simple carbonyl counterparts.

Formation of hydrazones and stabilized boron-nitrogen heterocycles in aqueous solution from carbohydrazides and ortho-formylphenylboronic acids.

A recent addition to the suite of fast bioorthogonal reactions combines hydrazines and hydroxylamines with ortho-carbonyl substituted phenylboronic acids. Carbohydrazides are easily incorporated into biomolecules, making them appealing substrates in these reactions. Here we show that simple alkyl carbohydrazides form a single product with ortho-formylphenylboronic acid in an organic solvent and in the solid state.

Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells.

Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are nontoxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation.

Intramolecular Catalysis of Hydrazone Formation of Aryl-Aldehydes via ortho-Phosphate Proton Exchange.

Bioorthogonal site-specific chemical reaction to label biomolecules and in living cells is one of the most powerful and convenient tools in chemical biology. A reactive pairs frequently used for chemical conjugation are aldehydes/ketones with hydrazines/hydrazides/hydroxylamines. Although the reaction is generally specific for the two components, even in a cellular environment, the reaction is very slow under physiological conditions.

Turn on Fluorescent Probes for Selective Targeting of Aldehydes.

Two different classes of fluorescent dyes were prepared as a turn off/on sensor system for aldehydes. Amino derivatives of a boron dipyrromethene (BDP) fluorophore and a xanthene-derived fluorophore (rosamine) were prepared. Model compounds of their product with an aldehyde were prepared using salicylaldehyde.

Rapid formation of a stable boron-nitrogen heterocycle in dilute, neutral aqueous solution for bioorthogonal coupling reactions.

Combining 2-formylphenylboronic acid with 4-hydrazinylbenzoic acid in neutral aqueous solution at low, equimolar concentrations of the reagents results in a single, stable product, a 1,2-dihydro-1-hydroxy-2,3,1-benzodiazaborine, in a matter of minutes with no side products. Application of this reaction to protein conjugation demonstrates that the reaction is orthogonal to protein functional groups, and the resulting conjugate withstands SDS-PAGE analysis. This reaction should be particularly useful for couplings that must be performed with low concentrations of reagents under physiologically compatible conditions.

Postpartum Exercise and Lactation.

Many women who are breastfeeding also want to participate in exercise, but have concerns about the safety of their newborn. The following chapter reviews issues related to postpartum exercise and lactation. The goals of the chapter are to help clinicians understand the benefits of exercise, examine the impact of postpartum exercise on breastfeeding, and provide practical recommendations for exercise during the postpartum period in women who are breastfeeding.

Detection of oxidative stress-induced carbonylation in live mammalian cells.

Oxidative stress is often associated with etiology and/or progression of disease conditions, such as cancer, neurodegenerative diseases, and diabetes. At the cellular level, oxidative stress induces carbonylation of biomolecules such as lipids, proteins, and DNA. The presence of carbonyl-containing biomolecules as a hallmark of these diseases provides a suitable target for diagnostic detection.

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

Aims: Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription.

Results: Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation.

Synthesis of isotopically labeled epothilones.

The epothilones, including epothilones B and D, are macrocyclic lactones, which have potent cytotoxicities and promote the polymerization of tubulin to mictotubules by binding to and stabilizing the tubulin polymer. They have a very similar mechanism of action to paclitaxel (Taxol®). The determination of the microtubule-binding conformation of the epothilones is an important piece of information in designing improved analogs for possible clinical use, and internuclear distance information that will assist the determination of this conformation can be obtained by rotational echo double resonance (REDOR) NMR studies of microtubule-bound epothilones with appropriate stable isotope labels.

Measurement of in vitro microtubule polymerization by turbidity and fluorescence.

Tubulin polymerization may be conveniently monitored by the increase in turbidity (optical density, or OD) or by the increase in fluorescence intensity of diamidino-phenylindole. The resulting data can be a quantitative measure of microtubule (MT) assembly, but some care is needed in interpretation, especially of OD data. Buffer formulations used for the assembly reaction significantly influence the polymerization, both by altering the critical concentration for polymerization and by altering the exact polymer produced-for example, by increasing the production of sheet polymers in addition to MT.

Site-specific fluorescent labeling of tubulin.

Fluorescent tubulin can be prepared in which a fluorophore is covalently bound to the protein at only the carboxy terminus of the α-subunit of the αβ-tubulin dimer. This two-step procedure consists of an enzymatic reaction followed by a bioorthogonal chemical reaction. In the first step of the process, the enzyme tubulin tyrosine ligase is used to attach a reactive tyrosine derivative, 3-formyltyrosine, to the protein.

Dissecting paclitaxel-microtubule association: quantitative assessment of the 2'-OH group.

Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to β-tubulin in assembled microtubules. The 2'-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated.

Synthesis, cytotoxic properties and tubulin polymerization inhibitory activity of novel 2-pyrazoline derivatives.

A series of novel 1-(3',4',5'-trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives were synthesized and evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization inhibitory activity. Compounds 6d and 6e exhibited remarkable cytotoxic activity against different cancer cell lines with good tubulin polymerization inhibitory activity. Compound 6d exhibited moderate selectivity toward renal cancer and breast cancer subpanels with selectivity ratios of 3.

C6-C8 bridged epothilones: consequences of installing a conformational lock at the edge of the macrocycle.

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D.