Homodimerization of the PAS-B Domains of Hypoxia-Inducible Factors

The Per–Arnt–Sim (PAS) domains of hypoxia-inducible transcription factors (HIF) mediate heterodimer formation between the HIF-α forms that are induced in the event of cellular hypoxia and the constitutive HIF-β variants. Previous efforts toward structural characterization of the HIF-1α PAS domains were limited by protein stability. Using homology modeling based on the published crystal structure of the PAS-B domain of the homologous protein HIF-2α in complex with the partner HIF-β (also known as ARNT), we have identified a variant of HIF-1α with improved solubility, monodispersity, and stability. Purified solutions of the PAS-B domains of HIF-1α and HIF-2α differ in their propensity for homodimer formation. In an attempt to understand the structural basis for this difference, and to document the structural changes that accompany homodimer formation, we have undertaken a comparative NMR study of the PAS-B domains of HIF-1α and HIF-2α and mutants of HIF-1α that mimic the behavior of HIF-2α. The NMR spectra of all of these domains are very similar, consistent with the similarity of their amino acid sequences. However, the greater propensity of the HIF-1α PAS-B domain to form dimers as the concentration was increased allowed us to determine the site of homodimerization and pointed toward possible sequence changes in HIF-1α that might discourage the formation of homodimers

Discovery of a Novel Class of Exquisitely Selective Mesenchymal-Epithelial Transition Factor (c-MET) Protein Kinase Inhibitors and Identification of the Clinical Candidate 2‑(4-(1-(Quinolin-6-ylmethyl)‑1<i>H</i>‑[1,2,3]triazolo[4,5‑<i>b</i>]pyrazin-6-yl)‑1<i>H</i>‑pyrazol-1-yl)ethanol (PF-04217903) for the Treatment of Cancer

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit <b>6</b> was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor <b>10</b> with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1<i>H</i>-[1,2,3]­triazolo­[4,5-<i>b</i>]­pyrazin-6-yl)-1<i>H</i>-pyrazol-1-yl)­ethanol (<b>2</b>, <b>PF-04217903</b>), an extremely potent and exquisitely selective c-MET inhibitor. <b>2</b> demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. <b>2</b> progressed to clinical evaluation in a Phase I oncology setting