Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe

Abstract

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.gene microarray ͉ homology model ͉ osteoporosis ͉ bone

Biomimetic Screening of Class-B G Protein-Coupled Receptors

“…Our results are inconsistent with a necessary allosteric change of the extracellular domain which had been postulated as the initiating step of signal transduction for other class B GPCRs (Dong et al 2006;Dong et al 2008). CRHR 1 therefore resembles more closely the parathyroid hormone receptor 1, the only other class B GPCR for which juxtamembrane-specific agonists have been described (Carter et al 2007;Shimizu et al 2000).…”

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

“…Along similar lines, PTHR1 antagonists have potential therapeutic applications in disorders of calcium metabolism, such as hyperparathyroidism and malignancy-associated hypercalcemia. Indeed, the utility of the information obtained from cross-linking approaches has recently been shown in a report describing a small molecule antagonist of PTHR1 (25). Using knowledge gleaned from previous Bpa-mediated cross-linking of PTH to PTHR1, the authors used molecular modeling to manually dock this small molecule into a pocket between the tops of TMs 3, 4, 5, and 6.…”

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

“…Several small-molecule ligands have been identified for the PTHR1 but most of these function as weak antagonists (Carter et al, 2007;McDonald et al, 2007), and only one compound, called AH-3960 (dibutyl-diaminomethylenepyrimidine-2,4, 6-trione), exhibited agonist activity, albeit the potency of the compound for stimulating cAMP formation in cells was in the micromolar range compared with the low-nanomolar potency observed for PTH(1-34) (Rickard et al, 2006) (Table 2). The mechanisms of action of the compound ligands identified so far for the PTHR1 have not been determined, and so it is not clear whether they bind to the same or overlapping binding sites in the receptor as that used by PTH peptide ligands or to some other site and thereby function as allosteric modulators (Hoare, 2007) H)-one], was found by screening for the capacity to inhibit binding of a modified PTH(1-14) radioligand analog to the PTHR1, and so this compound likely binds to the same TMD receptor site used by the N-terminal pharmacophore of the PTH agonist ligand (Carter et al, 2007). The findings with SW106 thus suggest that at least some portion of the true agonist ligand binding site in the TMD region of the receptor is at least accessible to small-molecule ligands.…”