Name: cellular membrane affinity chromatography columns to identify specialized plant metabolites interacting with immobilized tropomyosin kinase receptor b
Uploaded: 2022-01-19T12:30:00
Description: Watch this Scientific Journal Video about Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B at JoVE

Z.C.A. was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) 2219- International Postdoctoral Research Fellowship Program. Research reported in this publication was supported by the National Center for Complimentary and Integrative Medicine of the National Institutes of Health under award number 1R41AT011716-01. This work was also partially supported by American Society of Pharmacognosy Research Starter Grant, Regis Technologies grant to L.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Cell culture of SH-SY5Y neuroblastoma cells (TrkB and TrkB-NULL (parental) cell lines)
NOTE: Cell lines (SH-SY5Y Cell Line (TrkB, BR6) and SH-SY5Y Parental Cell Line (TrkB NULL))49,50 were purchased from Kerafast. Cultured cells are used as a source of the transmembrane receptors to be immobilized for the preparation of CMAC columns. The following steps describe how to obtain cell membrane fragments and assemble funct...

Identification of active compounds present in complex mixtures of specialized metabolites is a very challenging task23. Traditionally, individual compounds are isolated, and their activity is tested in different assays. This approach is time-consuming and costly and often leads to isolation and identification of the most abundant and well-characterized compounds23.&#160;Currently used high-throughput screening assays rely heavily on screening combinatorial chemistry librari...

Botanical mixtures are rich in pharmacologically active compounds1, serving as a good source for the identification of new drug hits and leads2,3,4,5.&#160;The discovery of new medicines from natural products has been a fruitful approach and many currently approved drugs originated from compounds first identified in nature. The chemical diversity of natural compounds is hard to be matched by man-made libraries of chemically synthesized molecules. Many natural compounds interact with and modulate human protein targets and can be considered evolutionarily optimized drug-like molecules6. These natural compounds are particularly well suited for drug lead identification to use in neurological disorders6. Two of the currently FDA-approved drugs for the management of Alzheimer&#39;s disease (AD) are derived from natural alkaloids, namely: galantamine and rivastigmine (a derivative of physostigmine)6. L-DOPA, presently the most commonly prescribed drug for Parkinson&#39;s disease, was first identified from the broad bean (Vicia faba L.)7. Pergolide and lisuride, dopaminergic receptor agonists are the derivatives of natural ergot alkaloids from the parasitic fungus Claviceps purpurea8. Reserpine, an alkaloid isolated from Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz)&#160;was one of the first antipsychotic drugs9. Recently, dysregulated immune response and systemic inflammation have been linked to the development of numerous neurological ailments, such as major depressive disorder or neurodegenerative diseases10. A plant-based diet together with other lifestyle interventions has been found to improve cognitive and functional abilities in the elderly11,12,13,14,15,16,17,18,19,20,21. Certain electrophilic molecules belonging to triterpenes and polyphenols have been found to modulate inflammatory responses in both in vitro and in vivo models12. For instance, natural compounds containing &#945;,&#946;-unsaturated carbonyl (e.g., curcumin, cinnamaldehyde), or isothiocyanate group (e.g., sulforaphane) interfere with Toll-like receptor-4 (TLR4) dimerization inhibiting the downstream synthesis of pro-inflammatory cytokines in a murine interleukin-3 dependent pro-B cell line12,22. Epidemiological evidence points strongly that dietary phytochemicals, present in complex food matrices, may also constitute a viable source of new drug leads6.
One of the major obstacles in the identification of biologically active molecules present in plant extracts, including plant-based food, is the complexity of the investigated samples. Traditionally, the individual compounds are isolated, purified, and subsequently tested for biological activity. This approach usually leads to the identification of the most abundant and well-characterized compounds. Phenotypic drug discovery approaches without a defined molecular target rely on the bio-guided-fractionation of complex mixtures23. In this approach, an extract is fractionated into less complex sub-fractions that are subsequently tested in phenotypic assays. The isolation and purification of active compounds are guided by biological activity verified in the assay. The knowledge of the identity of a specified drug target may significantly speed up the identification of pharmacologically active compounds present in complex mixtures. Those approaches are usually based on the immobilization of the molecular target, for example, an enzyme, on a solid surface, like magnetic beads23. The immobilized targets are subsequently used in the screening experiments resulting in the isolation of compounds interacting with the target. While this approach has been extensively used in the identification of compounds targeting cytosolic proteins, it has been less commonly applied in the identification of chemicals interacting with transmembrane proteins (TMPs)23. An additional challenge in the immobilization of TMPs stems from the fact that the activity of the protein depends on its interaction with cell membrane phospholipids and other molecules in the bilayer such as cholesterol23,24. It is important to preserve these subtle interactions between proteins and their native phospholipid bilayer environment when attempting to immobilize the transmembrane target.
In cellular membrane affinity chromatography (CMAC) cell membrane fragments, and not purified proteins, are immobilized on the artificial membrane (IAM) stationary phase particles23. IAM stationary phases are prepared by covalently bonding phosphatidylcholine analogs onto silica. Recently novel classes of IAM stationary phases have been developed in which free amine and silanol groups are end-capped (IAM.PC.DD2 particles). During CMAC columns preparation cell membrane fragments are immobilized onto the surface of IAM particles through adsorption.
CMAC columns have been used to date to immobilize different classes of TMPs including ion channels (e.g., nicotinic receptors), GPCRs (e.g., opioid receptors), protein transporters (e.g., p-glycoprotein), etc.24. The immobilized protein targets have been used in the characterization of pharmacodynamics (e.g., dissociation constant, Kd) or determining binding kinetics (kon and koff) of small molecule ligands interacting with the target as well as in the process of identification of potential new drug leads present in complex matrices24. Here we present the preparation of CMAC columns with the immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders.
Previous studies showed that the activation of the brain-derived neurotrophic factor (BDNF)/TrkB pathway is associated with the improvement of certain neurological ailments, such as AD or major depressive disorder25,26,27,28. It was reported that BDNF levels and its receptor TrkB expression decrease in AD, and similar reductions impair hippocampal function in animal models of AD29. Decreased levels of BDNF were reported in serum and brain of AD patients30,31,32. Tau overexpression or hyperphosphorylation were found to down-regulate BDNF expression in primary neurons and AD animal models33,34,35. Additionally, BDNF was reported to have protective effects on &#946;-amyloid induced neurotoxicity in vitro and in vivo36. Direct BDNF administration into the rat brain was shown to increase learning and memory in cognitively impaired animals37. BDNF/TrkB emerged as a valid target for ameliorating neurological and psychiatric disorders including AD28,38. Targeting the BDNF/TrkB signaling pathway for the development of therapies in AD will potentially enhance our understanding of the disease39. Unfortunately, BDNF itself cannot be used as a treatment because of its poor pharmacokinetic properties and adverse side effects40. Small molecule activators of TrkB/BDNF pathways have been explored as potential TrkB ligands41,42,43. Among tested small molecule agonists, 7,8-dihydroxyflavone (7,8-DHF), has been shown to activate the BDNF/TrkB pathway41,44,45,46. A derivative of 7,8-DHF (R13; 4-Oxo-2-phenyl-4H-chromene-7,8-diyl bis(methylcarbamate)) is currently under consideration as a possible drug for AD47. Recently, it was shown that several antidepressants work through directly binding to TrkB and promoting BDNF signaling, further stressing the importance of pursuing TrkB as a valid target to treat various neurological disorders48.
The protocol describes the process of assembling functional TrkB column and TrkB-NULL negative control column. The columns are characterized using a known natural product small-molecular ligand: 7,8-DHF. Additionally, we describe the process of screening complex matrices, using plant extract as an example, for the identification of compounds interacting with TrkB.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-8 Dihydroxyflavone hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>D5446-10 mg</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate (ATP) disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A2383-1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>VWR Chemicals BDH</td>
			<td>BDH9204-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF antibody</td>
			<td>Invitrogen</td>
			<td>PA5-15198-400 &#956;L</td>
			<td>Primary antibody; 2 mg/mL of concentration</td>
		</tr>
		<tr>
			<td>Benzamidine hydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>B6506-25 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF) human</td>
			<td>Sigma-Aldrich</td>
			<td>B3795-10 &#956;g</td>
			<td>Recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR Analytical</td>
			<td>BDH9224-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid sodium salt</td>
			<td>Alfa Aesar</td>
			<td>J62050-100 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>71000-516</td>
			<td>40 mL, 285 mm (overall lenght), 32 x 140 mm (O.D. x H)</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>493511</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>VWR Analytical</td>
			<td>BDH-9232-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500 mL</td>
			<td>sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>G418 disulfate salt solution</td>
			<td>Sigma-Aldrich</td>
			<td>G8168-100 mL</td>
			<td>50 mg/mL in H2O, 0.1 &#956;m filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR Life Science</td>
			<td>E520-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized artificial membrane (IAM.PC.DD2)</td>
			<td>Regis Technologies, Inc.</td>
			<td>1-771050-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>VWR Analytical</td>
			<td>BDH9244-500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322425</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Plan Fluor</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal laser scanning microscope</td>
		</tr>
		<tr>
			<td>Normal goat serum (10%)</td>
			<td>Life Technologies</td>
			<td>50197Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Thermo Scientific</td>
			<td>36978-5 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>VWR Life Science</td>
			<td>K812-500 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>VWR Chemicals BDH</td>
			<td>0395-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>VWR Life Science Ambreso</td>
			<td>M221-1 mL</td>
			<td>Proteomics grade, containing 50 mM AEBSF, 30 &#181;M aprotonin, 1 mM bestatin, 1 mM E-64 and 1 mM leupeptin</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758-500 mL</td>
			<td>with L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Secondary antibody goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen Alexa Flour Plus 488</td>
			<td>A32731</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cell lines expressing Trk-B</td>
			<td>Kerafast</td>
			<td>ECP007</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Trk-NULL cell line</td>
			<td>Kerafast</td>
			<td>ECP005</td>
			<td></td>
		</tr>
		<tr>
			<td>Snake skin dialysis tubing</td>
			<td>Thermo Scientific</td>
			<td>88245</td>
			<td>10K MWCO, 35 mm dry I.D.</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH VWR Analytical</td>
			<td>BDH9286-2.5 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricorn 5/20 column</td>
			<td>GE Healthcare</td>
			<td>24-4064-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>VWR Life Science</td>
			<td>0497-1 kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500 mL</td>
			<td>0.25%, sterile-filtered, suitable for cell culture, 2.5 g porcine trypsin and 0.2&#160;g EDTA</td>
		</tr>
	</tbody>
</table>

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, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

Following the protocol, two CMAC chromatographic columns were assembled: one with the immobilized SH-SY5Y neuroblastoma cell membrane fragments with overexpressed TrkB and one with SH-SY5Y TrkB-NULL cell membrane fragments. The correctly assembled CMAC column is presented in Figure 1 and the steps involved in cell membrane fragment immobilization are presented in Figure 2.
Since the immobilization of TrkB receptors on IAM.PC.DD2 chrom...

Watch this Scientific Journal Video about Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B at JoVE

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

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

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

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