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What are PROTACs?

Proteolysis Targeting Chimeric (PROTAC) drugs are innovative molecules that utilize the ubiquitin-proteasome system to selectively degrade specific proteins within cells. PROTACs are one of the most promising methods for targeted protein degradation (TDP), which is used to remove disease-causing or disease-associated proteins. PROTAC drugs consist of two components: a ligand for the target protein and a ligand for E3 ubiquitin ligase, connected by a chemical linker. In PROTAC therapy, the target protein is linked to an E3 ligase enzyme, which facilitates ubiquitination and subsequent degradation of the protein by the proteasome. This approach can be used to target harmful proteins associated with conditions like tau-protein diseases and viruses, as well as protein complexes that hinder specific downstream activities. Several PROTAC small molecules are currently undergoing clinical trials and have shown promising results, highlighting the potential of this technology for therapeutic applications.

Figure 1 Depiction of the PROTAC recruiting an E3 ubiquitin ligase to a protein of interest (POI) to tag it with ubiquitin. This leads to the degradation of the POI.

 

Types of targeted protein degradation (TPD) technologies.

Targeted protein degradation technologies can be broadly classified into two categories: 1) targeted protein degradation via the ubiquitin-proteasome pathway (e.g. PROTAC), and 2) targeted protein degradation via the endosome-lysosome system (e.g., AbTAC, LYTAC, etc.) or autophagy-lysosome system (e.g., AUTOTAC, ATTEC, etc.) ⁽¹⁾ .

 

What are Potency Assays?

The quantitative measurement of biological activity of a given substance/product, like a drug or a component of a drug, is known as potency. The potency of a product can be assessed in a multitude of ways, including animal models (in-vivo), cell-based assays (in-vitro), and molecular biological assays. Ideally, these data will identify the minimum amount of the product required to elicit a biological response through its mechanism of action. In the pursuit of drug discovery and clinical trial advancement, cGMP potency assays are required for products to pass critical analysis as they ensure compliance with Good Manufacturing Practices (GMP) as determined by the FDA.

US FDA definition:

“Potency is defined as ‘the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result.’ (21 CFR 600.3(s)).” Source: FDA Guidelines ⁽²⁾.

FDA regulations offer flexibility in determining potency measurements for each product, considering its unique attributes, which requires individual evaluation of potency tests. However, when conducting release testing for licensed biological drug products, adherence to applicable biologics and cGMP regulations is mandatory for all potency assays.

Considerations in designing cGMP potency assay for PROTAC drugs.

In the context of PROTACs, developing effective cGMP potency assays is crucial for evaluating the performance and therapeutic potential of these innovative molecules. Since PROTACs utilize the ubiquitin-proteasome system to selectively degrade specific proteins, potency assays for PROTACs require tailored strategies. These strategies may assess the degradation efficiency of the target protein, monitor the formation of PROTAC-induced protein complexes, or measure downstream effects of target protein degradation. By employing these specialized assays, researchers can gain insights into the potency of PROTACs, optimize their design, and identify the most effective candidates for further development and clinical trials. Among these methods are cytotoxic assays, flow cytometry, ELISA (enzyme-linked immunosorbent assay), cell-based assays, and reporter assays.

Cytotoxic Assays for screening potency of PROTAC drugs

The cytotoxic potency assay for PROTAC development involves the following steps: cells expressing the target protein are plated and treated with various concentrations of the PROTAC compound, incubated to allow PROTAC-mediated degradation, supplemented with a luciferase substrate, and subjected to luminescence measurement ⁽³⁾. The luminescent signal is proportional to cell viability, enabling the determination of the cytotoxic potency of the PROTAC compound. This assay provides valuable insights into the efficacy of PROTACs in inducing cell death and aids in the selection and optimization of potential candidates for further development.

Flow Cytometry in testing potency of PROTAC drugs

Flow cytometry is a powerful analytical technique used to analyze and quantify characteristics of individual cells or particles within a heterogeneous sample. It involves the suspension of cells or particles in a fluid and their passage through a flow cytometer, which measures and analyzes various parameters simultaneously. This information is used to characterize and sort cells based on their size, shape, granularity, and fluorescence. Moreover, flow cytometry can also be used to investigate downstream effects of target protein degradation. By examining changes in cell surface markers, signaling molecules, or functional assays, researchers can gain insights into the functional consequences of protein degradation induced by PROTACs. This information aids in understanding the impact of PROTACs on cellular pathways, validating target engagement, and optimizing PROTAC design for therapeutic applications.

Flow cytometry assays for PROTAC potency can include (Fig. 2), but are not limited to:

1. Use of antibodies to monitor the degradation of surface and intracellular target proteins ⁽⁴⁾.
2. Analysis of cytokine up- or down-regulation in response to a PROTAC drug ⁽⁵⁾.
3. Study of cell cycle inhibition using DNA-flow cytometry analysis ⁽⁶⁾ .
4. Measurement of apoptosis (e.g., Annexin V-FITC/DAPI assay) ⁽³⁾.

 

Figure 2 Applications of flow cytometry in developing potency assays for PROTAC drugs.

ELISA for testing potency of PROTAC drugs and pharmacokinetics (PK) studies

ELISA is a widely used technique in PROTAC development for evaluating target protein levels and assessing PROTAC-induced protein degradation. In this assay, a capture antibody specific to the target protein is immobilized on a plate, and lysates from PROTAC-treated cells or tissues are added ⁽⁷⁾ . A detection antibody labeled with an enzyme is then introduced, followed by a substrate that produces a measurable color, fluorescent or luminescent reaction. The color or light intensity is proportional to the target protein concentration, allowing researchers to quantify its levels and assess the efficacy of PROTAC-induced degradation. ELISA is a valuable tool for understanding the potency and selectivity of PROTAC compounds and guiding their optimization for therapeutic applications.

Cell-Based Potency Assays for PROTAC drugs

Cell-based cGMP assays are essential for the development of PROTACs, allowing researchers to evaluate their efficacy and potential therapeutic applications ⁽⁷⁾. These assays include target protein degradation assays, which measure the reduction of a reporter-tagged protein upon PROTAC treatment; proximity-based assays, which detect and quantify the formation of protein-protein complexes induced by PROTAC binding, providing insights into target engagement; cellular phenotypic assays, which assess the impact of PROTAC-induced target protein degradation on cell viability, apoptosis, or cell cycle progression; and cytokine assays, which evaluate the immunomodulatory effects of PROTACs by measuring changes in cytokine production or release. These cell-based assays provide valuable information on the potency, selectivity, and functional consequences of PROTAC-induced protein degradation, guiding their optimization and potential therapeutic applications.

Reporter Assays for testing the potency of PROTAC drugs

In the development of PROTACs, a gene reporter assay, as part of cGMP potency assays, can be employed to evaluate the modulation of transcriptional activity ⁽³⁾. For example, Makkonen, et al. ⁽⁸⁾ showed how various reporter assays could measure the activity of androgen receptors (ARs). The assay involves constructing a reporter gene containing androgen response elements (AREs) upstream of a reporter gene, and cells expressing this construct. A hypothetical PROTAC could be designed to target the degradation of the AR which, if successful, would lead to a decrease in the transcriptional activity of the reporter gene, indicating inhibition of AR-mediated transcription. The gene reporter assay provides quantitative measurements and dose-response analysis of the PROTAC’s effects on AR activity, enabling assessment of selectivity and guiding the optimization of PROTACs targeting the androgen receptor for potential therapeutic applications.

Conclusion

PROTACs offer a promising approach for targeted protein degradation, holding immense potential for therapeutic applications. Developing effective cGMP potency assays is crucial for evaluating the performance and therapeutic potential of PROTACs. Cytotoxic assays, flow cytometry, ELISA, cell-based assays, and reporter assays are valuable tools for assessing the potency, selectivity, and downstream effects of PROTAC-induced protein degradation. By employing these tailored strategies, researchers can optimize PROTAC design, identify the most promising candidates for further development, and ultimately contribute to the advancement of targeted protein degradation-based therapies.

 

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