Apoptosis Pathway Assays
Target-specific Assays

Apoptosis Pathway Assay Services

Cancer cells have to overcome a variety of protective mechanisms of the body to form tumors. One of them is apoptosis which is a highly regulated form of programmed cell death that is needed to remove damaged or aged cells. Modulating apoptosis has been exploited to fight malignancies for example by overcoming treatment resistance.

Reaction Biology offers apoptosis assays for the investigation of test molecules targeting the apoptosis pathway. The apoptosis assay technology measures the binding of the apoptotic protein such as BCL2 to a substrate peptide such as BAK. The interference of the binding by a test molecule can be detected via a fluorescence readout.

  • Low scale, large scale screening and high-throughput options as well as hit to lead screening options are available
  • Please ask us for apoptosis assay development and custom-tailored screening options
  • The apoptosis pathway proteins are produced in house and available for purchase

Targeting the Apoptosis Pathway

Tumors receiving chemotherapy often suppress apoptosis resulting in treatment resistance. Anti-apoptotic Bcl-2 is often overexpressed in tumors suppressing Bax/Bak oligomerization in the mitochondrial outer membrane (MOM) which is a prerequisite for cytochrome C release and cell death (left panel).

Resistance may also be mediated by IAPs (inhibitors of apoptosis) which interfere with the caspase cascade to inhibit the execution of apoptosis (right panel).

Inhibiting anti-apoptotic proteins such as IAPs or Bcl-2 by administering drug combinations to patients can skew the balance leading to cell death for the successful treatment of many types of cancer.

BCL2 overexpression in tumors can result in treatment resistance as can caspase inhibition by IAP. drugs inhibiting BCL2 and IAP may help to overcome resitstance to chemotherapy.

List of Targets

PPI  … Protein:Protein Interaction

Apoptosis Assay Details

  • Assay Format:HTRF
  • Assay Format: Fluorescence Polarization
  • Assay Setup
Assay Format:HTRF

HTRF (Homogeneous Time-Resolved Fluorescence) is an assay technology that combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR) for measuring molecular interactions in a homogeneous format.

Example of HTRF assay principle for screening compounds inhibiting the binding of BCL-2 to Bak.  Antibodies recognizing the GST tagged-BCL-2 protein are coupled with the donor fluorophore (Terbium-chelate). The Bak peptide substrate is coupled to an acceptor fluorophore (FAM). The binding of BCL-2 to Bak  cause a proximity of acceptor and donor fluorophore enabling FRET upon excitation.

Assay Format: Fluorescence Polarization

The fluorescence polarization assay is based on the principle that the degree of excitation by polarized light of a small fluorescently labeled peptide changes with the degree of rotation. The change of motion after binding to a larger protein is the basis for this homogenous and reproducible competition binding assay.

Example of fluorescence polarization assay principle for screening compounds targeting cIAP.  A substrate peptide binding to the BIR3 domain of cIAP coupled to FAM fluorophore is used as tracer. The fluorescence polarization signal decreases with the addition of compound that interrupts cIAP interaction to the fluorescent substrate.

Assay Setup

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

Controls: No inhibitor (DMSO vehicle) control and for every assay, one target-specific control compound is tested in 10-dose IC50 format.

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project, 20 µl of a 10 mM DMSO stock or solid material is needed. Less material is needed for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.