Bidirectional modulation of HIF-2 activity through chemical ligands

Abstract

Hypoxia‐inducible factor‐2 (HIF‐2) is a heterodimeric transcription factor formed through dimerization between an oxygen‐sensitive subunit HIF‐2α subunit and its obligate partner subunit ARNT. Enhanced HIF‐2 activity drives some cancers, while reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF‐2 activity via direct‐binding ligands could provide many new therapeutic benefits. Here, we explored HIF‐2α chemical ligands using combined crystallographic, biophysical, and cell‐based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF‐2α PAS‐B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first‐in‐class HIF‐2α agonists and found they significantly displaced pocket residue Y281. Its dramatic side‐chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF‐2α PAS‐B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF‐2α‐ARNT heterodimerization. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control

“…PT2385 binds to HIF-2α and has been shown to impair its dimerization with HIF-1β, thus preventing the formation of the DNA-binding complex and blocking HIF-2 transcriptional activity (12)(13)(14)31). Most of our findings are consistent with this mode of action.…”

“…The S305M mutation in HIF-2α confers resistance to PT2385 without altering HIF-2α protein stability or mRNA expression. The class of HIF-2α antagonists that includes PT2385 binds directly to a pocket within the PAS-B domain of the HIF-2α, but not HIF-1α, protein and allosterically weakens HIF-2α/HIF-1β heterodimer stability (10,11,31). To investigate the specificity of this action, the effect of PT2385 on HIF-2α bearing a point mutation located in the HIF-2α PAS-B domain (S305M in the mouse sequence; ref.…”

“…Plasmid construction and site-directed mutagenesis. Mouse HIF-2α PAS-B domain (GenBank accession AAH57870, residues 241-361) was cloned into pSJ2 vector, as previously described (31). Mutation of S305M was introduced using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).…”

“…Protein expression and purification. WT mouse HIF-2α PAS-B domain and the S305M mutation were both expressed and purified, as previously described (31). HIF-2α/HIF-1β heterodimers (WT and with the S305M mutation in the HIF-2α subunit) consisting of bHLH-PAS-A-PAS-B domains were expressed and purified using the coexpression constructs and procedures previously published (31).…”

“…Protein thermal shift. Experiments were carried out on a StepO-nePlus Real-Time PCR System (Applied Biosystems) using 2 μM of each protein construct in buffer containing 20 mM Tris pH 8.0 and 150 mM NaCl, as previously described (31). Data were analyzed using the DMAN, version 5.2 (47), to calculate ΔT m .…”