HaloTag as a substrate-based macroautophagy reporter.

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy.

Optimizing human α-galactosidase for treatment of Fabry disease.

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease.

Evaluation of a procaspase-3 activator with hydroxyurea or temozolomide against high-grade meningioma in cell culture and canine cancer patients.

Background: High-grade meningioma is an aggressive type of brain cancer that is often recalcitrant to surgery and radiotherapy, leading to poor overall survival. Currently, there are no FDA-approved drugs for meningioma, highlighting the need for new therapeutic options, but development is challenging due to the lack of predictive preclinical models.

Methods: To leverage the known overexpression of procaspase-3 in meningioma, PAC-1, a blood-brain barrier penetrant procaspase-3 activator, was evaluated for its ability to induce apoptosis in meningioma cells.

Adapting protein sequences for optimized therapeutic efficacy.

Therapeutic proteins alleviate disease pathology by supplementing missing or defective native proteins, sequestering superfluous proteins, or by acting through designed non-natural mechanisms. Although therapeutic proteins often have the same amino acid sequence as their native counterpart, their maturation paths from expression to the site of physiological activity are inherently different, and optimizing protein sequences for properties that 100s of millions of years of evolution did not need to address presents an opportunity to develop better biological treatments. Because therapeutic proteins are inherently non-natural entities, optimization for their desired function should be considered analogous to that of small molecule drug candidates, which are optimized through expansive combinatorial variation by the medicinal chemist.

Using sulfuramidimidoyl fluorides that undergo sulfur(VI) fluoride exchange for inverse drug discovery.

Drug candidates that form covalent linkages with their target proteins have been underexplored compared with the conventional counterparts that modulate biological function by reversibly binding to proteins, in part due to concerns about off-target reactivity. However, toxicity linked to off-target reactivity can be minimized by using latent electrophiles that only become activated towards covalent bond formation on binding a specific protein. Here we study sulfuramidimidoyl fluorides, a class of weak electrophiles that undergo sulfur(VI) fluoride exchange chemistry.

Synergistic and targeted therapy with a procaspase-3 activator and temozolomide extends survival in glioma rodent models and is feasible for the treatment of canine malignant glioma patients.

Purpose: Glioblastoma is a deadly brain cancer with a median survival time of ∼15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics.

Small-Molecule Procaspase-3 Activation Sensitizes Cancer to Treatment with Diverse Chemotherapeutics.

Conventional chemotherapeutics remain essential treatments for most cancers, but their combination with other anticancer drugs (including targeted therapeutics) is often complicated by unpredictable synergies and multiplicative toxicities. As cytotoxic anticancer chemotherapeutics generally function through induction of apoptosis, we hypothesized that a molecularly targeted small molecule capable of facilitating a central and defining step in the apoptotic cascade, the activation of procaspase-3 to caspase-3, would broadly and predictably enhance activity of cytotoxic drugs. Here we show that procaspase-activating compound 1 (PAC-1) enhances cancer cell death induced by 15 different FDA-approved chemotherapeutics, across many cancer types and chemotherapeutic targets.

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed.

Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals that Raptinal-induced apoptosis proceeds with unparalleled speed.

Removal of Metabolic Liabilities Enables Development of Derivatives of Procaspase-Activating Compound 1 (PAC-1) with Improved Pharmacokinetics.

Procaspase-activating compound 1 (PAC-1) is an o-hydroxy-N-acylhydrazone that induces apoptosis in cancer cells by chelation of labile inhibitory zinc from procaspase-3. PAC-1 has been assessed in a wide variety of cell culture experiments and in vivo models of cancer, with promising results, and a phase 1 clinical trial in cancer patients has been initiated (NCT02355535). For certain applications, however, the in vivo half-life of PAC-1 could be limiting.

Dual small-molecule targeting of procaspase-3 dramatically enhances zymogen activation and anticancer activity.

Combination anticancer therapy typically consists of drugs that target different biochemical pathways or those that act on different targets in the same pathway. Here we demonstrate a new concept in combination therapy, that of enzyme activation with two compounds that hit the same biological target, but through different mechanisms. Combinations of procaspase-3 activators PAC-1 and 1541B show considerable synergy in activating procaspase-3 in vitro, stimulate rapid and dramatic maturation of procaspase-3 in multiple cancer cell lines, and powerfully induce caspase-dependent apoptotic death to a degree well exceeding the additive effect.

Haploinsufficiency of retinaldehyde dehydrogenase 2 decreases the severity and incidence of duodenal atresia in the fibroblast growth factor receptor 2IIIb-/- mouse model.

Background: Homozygous null mutation of the fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) gene in mice results in 42% of embryos developing duodenal atresias. Retinaldehyde dehydrogenase 2 (Raldh2, a gene critical for the generation of retinoic acid) is expressed in the mouse duodenum during the temporal window when duodenal atresias form. Raldh2 is critical for the normal development of the pancreatoduodenal region; therefore, we were interested in the effect of a Raldh2 mutation on duodenal atresia formation.

Formation of duodenal atresias in fibroblast growth factor receptor 2IIIb-/- mouse embryos occurs in the absence of an endodermal plug.

Purpose: Duodenal atresia in humans has been hypothesized to arise from a failure of the duodenal lumen to recanalize after formation of an endodermal plug. Recently, mutations in the fibroblast growth factor receptor 2 gene (Fgfr2IIIb) have been shown to cause atretic defects of the duodenum in mice. However, work in rats suggests that murine species do not form an endodermal plug during normal duodenal development.

Formation of intestinal atresias in the Fgfr2IIIb-/- mice is not associated with defects in notochord development or alterations in Shh expression.

Purpose: The etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen, and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacologic animal model of intestinal atresia.

Parallel synthesis and biological evaluation of 837 analogues of procaspase-activating compound 1 (PAC-1).

Procaspase-Activating Compound 1 (PAC-1) is an ortho-hydroxy N-acyl hydrazone that enhances the enzymatic activity of procaspase-3 in vitro and induces apoptosis in cancer cells. An analogue of PAC-1, called S-PAC-1, was evaluated in a veterinary clinical trial in pet dogs with lymphoma and found to have considerable potential as an anticancer agent. With the goal of identifying more potent compounds in this promising class of experimental therapeutics, a combinatorial library based on PAC-1 was created, and the compounds were evaluated for their ability to induce death of cancer cells in culture.

A more efficient method to generate null mutants using Hprt-Cre with floxed alleles.

Purpose: The generation of nonviable homozygous null mouse embryos from heterozygote null/+ breedings can be highly resource consuming, with only 25% of the embryos in the litter being null mutants. We hypothesized that (1) we could double the number of homozygous null mouse embryos in a litter without reducing litter size using Hypoxanthine-guanine phosphoribosyltransferase-Cre (Hprt)-Cre (which is active in the female germ line at the time of fertilization), and (2) these homozygous null mutants would be identical to mutants generated through traditional null/+ breedings.

Methods: To test this hypothesis, we used a conditional allele Fgfr2IIIb(flox).

Humans, mice, and mechanisms of intestinal atresias: a window into understanding early intestinal development.

Introduction: Intestinal atresias have long been hypothesized to result from either failure of recanalization of the intestinal lumen or in utero vascular accidents. Recent work in animal models is now calling for a reassessment of these widely held paradigms.

Purpose: In this review, we will examine the data that led to the original hypotheses and then evaluate more recent work challenging these hypotheses.

Preparation of the caspase-3/7 substrate Ac-DEVD-pNA by solution-phase peptide synthesis.

This protocol describes the gram-scale solution-phase synthesis of the colorimetric caspase-3/7 substrate Ac-DEVD-pNA. The caspase enzymes are integral to cellular inflammation and apoptotic cascades, and are commonly studied by cell biologists, medicinal chemists and chemical biologists. In particular, the assessment of caspase enzymatic activity is a standard method to evaluate cell death pathways and new apoptosis-modulating agents.

Experimental estimation of the role of P-Glycoprotein in the pharmacokinetic behaviour of telithromycin, a novel ketolide, in comparison with roxithromycin and other macrolides using the Caco-2 cell model.

Purpose: The aim of this study was to examine the transport mechanism of telithromycin in comparison with erythromycin, azithromycin, clarithromycin and roxithromycin.

Methods: These antibiotics were examined in Caco-2 cell monolayers in order to demonstrate the potential involvement of P-GP in the absorption process, using verapamil as a P-GP competitor. A model using concentration equilibrium conditions was developed to delineate passive and active permeability components of telithromycin and roxithromycin transport in order to predict absorption in humans.

HT29-MTX and Caco-2/TC7 monolayers as predictive models for human intestinal absorption: role of the mucus layer.

The permeability of 19 compounds in both the Caco-2/TC7 and HT29-MTX models was determined, and the ability of each model to predict intestinal absorption in humans was compared. Similar apparent permeability values (log P(app)) were obtained in both models for the majority of compounds tested, and plots of log P(app) versus fraction absorbed in humans gave comparable sigmoidal curves. A linear correlation was also observed between the log P(app) values derived from these two models, which suggests that HT29-MTX is an alternative model for absorption prediction in humans.

Absorption of angiotensin II antagonists in Ussing chambers, Caco-2, perfused jejunum loop and in vivo: importance of drug ionisation in the in vitro prediction of in vivo absorption.

The aims of this study were (i) to compare the absorption of three closely related inhibitors of angiotensin II, RU60018, RU60079 and HR720, in various in vitro and in vivo models, and (ii) to explain the differences in the results and to assess the importance of drug ionisation to predict absorption. Drug absorption was investigated in Ussing chambers, Caco-2 cell monolayers, perfused rat jejunum loops and in vivo after oral, intraduodenal or intravenous administration. In Ussing chambers, the analogues showed the same site-related absorption profile and a common mechanism involving the paracellular pathway.

The development of a test system for investigating the performances of personal aerosol samplers under actual workplace conditions.

The performances of new "total" aerosol samplers for use in workplaces are required to match the inhalability criteria as contained in the latest recommendations of the International Standards Organization (ISO) and the American Conference of Governmental Industrial Hygienists (ACGIH). In the past, practical evaluations have been carried out under idealized conditions in wind tunnels, and there is now the need to extend these to more realistic workplace conditions. This paper describes a new test system that was designed and built for this purpose.

Scale model studies of the transport of airborne pollutants on coalfaces.

Studies have been carried out to investigate airflows in coalmine models, with special regard to the transport of airborne pollutants, and to examine how they relate to what happens at full-scale in an actual underground mine. If such models can be shown to provide data representative of actual mine ventilation engineering, then they can provide cost-effective alternatives to full-scale investigations. The work set out in the first instance to identify the properties of: (a) the bulk airflow and associated transport of airborne pollutants along a longwall coalface; and (b) the transport of material out of regions that were partially enclosed or poorly ventilated (e.

Field comparison of personal samplers for inhalable dust.

The performances of two designs of personal inhalable fraction sampler, which have been shown to differ in laboratory wind tunnel studies, were assessed in 23 real industrial environments, relative to the collection of dust by a simulated worker. This took the form of a mobile, articulated, breathing robot which was fitted with an oral filter, and could be moved around the workplace to follow and mimic real workers. Statistical analysis of the resulting data show that if a small correction for bias is applied there is no significant difference either between the personal samplers as predictors of the 'real' worker exposure, or in the reproducibility obtained with the two personal samplers.

Cholinergic receptors on cultured neurones from the central nervous system of embryonic cockroaches.

Cultured neurones from the cockroach, Periplaneta americana, have been used to investigate putative acetylcholine receptors. Ligand-binding experiments revealed that these neurones possessed an alpha-bungarotoxin binding site that was saturable, had an apparent affinity constant of 3.51 nM and was predominantly nicotinic in nature.