Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

[¹²5I]YP20: a novel radioligand specific for the extracellular domain of the CRF1 receptor.

The peptide corticotropin-releasing factor (CRF) binds to the CRF1 receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or "J"-site. Known small molecule antagonists bind to the J-site while known CRF1 receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF1 receptor. This ligand, which we named [¹²5I]Yamada peptide 20 ([¹²5I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [¹²5I]CRF binding site on the hCRF1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF1-CHO cells. Under optimized conditions, 20 pM [¹²5I]YP20 reproducibly bound to hCRF1-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²5I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²5I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t(1/2)=0.92 min and t(1/2)=11.7 min). While [¹²5I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF1 receptor.