Wednesday, June 30, 2010

Automated Kd, Ki determinations for CXCR4-SDF1

The chemokine CXCR4 resides on some cell membranes and selectively binds the SDF1 ligand signaling a pathway that has multiple critical functions in normal physiologies including embryonic development of the cardiovascular, hemopoietic and central nervous systems. CXCR4 also plays a key role in breast cancer metastasis, and has been implicated as a co receptor in HIV-1 cell penetration, and a primary receptor for HIV-2 cell penetration. Its function in these and other disease pathologies are fueling the search for small molecule CXCR4 antagonists as a means for intervention (1).

The Product Specialist for the Cisbio Tag-lite assay line visited BioTek last week. He was here to collaborate with us on validating the automation of the ‘Chemokine CXCR4 receptor ligand binding assay’ using BioTek liquid handling and detection instrumentation. Using HTRF technology based on the competition between fluorescently labeled acceptor ligand and non-labeled compounds to bind with a CXCR4 donor labeled receptor site on a cell membrane, this robust assay offers a simple and confident way of determining CXCR4 antagonist properties of possible drug candidate compounds via Kd, Ki/IC50 and Z’ values. These values are commonly used to validate assay performance and determine binding affinity of known and unknown agonists and antagonists in protein-ligand reactions.

The assay is very user friendly and proved to be automation friendly as well. There are only a few distribution steps to the assay well: cells, compounds, and tracer. The cells and tracer are added to the wells in a single step using the same volume for all wells on the plate. The BioTek MicroFlo Select equipped with a 1 ┬Ál cassette was used for cell and tracer dispensing, and in the Z’ experiment also performed a simultaneous 4-compound dispense in addition to the cell and tracer (the Z’ test was fully automated using MicroFlo Select). Although the serial dilutions for the Kd determination and Ki/IC50 determination have different start volumes and dilution ratios, their end volumes are the same and they are both done as 11-point serial dilutions with a 12th point assay control and depending on the test were dispensed in either duplicate or quadruplicate to a 384-well low volume Greiner BioOne white plate. Automation of the serial dilution steps and loading the serial dilutions to the assay plate was done using BioTek's Precision XS. After all dispenses to the assay plate are complete, there is a 1-2 hour room temperature incubation step, followed by detection of the fluorescence intensity. An HTRF ratio of the fluorescence intensities was used to calculate Kd, Z’, and IC50/Ki values. Data generated by automating the assay to that generated by performing the assay manually was used to judge viability of the instrumentation in performing the assay. The results of this successful collaboration will be highlighted by both an upcoming Application Note and a poster presentation at the 2010 MipTec conference in the early fall.

[1] H. Tamamura et al. (2008). “A future perspective on the development of chemokine receptor CXCR4 antagonists”, Expert Opinion on Drug Discovery, 3(10), pp. 1155-1166.

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