High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology.

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi.

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2.

A high-throughput-compatible FRET-based platform for identification and characterization of botulinum neurotoxin light chain modulators.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin.

Identification of clinically viable quinolinol inhibitors of botulinum neurotoxin A light chain.

Botulinum neurotoxins (BoNT) are the most potent toxins known and a significant bioterrorist threat. Few small molecule compounds have been identified that are active in cell-based or animal models, potentially due to toxin enzyme plasticity. Here we screened commercially available quinolinols, as well as synthesized hydroxyquinolines.

The proinflammatory cytokines interleukin-1α and tumor necrosis factor α promote the expression and secretion of proteolytically active cathepsin S from human chondrocytes.

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated.

Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A: extending towards the β-exosite.

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to man, exposure to which results in flaccid paralysis. Given their extreme potency, these proteins have become studied as possible weapons of bioterrorism; however, effective treatments that function after intoxication have not progressed to the clinic. Here, we have reexamined one of the most effective inhibitors, 2,4-dichlorocinnamyl hydroxamate, in the context of the known plasticity of the BoNT/A light chain metalloprotease.

A coincidence detector triggers botulinum neurotoxin translocation.

Botulinum neurotoxins (BoNTs) are the deadliest poisons known to man. They possess a particular duality, rapidly increasing clinical utility for a wide range of disorders and large concern as a possible weapon of bioterrorism. While great strides have been made in the structural and biochemical understanding of the mechanism of intoxication, the specific molecular details behind BoNT translocation out of endosomes remain elusive.

Silver nanoparticles on titanate nanobelts via the self-assembly of weak polyelectrolytes: synthesis and photocatalytic properties.

A weak-polyelectrolyte multilayer on a surface of titanate nanobelts (Ti-NBs) was utilized as a template for in situ Ag nanoparticle formation in the fabrication of Ag-loaded Ti-NBs nanocomposites. The polyelectrolyte multilayer (PEM) was fabricated using layer-by-layer self-assembly of poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) on the surface of high-surface-area titanate nanobelts (Ti-NBs) synthesized using a hydrothermal procedure. The concentration of Ag nanoparticles in the PEM was controlled by repeating the ion-loading/reduction cycle.

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation.

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of 'Reverse Design' represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation.

Selective and sensitive monitoring of caspase-1 activity by a novel bioluminescent activity-based probe.

The role of caspase-1 in inflammation has been studied intensely over recent years. However, the research of caspase-1 has remained difficult mainly due to the lack of sensitive and selective tools to monitor not only its abundance but also its activity. Here we present a bioluminescent activity-based probe (ABP) for caspase-1, developed by the Reverse Design concept, where chemically optimized protease inhibitors are turned into selective substrate ABPs.

Expression and activity profiling of selected cysteine cathepsins and matrix metalloproteinases in synovial fluids from patients with rheumatoid arthritis and osteoarthritis.

Cysteine cathepsins and matrix metalloproteases are considered to play important roles in the development of arthritic diseases. Their accumulation in synovial fluid of primarily rheumatoid arthritis patients is also well documented. However, a detailed comparison between the protease levels and activities between rheumatoid arthritis samples and osteoarthritis samples has never been made.

Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.

Autocatalytic processing of procathepsin B is triggered by proenzyme activity.

Cathepsin B (EC 3.4.22.

Murine and human cathepsin B exhibit similar properties: possible implications for drug discovery.

Validation of drug targets and subsequent preclinical studies are usually carried out on animal disease models, with mouse being the most commonly used. However, results from mouse models cannot always be directly related to human disease. Major discrepancies between the properties of murine and human variants were observed during the evaluation of compounds targeting cathepsins S and K.

Glycosaminoglycans facilitate procathepsin B activation through disruption of propeptide-mature enzyme interactions.

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B.