Caspases as pharmaceutical targets – screening for inhibitors?

Caspases (cysteine-dependent aspartate-directed proteases) belong to the family of cysteine proteases and are involved in networks controlling cell death (apoptosis and necrosis) and inflammation. 12 human caspases have been described so far (1.). Human Caspases have been classified according to their roles in apoptosis (Caspase-3, -6, -7, -8, and -9) and inflammation (Caspase-1, -4, -5, and -12). Caspase-2, -10, and -14 can be less easily classified concerning the function (for an overview see 2.).

So let’s take a further look at their role, and some of the tools available to investigate and screen compounds modifying Caspase activities.

Fotolia_71744424_XSCaspases in Apotosis

Caspases involved in apoptosis can be further divided into Initiator Caspases (Caspase-8, and -9) and Executioner Caspases (Caspase-3, -6, and -7). Initiator Caspases are activated by dimerization of  inactive procaspase monomers, while Executioner Caspases have to be cleaved and thus activated by Initiator Caspases. Executioner Caspase exist as procaspase dimers which upon cleavage and subsequent conformational change results in the mature and active enzyme. The activated Executioner Caspases finally lead to destruction of key structural proteins and activate other enzymes.

Caspases in Inflammation

The above mentioned Caspases (Caspase-1, -4, -5, and -12; in mice Caspase-1, -11, and -12 serve the same function) play a critical role as mediators of innate immune responses. These caspases are generated as inactive procaspases as well and are activated after cellular stimulation and the formation of the inflammasome complex. Finally activated Caspase-1 cleaves the proinflammatory  cytokines IL-1β, pro-IL-18, and pro-IL-33, thus facilitating their secretion which leads to  inflammation (3.).

Caspases involved in human diseases

Defective Caspase activities can be involved in the pathology of a number of human diseases.

  • Cancer: Non-conforming Caspase activation and consequential inadequte cell death can lead to tumorigenesis.
  • Neurodegenerative diseases: Hyper activation of Caspases and resulting excessive cell death is able to promote neurodegenerative conditions.
  • Infection/Inflammation: Increased sensitivity to infection can be caused by insufficient Caspase activation, while over-activation of the same Caspases can lead to inflammatory conditions.

Tools to investigate and screen compounds modifying Caspase activities

Looking at the roles of Caspases in diverse human diseases, it becomes obvious that both Caspase inhibitors as well as Caspase activitors can be considered as potential drug candidates. Here, I’ll summarize some of the tools which are available to measure Caspase activity and/or inhibitors effects of compounds on Caspase activity.

Caspase BPS graphs

Inhibition of Caspase-3 enzyme activity using XIAP Bir2 domain (upper left), Caspase-7 enzyme activity using XIAP Bir2 domain (upper right), Caspase-6 enzyme activity using Ac-IETD-CHO, and Caspase-8 enzyme activity using Ac-IETD-CHO using the homogeneous Caspase sssays mentioned in this blog. Fluorescence intensity was measured using a Tecan fluorescent microplate reader.

BPS Biosciences recently launched homogenous assays to measure the activity of:

All these kits kits allow you to measure enzyme activity and effects of Caspase modulators. They come in a convenient 96-well format, with all the reagents necessary for 100 fluorescent Caspase activity measurements. In addition, the kits include purified Caspases enzyme and a potent Caspase inhibitor for use as a positive and negative control. The fluorogenic substrate in the kit is incubated with purified Caspase and the enzymatic activity releases AFC fluorophore that can then be measured using a fluorescence reader.

Caspases Anaspec graph

Jurkat cells were seeded to 96-well plates. Apoptosis was induced with 1 µg/mL staurosporine for 3 h. The identical population of cells after induction of apoptosis was incubated with caspase-8 specific inhibitor Ac-IETD-CHO at 25 µM final concentration. Fluorescence readings were taken 60 min. after addition caspase-8 substrate.

Furthermore, Anaspec offers kits as well to measure activity and inhibition of:

Again, these homogenous kits are meant to screen for specific Caspase inhibitors and to measure the activity of the enzymes.  The read-out can be done with a fluorescence plater reader as well.



Last (but not least of course!), you can also explore a range of:

I’ll especially come back to cell based activity assays (to detect active Caspases in cells) and the Caspase modulators in a future blog.

If you’re interested in more information or would like to leave a comment, feel free below!



1. HUGO Gene Nomenclature Committee:

2. David R. McIlwain, Thorsten Berger, and Tak W. Mak, Caspase Functions in Cell Death and Disease, Cold Spring Harb Perspect Biol 2013; 5:a008656.

3. F. Martinon, K. Burns, and J. Tschopp, The inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-β, Mol Cell 10: 417–426 (2002)




  1. […] tools to measure Caspase activity and inhibition by compounds in homogenous biochemical assays: Caspases as pharmaceutical targets – how to screen for inhibitors? Today I would like to give you an overview about a method and kits which allow to detect active […]

  2. […] of reactions regulated by specific proteases called caspases (take a look at previous posts on Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety […]

  3. […] involves a complex cascade of reactions regulated by specific proteases called caspases (see Caspases as pharmaceutical targets – how to screen for inhibitors?), and results in DNA degradation. Apoptotic processes have been researched in an extensive variety […]

  4. […] In a previous post, I discussed Caspases as pharmaceutical targets – how to screen for inhibitors? […]

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