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

Summary

This protocol introduces a fluorometric screening methodology optimized for identifying natural product/small molecule inhibitors of protein synthesis. Its practicality makes it suitable for both undergraduate instruction in drug discovery techniques and implementation in medicinal chemistry campaigns.

Abstract

Natural products represent a rich source of therapeutic compounds with applications across multiple diseases, particularly cancer. By leveraging these biomolecules as structural templates in drug discovery, researchers have achieved both faster development timelines and improved biological effectiveness compared to fully synthetic compounds. The protein synthesis pathway has emerged as a compelling target for natural products, especially given its involvement in hard-to-treat cancers. The potent protein synthesis inhibitor cycloheximide, derived from Streptomyces griseus, exemplifies the potential of natural compounds in this space. As these natural product derivatives are developed into drug-like molecules, confirming their protein synthesis inhibitory activity remains a crucial validation step. The primary aim is to provide an undergraduate-friendly protocol for rapid evaluation of compound inhibition activity. The protocol employs a fluorescent substrate and advanced fluorescence microscopy techniques to systematically detect and measure the inhibitory capabilities of these natural products. The study examined compounds derived from two plant species: Pinus sylvestris and Psoralea corylifolia. Using cycloheximide as a reference inhibitor, both natural products showed varying levels of protein synthesis inhibition.

Introduction

Protein synthesis involves highly regulated processes: transcription and translation. During transcription, DNA sequences of genes are copied, producing messenger RNA (mRNA). The enzyme RNA polymerase works to separate the DNA double helix and uses one strand as a guide to build a matching mRNA molecule. Once the mRNA is fully formed, it travels from the nucleus out to the cytoplasm for translation. Translation occurs in the ribosome, wherein mRNA is used as a template to assemble amino acids into a polypeptide chain. This polypeptide chain, upon leaving the exit tunnel of the ribosome, is folded into a functional protein. 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 effectively¹.

The vast majority of human cancers—approximately 85%—manifest as solid tumors². While these tumors can vary dramatically in their specific features and behaviors, they tend to share certain common physiological traits and patterns, such as upregulated protein synthesis regardless of their etiology or anatomical position³. 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 pathways⁴. In tumor cells, the machinery for making ribosomes goes into overdrive, enabling increased protein production and rapid cell division. Since this enhanced protein synthesis is crucial for cancer survival, treatments that target and block this process show promise as potential therapies⁵. Therefore, research focusing on cancer-specific translation factors, unique ribosomal compositions, or altered signaling pathways that regulate protein synthesis could yield more targeted inhibitors with improved therapeutic windows.

Because natural products (NPs) are known for their wide array of biological activity, the development of selective protein synthesis inhibitors for cancer treatment using their scaffolds is a promising approach^6,7. NPs can target different components of the protein synthesis machinery, from ribosome binding to interference with initiation factors or elongation processes, making them invaluable starting points in drug development for protein synthesis inhibitors in solid tumors⁶. The capacity to rapidly screen compounds for desired activity serves as a crucial entry point for drug discovery platforms seeking to efficiently identify NP hits. Typically, high-throughput screening of compounds utilizes fluorescence assays to quickly narrow down lead candidate molecules⁸.

Scientists worldwide rely on fluorescence-based techniques as essential tools for analyzing biological systems. These approaches are widely used to study cellular processes, allowing researchers to track biomolecular interactions in real time⁹. When combined with bio-orthogonal click-chemistry reactions, these methods can enable precise visualization of protein synthesis¹⁰. The presented fluorescent protein synthesis assay is a non-radioactive technique for assessing protein synthesis that utilizes O-propargyl-puromycin (OPP), a cell-permeable alkyne-containing puromycin analog. Following cellular treatment, OPP becomes incorporated at the C-terminus of elongating polypeptide chains, causing translation termination. These alkyne-labeled truncated proteins are then detected using copper-catalyzed click chemistry coupled with 5 FAM-azide.

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 in modern drug discovery techniques and accelerates the drug discovery process. As the demand for new protein synthesis inhibitors grows, this approach equips undergraduates with meaningful research opportunities while contributing to advancements in cancer therapeutics.

Protocol

1. Reagent preparation from kit

NOTE: Adequately mix, spin down, and keep all reagents on ice until use.

1. Organize and obtain the protein synthesis inhibition kit, PBS buffer, cell culture medium, cell culture treated plates, 15 mL conical sterile polypropylene centrifuge tubes, 2 mL centrifuge tubes, p10, p20, p200, and p1000 pipettes, and appropriate tips.
2. Prepare wash buffer.
   1. Thaw the 10x wash buffer at 37 °C by cupping the vial in the hand. Use the body heat in the hands to thaw and frequently check if thawing is complete.
   2. Dilute the 10x wash buffer stocks 1:10 in deionized water in a centrifuge tube. Mix by inversion or resuspension by pipetting up and down. Store on ice until use.
3. Thaw fixative solution at room temperature. Store on ice until use.
4. Prepare the permeabilization buffer.
   1. Thaw the 10x permeabilization buffer at 37 °C by cupping the vial in the hand. Use the body heat in the hands to thaw and frequently check if thawing is complete.
   2. Dilute the 10x stock 1:10 in deionized water in a centrifuge tube. Mix the components by inversion or resuspension by pipetting up and down. Store on ice until use.
5. Thaw 400x protein label, 100x fluorescent azide, 100x cycloheximide, DNA stain, and the 20x reducing agent at room temperature. Keep on ice in a dark environment while in use. Cover the dyes with foil or keep them in a 2 °C refrigerator when not in use.

2. Labeling controls and experimental cells

NOTE: This protocol was developed using stable adherent, epithelial breast cancer MDA-MB-231 cells. Any adherent cell lines can be used. All steps listed below use a 96-well plate, and all volumes and concentrations are calculated for two test compounds and two controls with a working volume of 100 µL.

1. Culturing adherent mammalian cells
   NOTE: Cells should be passed before reaching 80-90% confluency to maintain healthy, exponential growth and prevent contact inhibition or differentiation.
2. Thaw a vial of cryopreserved cells to room temperature, then dilute with appropriate medium.
3. Centrifuge the suspension to pellet the cells and aspirate the supernatant. Resuspend the pellet in fresh medium and transfer to a new flask at the appropriate cell density.
4. Once cells are 80% confluent, remove media and aspirate old growth medium from the flask. Then, rinse cells with sterile PBS to remove residual serum, which inhibits trypsin. Add trypsin-EDTA solution to cover the cell layer and incubate for 3-5 min or until cells detach at 37 °C.
5. Add complete medium containing serum to inactivate the trypsin, then transfer the cell suspension to a tube, mix gently, and count the cells.
6. Seed cells in screening plates-dilute cells at the appropriate split ratio (e.g., 1:3 or 1:5) into fresh flask(s) with new medium and incubate (typically 37 °C, 5% CO₂) to obtain the desired cell concentration.
   NOTE: For the experiment described here, the cell line was plated at 2 × 10⁴ per well (96-well plate) and incubated at 37 °C for 12 h.

3. Addition of compounds

1. Prepare master mix A.
   1. Label a centrifuge tube as "A". Pipette 598.5 µL of culture medium and 1.5 µL of protein label into "A". Resuspend the mixture to ensure homogeneity by pipetting up and down.
2. Prepare master mix B.
   1. Label a centrifuge tube as "B". Pipette 1.5 µL of protein label, 6.6 µL of cycloheximide, and 591.9 µL of culture medium into "B". Resuspend the mixture to ensure homogeneity by pipetting up and down.
3. Treat cells with desired compounds (Figure 1), positive, and negative controls.
   1. Remove cells from the incubator and remove the growth medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent disturbance of the cells.
   2. Pipette 1 µL of test compound into a well and 1 µL of test compound vehicle (0.1% v/v DMSO) into a well. For duplicate studies, add each sample to a second well and repeat this process. Add 99 µL of master mix A into these wells. Gently pipette at an angle into the side of the well to prevent kinetic disruption of cells.
      ​NOTE: The maximum DMSO concentration for compound screening depends on the cell type and assay, but general guidelines are: 0.1-0.5% DMSO-generally safe for most cell-based assays with minimal toxicity, and 1% DMSO-commonly used maximum and acceptable for many cell types in short-term assays.
   3. Pipette 100 µL of master mix B into two wells. Pipette at an angle into the side of the well to prevent disruption of cells.
   4. Gently tap the plate to achieve through incorporation of the desired components with the cells. Incubate cells at 37 °C for 0.5-2 h.

4. Termination of the experiment and washing of cells

1. Remove the culture medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent disturbance of the cells.
2. Add 100 µL of PBS buffer into each well and centrifuge the plate at 900 × g for 5 min.
3. Remove the PBS buffer via aspiration.

5. Fixation and permeabilization

1. Add 100 µL of fixative solution to each well. Incubate the cells at room temperature for 15 min in a dark environment.
2. Remove the fixative solution via aspiration. Tilt the plate and aspirate at the side of the well instead of the center.
3. Wash cells with 100 µL of wash buffer and remove buffer via aspiration. Repeat this step 2x.
4. Add 100 µL of permeabilization buffer to each well and incubate at room temperature for 10 min.
5. Remove the permeabilization buffer via aspiration. Tilt the plate and aspirate at the side of the well instead of the center.

6. Protein reaction and staining

1. Prepare the reaction cocktail master mix.
   1. Add 651 µL of PBS buffer, 7.0 µL of 100x copper reagent, 7 µL of fluorescent azide, and finally, 35 µL of reducing agent to a centrifuge tube. Resuspend this mixture to ensure homogeneity.
      NOTE: Follow this exact order when adding the reagents. Use within 15 min of preparation.
2. Add 100 µL of the reaction cocktail to each well. Incubate the cells for 30 min at room temperature in a dark environment to prevent photobleaching.
3. Centrifuge cells at 900 × g for 5 min. Balance the centrifuge by adding empty plates to distribute the weight evenly. Remove the mixture via aspiration.
4. Wash cells with 100 µL of wash buffer and remove buffer via aspiration. Repeat this step 2x.

7. DNA staining

1. Prepare a 1x dilution of total DNA stain and add 100 µL to each well. Incubate cells at room temperature for 20 min in a dark environment to prevent photobleaching.
2. Centrifuge the plate at 900 × g for 5 min. Balance the centrifuge by adding empty plates to distribute the weight evenly. Remove the mixture via aspiration.
3. Wash cells with 100 µL of wash buffer and remove buffer via aspiration. Repeat this step 2x.
4. Resuspend cells with 100 µL of chilled PBS buffer to prepare them for imaging.

8. Imaging

1. Insert the plate into the plate reader.
2. Set magnification to 20x.
3. Analyze nucleus stain with the GFP 469/525 nm channel. Use autobinning and autofocus to enhance imaging results.
4. Analyze active protein synthesis with the Texas Red 586/647nm channel. Use autobinning and autofocus to enhance imaging results.

Results

This assay utilized fluorescence to visualize and quantify different natural products' ability to inhibit protein synthesis. Cycloheximide (CHX) is a known protein synthesis inhibitor whose mode of action involves interfering with the translocation process during elongation¹¹. For this study, living cells were detected with a green, fluorescent nuclear dye. OPP is a derivative of puromycin that covalently binds to nascent proteins¹². The MDA-MB-231 cell line served as an ap...

Discussion

This protocol provides a fluorometric screening methodology for testing natural products and small molecules to evaluate their ability to inhibit protein synthesis. The simplicity of this protocol enables undergraduate researchers to implement it correctly, allowing them to make substantive contributions to scientific knowledge. The protocol demands a basic comprehension of biological methods and techniques, with steps requiring careful execution to ensure accurate and reliable results.

For in...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agilent Biotek Cytation 1 Cell Imaging Multi-Mode Reader	Agilent	BTCYT1V
Compound 1 (Dehydroabietylamine, CAS 1446-61-3)	Enamine	EN300-7409771
Compound 2 (Bakuchiol, CAS 10309-37-2)	ChemFaces	CFN99047
Heraeus Megafuge 16R	Thermo Scientific	75004271
Ibidi Gmbh-slide 96-well plate	Ibidi	80826
MDA-MB-231 Cell Lines	ATCC	ATCC HTB-26
Phosphate Buffered Saline pH 7.4	ATCC	ATCC 30-2200
Protein Synthesis Assay Kit (Green)	Abcam	Ab239725