Developing an Autophagy Assay

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Dysregulation of autophagy has been implicated in a myriad of human diseases, including cancer, muscular, and neurodegenerative disorders, highlighting its significance as a therapeutic target. To elucidate the intricacies of autophagy and facilitate drug discovery efforts, the development of robust assay platforms capable of accurately measuring autophagic flux is paramount. 

What is Autophagy?

Autophagy, an evolutionarily conserved lysosomal process, serves as a cornerstone in maintaining cellular homeostasis and is pivotal in cellular health under normal and stress conditions. The term “autophagy,” derived from the Greek “auto” (meaning self) and “phagy” (meaning eating), aptly describes its function as a self-degradation mechanism within cells. This dynamic process involves the degradation and recycling of superfluous or dysfunctional cellular components, including long-lived proteins and organelles, to sustain cellular integrity and adaptability. 

Autophagy assay development

Figure 1. Autophagy begins with the formation of the phagophore or isolation membrane (vesicle nucleation step). The concerted action of the autophagy core machinery proteins at the phagophore assembly site, is thought to lead to the expansion of the phagophore into an autophagosome (vesicle elongation). This can engulf bulk cytoplasm non-specifically, including entire organelles, or target cargos specifically. When the outer membrane of the autophagosome fuses with an endosome, forming an amphisome, or directly with a lysosome (docking and fusion steps), it forms an autophagolysosome. Finally, the sequestered material is degraded inside the autophagolyosome (vesicle breakdown and degradation) and recycled.

Challenges

Developing an autophagy assay for drug discovery presents significant challenges and complexities due to the intricate nature of autophagic processes and the need for reliable, sensitive detection methods. This endeavor requires careful selection of assay methodologies that can accurately measure autophagic activity and reliably distinguish between various compounds’ effects on this process. 

Utilizing assays such as NanoBiT autophagy reporter cell lines, AlphaLISA, high content imaging, and western blotting enables us to assess autophagy modulation in response to potential drug candidates. These assays provide valuable insights into the pharmacological effects of compounds on autophagy, aiding in the identification and characterization of novel therapeutic agents targeting autophagy pathways. 

By employing robust and versatile assay platforms, researchers can expedite the drug discovery process and enhance the development of treatments for diseases associated with autophagy dysregulation.

What We Did...

NanoBiT Autophagy Reporter Cell Line (Promega)

The utilization of NanoBiT autophagy reporter cell lines from Promega proved instrumental in our endeavor to develop a robust assay for measuring autophagy. This innovative approach involved tagging human LC3B with HiBiT, a 15 amino acid peptide exhibiting high affinity for LgBiT, a subunit of NanoBiT luciferase. The NanoBiT luciferase assay, designed for plate-based homogenous screening, offered exceptional sensitivity and stability, making it well-suited for high throughput screening applications.

By monitoring changes in luminescent signal following treatment with autophagy inducers or inhibitors, we were able to accurately assess alterations in autophagic flux. Notably, the NanoBiT assay demonstrated consistency across both 96 and 384 well formats, with pharmacological standards effectively inducing and inhibiting autophagy in a dose-dependent manner.

Autophagy assay development_HiBiT reporter assay_diagram

Figure 2. Principle of the Autophagy LC3 HiBiT reporter assay.

Our results reaffirmed the efficacy of the NanoBiT autophagy reporter cell line in detecting and quantifying changes in LC3B protein levels induced by various compounds. Autophagy enhancers AZD8055, PP242, and rapamycin exhibited dose-dependent reductions in LC3B levels, as evidenced by a decrease in NanoBiT signal (figure 3).

Autophagy assay development_HiBiT reporter assay_results graph

Figure 3. Pharmacology of Autophagic Enhancers. AZD8055, rapamycin and PP242 enhance autophagy by decreasing LC3B levels, as detected by a decrease in the NanoBiT signal, consistent with the reduction of HiBiT labelled LC3B within the cells. Note the consistency between 96 (left) and 384 well (right) formats and the agreement in the EC50 values for each of the compounds.

Conversely, autophagy inhibitors chloroquine and bafilomycin caused a dose-dependent increase in LC3B levels, resulting in a higher NanoBiT signal (figure 4).The consistent correlation between NanoBiT readouts and pharmacological responses underscores the reliability and accuracy of this assay platform.

Autophagy assay development_HiBiT reporter assay_results graph

Figure 4. Pharmacology of Autophagic Inhibitors. Chloroquin and bafilomycin inhibit autophagy by increasing LC3B levels, as detected by an increase in the NanoBiT signal, consistent with the increase of HiBiT labelled LC3B within the cells. Note the consistency between 96 (left) and 384 well (right) formats and the agreement in the EC50 values for each of the compounds.

Additionally, the assay’s performance in high throughput screening formats, with Z factors exceeding 0.5 and acceptable hit rates, highlights its suitability for large-scale compound screening endeavors. Overall, the NanoBiT autophagy reporter cell line from Promega emerged as a valuable tool for elucidating autophagy modulation and holds immense potential for accelerating drug discovery efforts targeting autophagy pathways.

AlphaLISA LC3B Assay (Revvity)

The integration of the AlphaLISA LC3B assay from Revvity represented a pivotal component of our comprehensive approach to developing an autophagy assay. This assay capitalizes on the interaction between LC3B in cell lysates and paired anti-LC3B antibodies on donor and acceptor beads, facilitating a proximity-based luminescent signal emission upon excitation of donor beads. 

Through this innovative methodology, we were able to reliably detect and quantify changes in LC3B protein levels induced by autophagy modulators. Our results demonstrated the dose-dependent accumulation of LC3B in response to autophagy inhibitors chloroquine and bafilomycin, as evidenced by an increase in AlphaLISA signal (figure 5).

Autophagy assay development_AlphaLisa LC3B assay_results graph

Figure 5. Comparison of AlphaLISA (left) versus HiBiT (right) for autophagic enhancers. AZD8055, rapamycin and PP242 all decrease LC3B levels. Both technologies readout EC50 values for the compounds that are comparable in 384 well format. However, it was not possible to detect a consistent AlphaLISA signal in native HEK-293 cells and, consequently, we used the NanoBiT cell line, which expressed enough LC3B to make the AlphaLISA possible.

Conversely, autophagy enhancers AZD8055 and PP242 elicited a dose-dependent decrease in LC3B levels, resulting in a reduction of AlphaLISA signal. Importantly, the AlphaLISA assay exhibited excellent correlation with NanoBiT luciferase readouts, further validating its effectiveness in assessing autophagic flux (figure 6). 

Autophagy assay development_AlphaLisa LC3B assay_results graph

Figure 6. Comparison of AlphaLISA (left) versus HiBiT (right) for autophagic inhibitors. Chloroquin and bafilomycin both increase LC3B levels. Both technologies readout EC50 values for compounds that are comparable in 384 well format. For comparison the NanoBiT cell line, which expresses LC3B, was used in the AlphaLISA assay.

Additionally, the assay’s compatibility with high throughput screening formats and its ability to generate comparable EC50 values to NanoBiT assays underscore its utility for large-scale compound screening endeavors (table 1). 

Overall, the AlphaLISA LC3B assay from Revvity emerged as a valuable complement to our autophagy assay toolkit, offering a sensitive, reliable, and high-throughput-compatible platform for elucidating autophagy modulation in drug discovery applications.

Compound 

Promega 96 well EC50 (nM)

Promega 384 well EC50 (nM)

AlphaLISA 384 well EC50 (nM)

AZ8055

66

58

32

Rapamycin

0.50

0.76

6.3*

PP242

425

590

430

Chloroquin

4900

6200

7200

Bafilomycin

4.9

5.3

3.0

Table 1. Comparison of EC50 values for autophagy enhancers and inhibitors between 96 and 384 well formats for NanoBiT and when compared with AlphaLISA. The latter required the use of the HEK-293 NanoBiT cell line to detect LC3B.
Note: * value is higher due to a lack of definition at the bottom of the AlphaLISA curve.

WES-based Western Blot

Employing WES-based Western blotting (Biotechne) provided valuable insights into the modulation of autophagy and validated our findings from other assay platforms.

HEK-293 cells expressing HiBiT-tagged LC3B were treated with autophagy inducers and inhibitors, followed by lysis in RIPA buffer. Subsequently, protein lysates were quantified and loaded onto the WES system for analysis. 

Our results revealed distinct changes in LC3B protein levels in response to pharmacological modulation of autophagy (figure 7). 

Autophagy assay development_Protein Levels_results graph

Figure 7. LC3B protein levels in HEK-293 cells treated for 24 hours with PP242 (10mM) or AZD8055 (0.1mM) or DMSO. Both PP242 and AZD8055 reduce the protein levels of LC3B. Left panel is the Chemo-gram of the amount of secondary antibody detected using horse radish peroxidase. LC3B in cells treated with DMSO is 12,000 chemi-luminescence units, whilst PP242 and AZD8055 reduce this by 50%. Right panel is the pseudo blot of this data.

Specifically, autophagy enhancers AZD8055, PP242, and rapamycin elicited a significant reduction in LC3B levels, consistent with decreased autophagic flux. In contrast, autophagy inhibitors chloroquine and bafilomycin induced a marked increase in LC3B protein levels, indicative of enhanced autophagic activity (figure 8).

These findings corroborated the results obtained from NanoBiT and AlphaLISA assays, confirming the efficacy of WES-based Western blotting in detecting alterations in autophagic flux. The ability of WES to provide quantitative data and visualize protein bands further strengthens its utility as a complementary assay platform for autophagy research in drug discovery endeavors.

Autophagy assay development_Protein Levels_results graph

Figure 8. LC3B protein levels in HEK-293 cells treated for 24 hours with chloroquin (25mM) or bafilomycin (1.0mM) or DMSO. Both chloroquin and bafilomycin induce LC3B protein levels. Left panel is the Chemo-gram of the amount of secondary antibody detected using horse radish peroxidase. LC3B in cells treated with DMSO is ~12,000, whilst chloroquin and bafilomycin induce LC3B by 10 fold and 4 fold respectively. Right panel is the pseudo blot of this data.

High Content Imaging

High content imaging played a crucial role in our comprehensive assessment of autophagy modulation, providing valuable visual insights into cellular responses to pharmacological interventions. Adherent cells were treated with autophagy inducers and inhibitors overnight, followed by fixation and permeabilization. Immunostaining with a rabbit polyclonal LC3B antibody allowed for the visualization of LC3B protein localization and abundance within the cells. Imaging was performed using the Cellinsight CX5 high content imager at 20x magnification, enabling detailed examination of cellular morphology and LC3B staining patterns.

Our results revealed distinct differences in LC3B staining between treated and untreated cells, indicative of changes in autophagic activity. Specifically, treatment with autophagy enhancers such as AZD8055 resulted in a marked reduction in LC3B staining, consistent with decreased autophagic flux. Conversely, autophagy inhibitors like chloroquine induced a punctate LC3B staining pattern, indicative of autophagosome accumulation (figure 9). 

These findings were consistent with the results obtained from other assay platforms, validating the effectiveness of high content imaging in assessing autophagy modulation. Overall, high content imaging proved to be a valuable tool for visualizing and quantifying cellular responses to autophagy-modulating compounds, contributing to our understanding of autophagy regulation in drug discovery applications.

Autophagy assay development_High content imaging

Figure 9. LC3B antibody staining (Alexa 647) in HEK-293 cells imaged on Cellinsight CX5 at 20x magnification (Hoescht stained nuclei in blue). Cells were treated (left to right) with DMSO showing diffuse cytoplasmic LC3B staining, chloroquin (25M) (centre), in which the LC3B forms perinuclear punctate staining of autolysosomes in the cytoplasm and AZD8055 (right) in which there is significant inhibition of LC3B. 

High Throughput Screening (HTS) Format

Implementing a high throughput screening (HTS) format was essential for evaluating the efficacy of our autophagy assay in large-scale compound screening endeavors. 

To conduct the screen, we utilized the HEK-293 NanoBiT assay in a 384-well format, screening 25 plates (8,000 compounds) for both autophagy enhancers and inhibitors. Each plate included controls (DMSO) and an EC80 concentration of AZD8055 or PP242 for enhancer screening, or appropriate inhibitors for inhibitor screening.

The screening process was automated to ensure consistency and efficiency. Impressively, our results demonstrated robust performance of the NanoBiT assay in HTS format, with Z factors exceeding 0.5 and acceptable hit rates. 

Moreover, a substantial number of compounds were selected for further evaluation based on EC50 determination using a 10-point dose-response curve. This successful implementation of HTS format underscores the suitability of the NanoBiT autophagy assay for large-scale compound screening applications, facilitating the identification and characterization of novel therapeutics targeting autophagy pathways.

Autophagy assay development_High throughput

Figure 10. Evaluation of the NanoBiT LC3B autophagy cell line in HTS format. Controls (DMSO) and an EC80 concentration of AZD8055 or PP242 (dependent on enhancer or inhibitor screening mode) were dispensed into rows 1, 2 and 23, 24 respectively. Compounds at 10M final screening concentration were dispensed into rows 3 through 22. Plates were set up and screened using automated equipment. Z factors of greater than 0.5 were obtained, suggesting that this is a robust assay for high throughput screening purposes.

Autophagy assay development_High throughput

Figure 11. High Throughput Screening data. Left – enhancers of autophagy (decreased LC3B): Setting a 32% cut-off resulted in 379 compounds taken forward for retest of which 102 went forward for EC50 based on a 2 out of 3 rule. Inhibitors of autophagy (increased LC3B) – setting at 45% cut-off resulted in 442 compounds for retest of which 88 were taken forward for EC50 again based on 2 out of 3 rule. Compounds were chosen for EC50 based on medicinal chemistry rules.

Summary

In conclusion, the development of a comprehensive autophagy assay for drug discovery purposes has yielded promising results, offering valuable insights into the modulation of autophagy pathways by pharmacological agents.

Through the integration of innovative assay platforms such as NanoBiT autophagy reporter cell lines, AlphaLISA, WES-based Western blotting, and high content imaging, we have successfully characterized the effects of autophagy enhancers and inhibitors on cellular autophagic flux. These assays have demonstrated robust performance, providing sensitive and reliable readouts that correlate well with each other. 

Moreover, the implementation of high throughput screening (HTS) format has facilitated the screening of large compound libraries, enabling the identification of potential lead compounds for further development.

Overall, our findings underscore the importance of employing diverse assay methodologies to comprehensively evaluate autophagy modulation in drug discovery efforts. Moving forward, these assay platforms hold great promise for accelerating the discovery and development of novel therapeutics targeting autophagy pathways, with the ultimate goal of addressing unmet medical needs in various disease contexts.

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