Development of in vitro Assays for Compound Screening (Subproject in P2: CYP11B2 inhibitors)
People involved: Katarzyna E. Schewe, Christina Ries
Fast and robust in vitro screening systems are crucial for discovery and development of new drugs. Firstly, the synthesized compounds have to be tested for their potency in a high-throughput system, such as S. pombe or mammalian cells expressing the human target enzyme. Based on the results compounds can be optimised for a specific inhibition. In the next step the potency of the lead compounds should be verified in an ex vivo test system using human tissues. Since rats are the most common model for in vivo studies during the pre-clinical phase, additional tests on rat enzyme expressed in mammalian cells and on rat tissues are essential.
1.
Stable expression of rat CYP11B2 (target enzyme) and CYP11B1 (control enzyme) in V79 cells
The mRNA was isolated from surgically removed rat adrenal gland using PCR, transcribed in cDNA, and CYP11B2 and CYP11B1 were cloned using PCR. As a vector we used the pcDNA3.1/V5-His TOPO TA. The stable expression will be performed in V79 hamster cells using a transfection method with a liposome formulation.
2.
In vitro Assay using rat / human adrenal glands
Classically, aldosterone is synthesised in the adrenal zona glomerulosa (Fig.1). There is some evidence that aldosterone can be synthesised in other tissues, like heart or central nervous system. However, adrenal glands are the main source of aldosterone and are best suited for measurement of aldosterone bioproduction.
Fig. 1 Adrenal cortex
The adrenal glands are architecturally complex tissues. The adrenal cortex is functionally and morphologically separated into distinct zones of steroidogenic activity. We could show that disruption of this tissue’s architecture decreases the steroid synthesis in rat adrenals. According to these results we developed an in vitro assay with whole rat adrenal glands. Using this assay we are able to show the potency of our compounds on rat enzyme and to determine the IC50 values (see example Fig. 2).
Fig. 2 Determination of IC50, showed is an average of four experiments
Now we concentrate on the development of an in vitro assay using human adrenal glands. For this reason, we collaborate with the Universitätsklinikum Homburg (Klinik für Urologie und Kinderurologie, Prof. M. Stöckle), which provides us with human adrenal tissue. The experimental procedures are in progress.
3. In vitro-heart failure model
One indication for the CYP11B2 inhibitor should be heart failure. The elevated levels of aldosterone stimulate the heart fibroblast to increase the production of collagen. Since heart is discussed as an aldosterone producing organ, it could be possible to develop an in vitro heart failure model using heart cell culture or heart slices. Primarily the aldosterone production as well as collagen formation in heart should be represented. Using CYP11B2 inhibitors a decreased collagen production could be expected.
4.
Aldosterone detection via LC/MS/MS