Study Of The Small Molecules Targeting Hypoxia Inducible Factor HIF-2 Through Allosteric Effects

Hypoxia-inducible factor(HIF)is a group of transcription factors that can regulate the sensing and adaptation of cells to oxygen availability.HIF with transcriptional activity is composed of an α-subunit and β-subunit for dimerization.In vertebrates,three HIF-α isoforms(HIF-1α,HIF-2α and HIF-3α)have been identified.HIF-2αregulates a variety of genes related to the development of solid tumors.The HIF-2αprotein is overexpressed in patients with clear cell renal cell carcinoma(ccRCC),thus reducing the activity of HIF-2α can potentially inhibit tumor growth.Belzutifan,a small molecule inhibitor targeting HIF-2α studied by MSD,is currently in phase Ⅲ clinical trials for the treatment of ccRCC.However,the crystal structure of HIF-2α-ARNT with Belzutifan is still not available,limiting the comprehensive understanding of the mechanism of Belzutifan.On the other hand,erythropoietin(EPO)is mainly produced in kidney,patients with chronic kidney disease(CKD)usually show symptoms of renal anemia due to insufficient EPO secretion,HIF-2α can up-regulate the expression of EPO,thus increasing the activity of the HIF-2 signaling pathway helps the treatment of anemia in CKD patients.Small molecule agonists M1001 and M1002 can directly target HIF-2α and increase the expression of HIF-2α downstream genes in 786-O ccRCC cell line.HIF-2α agonists has potential clinical value for the treatment of renal anemia.Therefore,we revealed the molecular mechanism how belsutifan inhibits HIF-2α based on the crystal structure,and obtained one HIF-2α agonist by screening a compound library.Our work is generally divided into two parts.In the first part we studied the molecular mechanism how Belzutifan inhibits HIF-2α.HIF-2α belongs to the bHLHPAS family of transcription factors,each of which contains a bHLH and tandem PAS(PAS-A and PAS-B)domains.We obtained the structure of HIF-2α-ARNT dimer in complex with Belzutifan at 2.76 (?) resolution.Belzutifan binds in the PAS-B domain pocket of HIF-2α and shares a hydrogen bonding network with residues His293,His298 and Asn341,stabilizing its conformation.Besides,Belzutifan forms a π-π interaction with residue Phe254 and numerous hydrophobic interactions with neighboring amino acids.The binding of Belzutifan forces the side chain of HIF-2α Met252 to flip out of the pocket,destroying the stability of the dimerization interface between HIF-2α PASB domain and ARNT PAS-B domain.Consistent with this hypothesis,a time-resolved fluorescence resonance energy transfer experiment showed that Belzutifan disrupted the dimerization of HIF-2α and ARNT with a Ki value of 40 nM.In addition,the affinity between Belzutifan and HIF-2α was determined to be 15.6 nM using an isothermal titration calorimetry assay.Belzutifan was indicated to inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM via a luciferase reporter assay.The second part describes the screening and optimization of HIF-2α agonists.We screened a small library of compounds designed and synthesized by Zhang Yinan’s group based on the reported HIF-2α agonists M1001 and M1002.The binding ability of these compounds with HIF-2α was characterized by a protein thermal shift assay.Two compounds,A93 and A98 were found with higher affinity to HIF-2α(ΔTmD>1℃).Subsequently,compounds were screened via a luciferase reporter assay using 786-O cells stably transfected with HRE or VEGF reporter gene.Compounds A44,A47,A69,A83,A86,A87 and A91 showed better transcriptional agonistic activity than M1001 on both reporters.Finally,those compounds with agonistic activity were rescreened using a quantitative PCR assay.Compound A64 was identified with stronger transcriptional agonistic activity than M1001 on HIF-2α downstream genes EPO and NDRG1.In summary,we solved the crystal structure of the HIF-2α-ARNT-Belzutifan complex,revealed the allosteric mechanism of Belzutifan inhibiting the transcriptional activity of HIF-2α,and discovered compounds with better transcriptional agonistic activity than M001.The work provides valuable information for the design of anticancer drugs(antagonists)and anti-anemia drugs(agonists)targeting HIF-2α.