cellular-membrane-affinity-chromatography-columns-to-identify

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

The University of Alabama

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity and the binding to voltage-gated sodium channels (VGSCs) and GABA receptors. Docking simulations allowed us to determine the mode of interactions between the investigated compounds and binding cavity of the human VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial membrane permeability assay (PAMPA), single cell gel electrophoresis (SCGE) and cytotoxicity evaluation in HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The combined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising agents in the development of new anticonvulsants.

Development of the 1,2,4-triazole-based anticonvulsant drug candidates acting on the voltage-gated sodium channels. Insights from in-vivo, in-vitro, and in-silico studies.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder with no cure. Despite intensive research, most of the currently available therapies are only effective in alleviating symptoms with no effect on disease progression. There is an urgent need for new therapeutics to impede disease progression. Natural products are valuable sources of bioactive compounds that can be exploited for novel therapeutic potential in PD pathogenesis. However, rapid screening of plant-derived natural products and characterization of bioactive compounds is costly and challenging. , commonly known as the fruit fly, has recently emerged as an excellent model for human neurodegenerative diseases, including PD. The high degree of conserved molecular pathways with mammalian models make  PD models an inexpensive solution to preliminary phases of target validation in the drug discovery pipeline. The present review provides an overview of drug discovery from natural extracts using  as a screening platform to evaluate the therapeutic potential of phytochemicals against PD.

Using  as a platform for drug discovery from natural products in Parkinson's disease.

Parkinson's disease is an age-associated neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons from the midbrain. Epidemiological studies have implicated exposures to environmental toxins like the herbicide paraquat as major contributors to Parkinson's disease etiology in both mammalian and invertebrate models. We have employed a paraquat-induced Parkinson's disease model in Drosophila as an inexpensive in vivo platform to screen therapeutics from natural products. We have identified the polymethoxyflavonoid, GardeninA, with neuroprotective potential against paraquat-induced parkinsonian symptoms involving reduced survival, mobility defects, and loss of dopaminergic neurons. GardeninA-mediated neuroprotection is not solely dependent on its antioxidant activities but also involves modulation of the neuroinflammatory and cellular death responses. Furthermore, we have successfully shown GardeninA bioavailability in the fly heads after oral administration using ultra-performance liquid chromatography and mass spectrometry. Our findings reveal a molecular mechanistic insight into GardeninA-mediated neuroprotection against environmental toxin-induced Parkinson's disease pathogenesis for novel therapeutic intervention.

GardeninA confers neuroprotection against environmental toxin in a Drosophila model of Parkinson's disease.

The use of natural products has been shown to be a fruitful approach in the discovery of novel pharmaceuticals. In fact, many currently approved drugs originated from compounds that were first identified in nature. Chemical diversity of natural compounds cannot be matched by man-made libraries of chemically synthesized molecules. Many natural compounds interact with and modulate regulatory protein targets and can be considered evolutionarily-optimized drug-like molecules. Despite this, many pharmaceutical companies have reduced or eliminated their natural product discovery programs in the last two decades. Screening natural products for pharmacologically active compounds is a challenging task that requires high resource commitment. Novel approaches at the early stage of the drug discovery pipeline are needed to allow for rapid screening and identification of the most promising molecules. Here, we review the possible evolutionary roots for drug-like characteristics of numerous natural compounds. Since many of these compounds target evolutionarily conserved cellular signaling pathways, we propose novel, early-stage drug discovery approaches to identify drug candidates that can be used for the potential prevention and treatment of neurodegenerative diseases. Invertebrate in vivo animal models of neurodegenerative diseases and innovative tools used within these models are proposed here as a screening funnel to identify new drug candidates and to shuttle these hits into further stages of the drug discovery pipeline.

Drug discovery from natural products - Old problems and novel solutions for the treatment of neurodegenerative diseases.

Brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) have been shown to play an important role in numerous neurological disorders, such as Alzheimer's disease. The identification of biologically active compounds interacting with TrkB serves as a drug discovery strategy to identify drug leads for neurological disorders. Here, we report effective immobilization of functional TrkB on magnetic iron oxide nanoclusters, where TrkB receptors behave as "smart baits" to bind compounds from mixtures and magnetic nanoclusters enable rapid isolation through magnetic separation. The presence of the immobilized TrkB was confirmed by specific antibody labeling. Subsequently, the activity of the TrkB on iron oxide nanoclusters was evaluated with ATP/ADP conversion experiments using a known TrkB agonist. The immobilized TrkB receptors can effectively identify binders from mixtures containing known binders, synthetic small molecule mixtures, and Gotu Kola () plant extracts. The identified compounds were analyzed by an ultrahigh-performance liquid chromatography system coupled with a quadrupole time-of-flight mass spectrometer. Importantly, some of the identified TrkB binders from Gotu Kola plant extracts matched with compounds previously linked to neuroprotective effects observed for a Gotu Kola extract approved for use in a clinical trial. Our studies suggest that the possible therapeutic effects of the Gotu Kola plant extract in dementia treatment, at least partially, might be associated with compounds interacting with TrkB. The unique feature of this approach is its ability to fast screen potential drug leads using less explored transmembrane targets. This platform works as a drug-screening funnel at early stages of the drug discovery pipeline. Therefore, our approach will not only greatly benefit drug discovery processes using transmembrane proteins as targets but also allow for evaluation and validation of cellular pathways targeted by drug leads.

Lukasz M. Ciesla

Department of Biological Sciences

The University of Alabama

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B