Abstract
C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Thus, CXCR4 represents a promising drug target and several CXCR4 antagonizing agents are in preclinical or clinical development. Important parameters in drug lead evaluation are determination of binding affinities to the receptor and assessment of their stability and activity in plasma or blood of animals and humans. Here, we designed a microtiter plate-based CXCR4 antibody competition assay that enables to measure inhibitory concentrations (IC50 values) and affinity constants (Ki values) of CXCR4 targeting drugs. The assay is based on the observation that most if not all CXCR4 antagonists compete with binding of the fluorescence-tagged CXCR4 antibody 12G5 to the receptor. We demonstrate that this antibody-competition assay allows a convenient and cheap determination of binding affinities of various CXCR4 antagonists in living cells within just 3 h. Moreover, the assay can be performed in the presence of high concentrations of physiologically relevant body fluids, and thus is a useful readout to evaluate stability (i.e. half-life) of CXCR4 ligands in serum/plasma, and even whole human and mouse blood ex vivo. Thus, this optimized 12G5 antibody-competition assay allows a robust and convenient determination and calculation of various important pharmacological parameters of CXCR4 receptor-drug interaction and may not only foster future drug development but also animal welfare by reducing the number of experimental animals.
Highlights
C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis
Two other orally available small drug candidates are Burixafor (TG-0054) that is a monocyclic CXCR4 antagonist und currently tested in a phase II study for stem cell mobilization[20,23], and the isothiourea compound IT1t that was used for crystallization of the CXCR4 receptor thereby revealing a distinct binding mode from AMD3100 within the receptor binding pocket[24,25,26]
It has previously been demonstrated that several CXCR4 antagonists, including AMD310042,43, EPI-X431, T14044, T13445, ALX40-4C46, and POL302647 compete with the monoclonal anti-CXCR4 12G5 antibody for binding to CXCR4
Summary
C-X-C chemokine receptor type 4 (CXCR4) is involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis, asthma and pulmonary fibrosis. Several peptide-based CXCR4 antagonists are being tested for multiple applications Those peptides are often based on naturally occurring ligands, e.g. the 17 amino acid CXCL12 analogue CTCE-990827, and the two cyclic peptides LY251092415 and BKT-14028 that are currently tested in phase II clinical trials determining their effect on different kinds of cancer and stem cell m obilization[20,29] (for a review see[30]). Another linear peptide drug candidate is EPI-X4 (Endogenous Peptide Inhibitor of CXCR4), a recently identified body own antagonist of CXCR431–33. We demonstrate that this assay allows a robust and convenient determination of ligand-receptor pharmacodynamics and to measure the stability of CXCR4 ligands in serum/plasma and even whole human and mouse blood
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