Cryo-EM structures of human arachidonate 12S-lipoxygenase bound to endogenous and exogenous inhibitors

• Published in: Blood Vol. 142; no. 14; pp. 1233 - 1242
• Main Authors: Mobbs, Jesse I., Black, Katrina A., Tran, Michelle, Burger, Wessel A. C., Venugopal, Hariprasad, Holman, Theodore R., Holinstat, Michael, Thal, David M., Glukhova, Alisa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.10.2023; The American Society of Hematology
• Subjects: Arachidonate 12-Lipoxygenase - metabolism; Arachidonic Acid - metabolism; Cryoelectron Microscopy; Humans; Platelet Activation; Platelets and Thrombopoiesis; Thrombocytopenia; United States; United States
• DOI: 10.1182/blood.2023020441
• ISSN: 0006-4971; 1528-0020

•The first full-length structure of human arachidonate 12-LOX reveal mechanisms of its oligomeric and conformational states.•The structures uncover the natural inhibitor of 12-LOX and reveal the binding site of inhibitor ML355. [Display omitted] Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme. The enzyme human arachidonate 12S-lipoxygenase (12-LOX) is expressed in platelets where it promotes activation of αIIbβ3, glycoprotein VI, and protease-activated receptor 4. Mobbs et al report on high resolution structures of 12-LOX obtained by cryogenic electron microscopy. The authors elucidate oligomeric states, conformational plasticity, and binding interactions, providing new knowledge that may accelerate the development of antithrombotic structure–based inhibitors.