A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources

A fluorescence-based protocol for preliminary screening of protein synthesis inhibitors from natural sources. Protein synthesis involves highly regulated processes, transcription and translation. Transcription is primarily regulated by transcription factors, chromatin modifications, and regulatory RNAs.

While translation is regulated by initiation factors, signaling pathways and RNA stability mechanisms. These regulatory systems allow cells to maintain cellular functions and respond to stress or damage effectively. Cancer cells dysregulate protein synthesis by hijacking translational machinery to support their high energy demands and uncontrolled growth.

This reprogramming is a result of dysregulation of nearly all major oncogenic signaling pathways, promoting cell survival, proliferation and metastasis while suppressing cell death pathways. Since this enhanced protein synthesis is crucial for cancer survival, treatments that target and block this process show promise as potential therapies. To identify protein synthesis inhibitors, the following protein synthesis inhibition fluorescence assay can be utilized.

Prior to starting the experiment, in order to obtain reliable results, compounds must be administered at an appropriate concentration beneath its toxicity threshold. To establish a starting concentration, a cytotoxicity assay should be performed to determine the compound's EC 50. If this is not feasible, compounds in this assay can be tested in a dose dependent manner to determine the most effective concentration parameters.

If the compounds concentration triggers cell death, testing at reduced concentrations becomes necessary. It is also important to protect dyes from light at all times when not in use. Extended light exposure will result in photobleaching, which leads to compromised data quality.

Step 3.1, prepare master mix A, label a centrifuge tube as A then pipette 598.5 microliters of culture medium, and 1.5 microliters of protein label into A.Resuspend this mixture to ensure homogeneity by pipetting up and down. Now prepare master mix B.Label a centrifuge tube as B, then pipette 1.5 microliters of protein label, 6.6 microliters of cycloheximide and 591.9 microliters of culture medium into B.Resuspend this mixture to ensure homogeneity by pipetting up and down. After pipetting one microliter of test compound and one microliter of vehicle into two wells, gently pipette 99 microliters of master mix A into both of these wells, pipette at an angle into the side of the well to prevent kinetic disruption of cells.

Pipette 100 microliters of master mix B into two untreated wells. Pipette at an angle into the side of the well to prevent kinetic disruption of cells. Gently tap the plate to achieve thorough incorporation of the desired components within the cells.

Incubate cells at 37 degrees Celsius for 30 minutes to two hours. Remove the cell culture medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent kinetic disturbance of cells.

Then add 100 microliters of PBS buffer into each well and centrifuge the plate at 900g for five minutes. After centrifuging, remember to remove the PBS buffer via aspiration. Step five, fixation and permeabilization.

Add 100 microliters of fixative solution to each well. Then incubate the cells at room temperature for 15 minutes in a dark environment. Then aspirate the cells.

Wash the cells with 100 microliters of wash buffer and remove via aspiration. Repeat this twice. Add 100 microliters of permeabilization buffer to each well.

After this, incubate at room temperature for 10 minutes. Remove the permeabilization buffer via aspiration. Step six, protein reaction and staining.

Prepare the reaction cocktail master mix. Add 651 microliters of PBS buffer to a centrifuge tube. Then add seven microliters of 100x copper reagent.

Then seven microliters of fluorescent azide. And finally, add 35 microliters of reducing agent to the centrifuge tube. Follow this exact order when adding the reagents, then add 100 microliters of the reaction cocktail to each well.

Incubate the cells for 30 minutes in a dark environment at room temperature to prevent photobleaching. Centrifuge the cells at 900g for five minutes. Aspirate, wash the cells and then aspirate again.

Prepare a 1x dilution of total DNA stain and add 100 microliters to each well. Incubate cells at room temperature for 20 minutes in a dark environment to prevent photobleaching. Centrifuge the plate at 900g for five minutes.

Wash the cells with 100 microliters of wash buffer, remove via aspiration. Repeat this step twice. Finally, resuspend the cells with 100 microliters of chilled PBS buffer to prepare them for imaging.

Step eight, imaging. Insert the plate into the plate reader and set magnification to 20 x. Analyze the nucleus stain with the GFP 469/525 nanometer channel.

Use auto-bending and auto-focus to enhance imaging results. Analyze active protein synthesis with the Texas Red 586/647 nanometer channel. Use auto-bending and auto-focus to enhance imaging results.

Fluorescent probes are sensitive to photobleaching, which is a phenomenon in which a radiation with light causes fluorophores to lose their fluorescent properties. Dyes should be protected from light at all times when not in use. Additionally, the number of scans within each well and time under irradiated light need to be limited.

Extended light exposure will result in photobleaching, which ultimately leads to compromised data quality. Let's look at the representative results. Live cells here are seen in green and active protein synthesis is indicated by red.

Known protein synthesis inhibitor cycloheximide results in a large decrease in protein synthesis as seen in figure 1B compared to the negative control DMSO in figure 1A, there is less red fluorescent light emitted. Treatment with compound one as seen in figure 1C resulted in either cell detachment or cell death. Thus, we cannot make any definitive claims on its protein synthesis inhibition activity.

This warrants further dose response studies to try and determine if it inhibits protein synthesis at lower concentrations. Compound two displays moderate protein synthesis inhibition as indicated in figure 1D. This fluorometric assay is a valuable research tool and an accessible chemical biology method for training undergraduate students.

By providing an accessible, efficient protocol with minimal data processing, it provides students with hands-on experience and modern drug discovery techniques and accelerates the drug discovery process. The protocol employs a fluorescent substrate and advanced fluorescent microscopy techniques to systematically detect and measure the inhibitory capabilities of natural product compounds one and two. Using cycloheximide as a reference inhibitor, both natural products showed varying levels of protein synthesis inhibition.

As the demand for new protein synthesis inhibitors grows, this approach equips undergraduates with meaningful research opportunities while contributing to advancements in cancer therapeutics.