IDENTIFYING ANTAGONISTS TOWARDS A G-PROTEIN COUPLED RECEPTOR USING THE STRUCTURE OF A HORMONE COMPLEXED TO A NEUTRALISING MONOCLONAL ANTIBODY
Galina Polekhina,a* William J. McKinstry,a* Hannelore Diefenbach-Jagger,a Patricia W.M. Ho,a Craig J. Morton,a Koh Sato, b Etsuro Onuma,b Matthew T. Gillespie,a
T. John Martin,a and Michael W. Parkera
aSt. VincentÕs Institute of Medical Research, 9 Princes Street, Fitzroy, VIC 3065, Australia; bPharmaceutical Research Laboratory, Chugai Pharmaceutical, Japan
(galinap@medstv.unimelb.edu.au)
*Both authors contributed equally to the work
G-protein coupled receptors (GPCR) are the most common receptor class in humans controlling critical cell functions and are thus an important class of drug targets. They are notoriously difficult to over-express and crystallize, and to date only one high resolution structure (bacterial rhodopsin) is available. We present a promising new method for discovering GPCR agonists and antagonists using structures of GPCR ligands bound to antibodies.
Parathyroid hormone related protein (PTHrP) was discovered as a hypercalcemia-causing factor of malignancy [1]
. Under normal circumstances PTHrP plays a key role in regulating
embryonic development of the skeleton and other tissues
. PTHrP as well as parathyroid hormone (PTH) act upon the same GPCR, parathyroid hormone receptor (PTH-R). PTHrP has also been shown to be one of the main culprits of metastasis into bone of human breast cancer cells causing bone breakdown.
We have crystallized and determined the structure of the complex between PTHrP and a neutralizing monoclonal antibody. The humanised version of this antibody is currently in clinical trials (Phase II) in Japan for treatment hypercalcemia, bone metastasis and cachexia [2]. The structure revealed that the residues of PTHrP involved in the interaction with the antibody are either strictly conserved among PTH and PTHrP sequences or have been implicated in the binding to the receptor. We therefore predicted that the binding pocket on the antibody resembles the receptorÕs. Using the structural data, we will screen the large library of small Òdrug-likeÓ molecules for the best fit into the PTHrP binding pocket of the antibody. The top hits will be screened in cellular assays for inhibition towards action of PTHrP on PTH-R. The structural biology results and those from the computational screen and cellular assays will be presented.
1 T.J. Martin (2002) The Journal of Clinical Investigations. 10, 1399-1401
2 K. Sato (1999) The Seventh International Forum on Calcified Tissue and Bone Metabolism. 32-37.