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...

Lukasz Ciesla collaborates with Regis Technologies, the provider of the IAM.PC.DD2 particles.

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, ...

This vertical presents important steps in assembling cell membrane affinity chromatography columns with functional transmembrane receptors. These columns can be used for the identification of small molecules present in complex extracts and interacting with immobilized transmembrane receptors. Cellular membrane affinity chromatography columns speed up the identification of biologically active compounds present in complex mixtures.The use of cellular membrane affinity chromatography columns can be of particular interest to those interested in the identification of pharmacologically active compounds present in complex natural matrices, such as plant, fungal, or bacterial extracts. To begin, prepare 200 millimolar benzamidine stock solution by dissolving 120 of benzamidine in five milliliters of ultrapure deionized water. Prepare a 10 millimolar PMSF stock solution by dissolving 0.017 grams of PMSF in 10 milliliters of ethanol in a fume hood.Prepare 100X proteases inhibitor cocktail by dissolving commercially available protease inhibitor mixture in one milliliter of ultrapure deionized water. Mix thoroughly and aliquot 200 to 300 microliters of the cocktail. Prepare 100 millimolar ATP stock solution by dissolving 55.114 milligrams of ATP disodium salt hydrate in one milliliter of the ionized ultrapure water.Mix thoroughly and aliquot 100 microliters of the mixture. Prepare buffer by dissolving 3.03 grams of Tris-HCL, 2.9 grams of sodium chloride, 0.22 grams of calcium chloride anhydrous, 0.2 grams of magnesium chloride hexahydrate, and 0.19 grams of potassium chloride in 500 milliliters of ultrapure deionized water. Prepare homogenization buffer by mixing 17.3 milliliters of the Tris buffer with 0.3 milliliters of benzamidine stock solution, 0.2 milliliters of PMSF stock solution, 0.2 milliliters of proteases inhibitor cocktail, 20 microliters of ATP stock solution, two milliliters of glycerol, and 0.029 grams of EDTA.Adjust pH to 7.4 using hydrochloric acid solution. Spin down the cells harvested at four degrees Celsius for five minutes at 400 x g and remove the supernatant and wash the remaining cell pellet with 10 milliliters of 1X pH 7.4 ice cold PBS. Discard the supernatant and add 20 milliliters of homogenization buffer.Place the conical tube on ice. Transfer the cell suspension into a 40 milliliter Dounce homogenizer tissue grinder, homogenize the suspension manually on ice using 40 up and down pestle strokes. Transfer homogenized cell suspension into a 50 milliliter conical tube, discard the pellet and transfer the supernatant to a new conical tube.Save the cell membrane pellet and discard the supernatant. Prepare solubilization buffer by mixing 8.7 milliliters of the buffer solution with 0.1 milliliters of PMSF stock solution, 0.15 milliliters of benzamidine stock solution, 0.1 milliliters of proteases inhibitor cocktail, 10 microliters of ATP stock solution, one milliliter of glycerol, and 0.2 grams of sodium cholate. Transfer the solubilization buffer into the conical tube with cell membrane fragments.And resuspend the pellet. Rotate the resulting mixture at 150 RPM at four degrees Celsius for 18 hours. After 18 hours, centrifuge the solubilization mixture at four degrees Celsius for 30 minutes at 47, 900 x g.Add 100 milligrams of IAM. PC.DD2 particles to the supernatant and rotate the resulting suspension mixture at 150 RPM at four degrees Celsius for one hour. Prepare dialysis buffer in four liters of ultrapure deionized water by adding 24 grams of Tris-HCl, 23.4 grams of sodium chloride, 0.06 grams of calcium chloride, 5.85 grams of EDTA, and 0.07 grams of PMSF.Dissolve PMSF first in a small volume of ethanol and then add slowly into the beaker with the buffer. Cut 10 centimeters of cellulose membrane dialysis tubing to prepare dialysis tube, and transfer the suspension containing IAM. PC.DD2 particles and cell membrane fragments into the dialysis tube.Close both ends of the dialysis tube with dialysis tubing clips. Place the dialysis tube in the dialysis buffer. After 24 hours, place the dialysis tubing in the freshly prepared dialysis buffer and continued dialysis for another 24 hours.Prepare 10 millimolar pH 7.4 ammonium acetate buffer that will be used to wash the IAM. PC.DD2 particles and in column characterization experiments by dissolving 0.778 grams of ammonium acetate in one liter of ultrapure deionized water. After 48 hours of dialysis, remove the dialysis tube from the dialysis buffer and transfer the content of the dialysis tube into a 15 milliliter conical tube.Discard the supernatant and wash the remaining pellet three times with 10 milliliters of ammonium acetate buffer. After the third wash, resuspend the remaining pellet in one milliliter of ammonium acetate buffer. Mix it thoroughly and use the resulting slurry to pack the 5 x 20 glass column to yield the CMAC chromatography column.To pack the column, place a bottom filter previously soaked with ammonium acetate buffer into the filter holder. Fit the filter holder in the glass column and screw the column cap to secure the holder's position. Place the column vertically in a finger clamp and secure it in the lab stand.Place a beaker below the column, transfer a small volume of the slurry into the glass column slowly with a single channel pipetter holding the pipette tip against the glass column wall. To speed up the packing process, remove the buffer from above the stationary bed with a micropipette between each step. Place a top filter and screw the adapter unit so that there is no remaining buffer above the stationary phase.Secure the position of the adapter unit with the adapter lock. Connect the column to a high performance liquid chromatography pump, and to wash the column overnight with ammonium acetate buffer, set the flow rate to 0.2 milliliters per minute. For longer storage, run the column with 0.05%sodium azide solution in ammonium acetate buffer, wearing a lab coat, safety glasses, and gloves when working with sodium azide and store at four degrees Celsius.IAM.PC.DD2 particles with immobilized neuroblastoma TrkB cell membrane fragments and in the presence of BDNF resulted in fluorescent particles. No fluorescence was observed in TrkB null cell membrane encapsulated IAM particles or when no BDNF was used as in TrkB cell membrane encapsulated IAM particles. A functional CMAC columns typical frontal affinity chromatogram of increasing 7, 8-DHF concentrations of one millimolar, 750 nanomolar, 500 nanomolar, and 300 nanomolar to the immobilized TrkB receptors are shown, which showed a concentration dependent change in retention time.A nonfunctional CMAC column showed the lack of concentration dependent changes in retention of the marker ligand. A CMAC negative control column was prepared by immobilizing cell membrane fragments, not expressing the targeted protein to rule out non-specific interactions. The addition of 0.2%aqueous Gotu kola extract resulted in a significant reduction of 7, 8-DHF retention, indicating the presence of competing ligands for the agonist binding site.The lack of reduction of 7, 8-DHF retention on the CMAC negative TrkB null column further confirmed the lack of functional TrkB receptors on this column. The missing peak chromatography approach identified a compound strongly retained on TrkB column, while eluting early on TrkB null column, indicating specific interaction. Its crucial to know your source of receptors for fast cellular membrane affinity chromatography column.Make sure to check the literature to properly design homogenization and solubilization buffers. Cellular membrane affinity chromatography column prepared in this protocol was used to identify new potential drug hits and to characterize the nature of the interaction between immobilized receptor and its natural ligands.

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