MetAP2 Inhibition Modifies Hemoglobin S (HbS) to Delay Polymerization and Improve Blood Flow in Sickle Cell Disease
Authors: Demers Melanie , Sturtevant Sarah , Guertin Kevin , Gupta Dipti , Desai Kunal , Vieira Benjamin , Hicks Alexandra , Ismail Ayman , Nakamura Yukio , Goncalves Bronner , Di Caprio Giuseppe , Schonbrun Ethan , Hansen Scott , Safo Martin K. , Wood David K. , Higgins John M. , Light David R. 
Published: November 2019

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet).
MetAP2 is the methionine aminopeptidase able to cleave iMet from Val1 on α-globin and βS-globin as the unfolded N-terminal peptides emerge from the ribosome. Enzyme kinetic studies with pure MetAP2 and N-terminal octapeptides showed that βS-globin peptide is a 5-fold better substrate than α-globin peptide. Lentiviral shRNA knock-down of MetAP2 in differentiating erythroid HUDEP cells in vitro confirmed that α-globin is more extensively modified than βS-globin, consistent with the enzyme kinetic data. Selective MetAP2 inhibitors used to treat cultured human erythroid cells (HUDEP and PBMC derived CD34+) and Townes SCD mice in vivo confirmed that both α-globin and βS-globin domains of HbS are extensively modified by N-terminal iMet and acetyl-iMet.
N-terminal retention of iMet and subsequent acetylation creates a mixture of modified HbS tetramers with combined modifications on both globins. Cation exchange chromatography separated nine different modified HbS variants from unmodified HbS as identified by LCMS. Purified samples of HbS modified by N-terminal iMet and acetyl-iMet had increased oxygen affinity as measured by decreased P50. Modified HbS containing the acetyl-iMet-βS-globin were found to have delayed polymerization under complete hypoxia (sodium metabisulfite triggered hypoxia in 1.8 M phosphate). Two modified HbS variants were further purified for X-ray crystallography studies (βS-globin / iMet-α-globin and acetyl-iMet-βS-globin / iMet-α-globin). Oxyhemoglobin structures of both modified HbS variants were in the R2-state previously described in structures of aldehyde modified HbS. This R2-state stabilizes the oxygenated R-state of HbS from conversion to the deoxygenated T-state that initiates HbS polymerization in sickle RBC.
Treatment by selective irreversible covalent or reversible MetAP2 inhibitors resulted in high levels of HbS modification (>75%) in cultured erythroid cells (HUDEP and CD34+ cells). Dose dependent modification of HbS was observed in Townes sickle cell mouse blood RBC in vivo with total modification of HbS approaching 50%. In whole blood ex vivo studies, modification of HbS decreased RBC sickling under hypoxia (4% O2) and significantly increased the affinity of RBC for oxygen (decreased P50). Blood samples from MetAP2 inhibitor treated mice were analyzed for single-cell O2 saturation of the RBC and for the fractional flow velocity drop in whole blood rheology under decreasing partial oxygen pressures. In blood from vehicle treated sickle mice, a low-saturation peak of deoxy-HbS was observed in 7.8% O2, in contrast to blood from MetAP2 inhibitor-treated mice where the low-saturation peak was only observed in 6.4% O2. Similarly, in an assay of O2 dependent blood flow rheology, the half-maximum fractional velocity drop occurred at 5% O2 in control blood decreasing to 2% O2 in MetAP2 inhibitor treated blood.
Our studies show that MetAP2 inhibition results in retention of iMet on βS-globin and α-globin and allows further acetylation of the retained iMet to create a mixture of N-terminal modified HbS tetramers. These modified HbS variants resist polymerization and RBC sickling under conditions of low O2 by delaying HbS polymerization and increasing O2 affinity. Our data suggests that MetAP2 may warrant further study as a potential therapeutic target for sickle cell disease.

Disclosures
Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.