Name: functionalized spirocyclic heterocycle synthesis and cytotoxicity assay
Uploaded: 2021-02-09T13:30:00
Description: Watch this Scientific Journal Video about Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay at JoVE.com

This work was funded by a grant from the Faculty Research Council to K.S.H. (Office of Research and Grants, Azusa Pacific University-USA). A.N.G. and J.F.M. are recipients of the Scholarly Undergraduate Research Experience (SURE) Fellowship. S.K.M. and B.M.R. are recipients of the STEM Research Fellowship Grants (Center for Research in Science, Azusa Pacific University-USA). We are grateful to Dr. Matthew Berezuk and Dr. Philip Cox for guidance on the bioassays.

NOTE: Several chemicals and biological reagents used in this protocol are toxic and carcinogenic. Consult relevant material safety data sheets (MSDS) prior to use. Use appropriate personal protective gears (Occupational Safety and Health Administration-approved safety goggles, proper gloves, lab coats, full-length pants, and closed-toe shoes) prior to starting the experiment. In addition, adopt appropriate safety practices when performing synthesis and handling toxic chemicals and reagents (fume hood).
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<ol>
	<li>Shangary, S., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+to+reactivate+p53+function:+a+novel+approach+for+cancer+therapy.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction to reactivate p53 function: a novel approach for cancer therapy.</a> Annual Review of Pharmacology and Toxicology. 49, 223-241 (2009).</li><li>Zhao, Y., Aguilar, A., Bernard, D., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+the+MDM2-p53+protein-protein+interaction+(MDM2+inhibitors)+in+clinical+trials+for+cancer+treatment.">Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 inhibitors) in clinical trials for cancer treatment.</a> Journal of Medicinal Chemistry. 58 (3), 1038-1052 (2015).</li><li>Paolo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+virtual+screening+protocol+to+identify+promising+p53-MDM2+inhibitors.">An effective virtual screening protocol to identify promising p53-MDM2 inhibitors.</a> Journal of Chemical Information and Modeling. 56 (6), 1216-1227 (2016).</li><li>Shieh, S. Y., Ikeda, M., Taya, Y., Prives, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage-induced+phosphorylation+of+p53+alleviates+inhibition+by+MDM2.">DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.</a> Cell. 91 (3), 325-334 (1997).</li><li>Hwang, B. J., Ford, J. M., Hanawalt, P. C., Chu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+p48+xeroderma+pigmentosum+gene+is+p53+dependent+and+is+involved+in+global+genomic+repair.">Expression of the p48 xeroderma pigmentosum gene is p53 dependent and is involved in global genomic repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (2), 424-428 (1999).</li><li>Oliner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncoprotein+MDM2+conceals+the+activation+domain+of+tumor+suppressor+p53.">Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53.</a> Nature. 362, 857-860 (1993).</li><li>Nag, S., Qin, J., Srivenugopal, K. S., Wang, M., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDM2-p53+pathway+revisited.">The MDM2-p53 pathway revisited.</a> Journal of Biomedical Research. 27 (4), 254-271 (2013).</li><li>Bond, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+nucleotide+polymorphism+in+the+MDM2+promoter+attenuates+the+p53+tumor+suppressor+pathway+and+accelerates+tumor+formation+in+humans.">A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans.</a> Cell. 119 (5), 591-602 (2004).</li><li>Isobe, M., Emanuel, B. S., Givol, D., Oren, M., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+gene+for+human+p53+tumor+antigen+to+band+17p13.">Localization of gene for human p53 tumor antigen to band 17p13.</a> Nature. 320 (6057), 84-85 (1986).</li><li>Gupta, A. K., Bharadwaj, M., Kumar, A., Mehrotra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiro-oxindoles+as+a+promising+class+of+small+molecules+inhibitors+of+p53-MDM2+interaction+useful+in+targeted+cancer+therapy.">Spiro-oxindoles as a promising class of small molecules inhibitors of p53-MDM2 interaction useful in targeted cancer therapy.</a> Topics in Current Chemistry. 375 (1), 1-25 (2017).</li><li>Griffin, S. A., Drisko, C. R., Huang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricyclic+heterocycles+as+precursors+to+functionalized+spirocyclic+oximes.">Tricyclic heterocycles as precursors to functionalized spirocyclic oximes.</a> Tetrahedron Letters. 58, 4551-4553 (2017).</li><li>Drisko, C. R., Griffin, S. A., Huang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+synthesis+of+[4.4]spirocyclic+oximes.">Solid-phase synthesis of [4.4]spirocyclic oximes.</a> Journal of Visualized Experiments. (144), e58508 (2019).</li><li>Lawrence, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linked+parallel+synthesis+and+MTT+bioassay+screening+of+substituted+chalcones.">Linked parallel synthesis and MTT bioassay screening of substituted chalcones.</a> Journal of Combinatorial Chemistry. 3 (5), 421-426 (2001).</li><li>. MTT assay protocol Available from: <a target="_blank" href="https://www.abcam.com/kits/mtt-assay-protocol">https://www.abcam.com/kits/mtt-assay-protocol</a> (2020)</li></ol>

The synthesis of the spirocyclic compounds was based on previous research conducted by this laboratory, but with some modifications (Figure 1)11,12. The progress of each reaction step was monitored by IR spectroscopy. Michael addition of the REM linker&#160;1&#160;with furfurylamine afforded polymer-bound&#160;2&#160;(IR 1722 cm-1&#160;&#8594; 1731 cm-...

Small-molecule inhibition of the interaction of E3 ubiquitin-ligase mouse double minute 2 homolog (MDM2) with p53 is known to restore p53-mediated induction of tumor cell apoptosis1,2,3. MDM2 is a negative regulator of the p53 pathway and is often overexpressed in cancer cells4,5,6,7,8,9. Recent crystallographic and biochemical studies have revealed that small molecules containing a spirocyclic framework can effectively inhibit MDM2-p53 interactions10. The spirocyclic framework (Figure 1, shaded in blue) is considered a privileged motif as derivatization of this rigid orthogonal ring system has led to the discovery of novel therapeutic drugs. Accessing this interesting architecture poses a challenge when using traditional organic synthesis techniques. Although the therapeutic effects of spirocyclic molecules in biological systems have been investigated, synthesis of these molecules is still a cumbersome process. Unwanted side products, using harsh conditions, and hazardous transition metals are often problematic.
The potential use of the spirocyclic motif in drug development led to the development of a protocol utilizing solid-phase synthesis to generate a library of molecules with the motif in addition to other interchangeable functional groups11,12. The separation of products and reactants between steps could be achieved by simply utilizing an REM linker attached to a resin bead and a solid-phase filter vessel. This would cut down steps and potentially increase yields. This synthetic approach could produce a large array of potential drug candidates. However, the effectiveness of these molecules in a biological system would require further investigation.
To determine the cytotoxicity of these spirocyclic compounds, the MTT assay13,14 was employed. This method measures cell viability and can be used to indirectly determine cell cytotoxicity. Different concentrations of the inhibitors were added to cultured cells in a 96-well plate, and the proportion of living cells was measured by colorimetric analysis of the extent of reduction of yellow MTT by mitochondrial dehydrogenases to the purple formazan compound (Figure 2). The activity is most often reported as an IC50 value-the concentration at which cell growth is inhibited by 50% relative to an untreated control. This paper describes the protocol for the MTT assay and the preliminary results of these novel spirocyclic molecules.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELLS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>COS-7 cells (ATCC CRL-1651)</td>
			<td>ATCC</td>
			<td>CRL-1651</td>
			<td>African green monkey kidney cells</td>
		</tr>
		<tr>
			<td>CHEMICALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Bromooctane</td>
			<td>Sigma-Aldrich</td>
			<td>152951</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Allylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>337528</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Benzylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Cayman Chemical</td>
			<td>13119</td>
			<td>Cytotoxicity control</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>276855</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, with L-glutamine</td>
			<td>Genesee Scientific</td>
			<td>25-500</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine serum)</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Furfurylamine</td>
			<td>Acros Organics</td>
			<td>119800050</td>
			<td>reagent&#160;</td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)</td>
			<td>EMD Millipore</td>
			<td>Calbiochem 475989-1GM</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Genesee Scientific</td>
			<td>25-507</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>REM Resin</td>
			<td>Nova Biochem</td>
			<td>8551010005</td>
			<td>Polymer support; 0.500 mmol/g loading</td>
		</tr>
		<tr>
			<td>trans-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>N26806</td>
			<td>Nitro-olefin reagent</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>244511</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td>Reagent for beta-elimination</td>
		</tr>
		<tr>
			<td>Trimethylsilyl chloride (TMSCl)</td>
			<td>Sigma-Aldrich</td>
			<td>386529</td>
			<td>Reagent; CAUTION - highly volatile; creates HCl gas</td>
		</tr>
		<tr>
			<td>GLASSWARE/INSTRUMENTATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL solid-phase reaction vessel</td>
			<td>Chemglass</td>
			<td>CG-1861-02</td>
			<td>Glassware with filter</td>
		</tr>
		<tr>
			<td>96 Well plate reader</td>
			<td>Promega (Turner Biosystems)</td>
			<td>9310-011</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>AVANCE III NMR Spectrometer</td>
			<td>Bruker</td>
			<td>N/A</td>
			<td>Instrument; 300 MHz; Solvents: CDCl3 and CD3OH</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nicole iS5</td>
			<td>Thermo Scientific</td>
			<td>IQLAADGAAGFAHDMAZA</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker</td>
			<td>Burrell Scientific</td>
			<td>757950819</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

functionalized spirocyclic heterocycle synthesis and cytotoxicity assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Spirocyclic oximes 6 and 7 were synthesized using a modified protocol (Figure 1). Michael addition of furfurylamine to an REM linker 1b afforded polymer-bound resin 2. The progress of the reaction was monitored by infrared (IR) spectroscopy by detecting the disappearance of the &#945;,&#946;-unsaturated ester at 1722 cm-1&#160;(Figure 3). Spirocyclic-bound resin 4 was formed from 2 via a transient intermediate 3. Methanolic hydrolysis of 4 produced 3-...

Watch this Scientific Journal Video about Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay at JoVE.com

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

This protocol utilizes a recyclable and modified method in producing spirocyclic compounds. The MTT assay helps answer the question pertaining to the cytotoxicity of these novel molecules. This technique producers novel modifiable molecules using a regenerating starting material.The MTT assay provides relatively quick, and easy to analyze biological results Begin by performing solid phase synthesis of spirocyclic hetero cycles six and seven, as previously demonstrated. Prepare 20 milliliters of a five milligrams per milliliter MTT solution using sterile PBS as the diluent. Filter and store the solution at minus 20 degrees Celsius.Then, prepare a one-to-one dilution of the MTT solution in DMEM. Prepare one milliliter each of stock solutions in 1.5 milliliter micro centrifuge tubes of test compounds in DMSO. Store them at minus 20 degrees Celsius.Prepare 200 microliters per dose of the working solutions of test compounds by diluting stock concentrations. One to 1000 in serum free medium. In the tissue culturehood seeds, COS seven cells in complete medium onto flat bottom tissue culture treated 96 well plates at a concentration of 4, 000 cells per 200 microliters per well.Using a multi-channel pipeter. Incubate the cells for 24 hours at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide. On the next day, aspirate the supernatant from the wells using a glass pasture pipette attached to a vacuum pump.Dose the cells in triplicate with the test compounds using the previously prepared working solutions. Then return the cells to the incubator. After 24 hours, aspirate the supernatant from the wells and add 200 microliters of MTT solution to each well.Incubate the plate for four hours. Gently aspirate the supernatant from the wells without disturbing the purple formazane crystals. Add 200 microliters of DMSO to each well to dissolve the crystals.Then incubate the plate at room temperature for 15 minutes. Measure absorbance for each well at 590 or 600 nanometers using a 96 well plate reader. Use wells with no cells as background and average the absorbance value.Spirocyclic gox seams six and seven were synthesized using a modified protocol. The progress of the reaction was monitored by detecting the disappearance of the alpha beta unsaturated Esther using infrared spectroscopy. The MTT assay was used for determining cell viability.Cisplatin, which is known to induce cell death at high concentrations, served as a positive control. As expected, the higher the concentration of cisplatin the lower is the cell viability. The MTT assay was then used to test the spirocyclic compounds six and seven and furfurylamine.Furfurylamine and spirocyclic gox seam six showed similar cytotoxicity. However, the toxicity of spirocyclic compound seven was noticeably greater than that of furfurylamine and six. When attempting this protocol, keep in mind that N-alkylation performed using bulky acyl halides was found to reduce the rate of beta elimination thus reducing the product yield.

functionalized-spirocyclic-heterocycle-synthesis-cytotoxicity

Research

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Biology

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...