Unlocking the Binding and Reaction Mechanism of Hydroxyurea Substrates as Biological Nitric Oxide Donors

Abstract

Hydroxyurea is the only FDA approved treatment of sickle cell disease. It is believed the primary mechanism of action is associated with the pharmacological elevation of nitric oxide in the blood; however, the exact details of this are still unclear. In the current work, we investigate the atomic level details of this process using a combination of flexible-ligand / flexible-receptor virtual screening coupled with energetic analysis that decomposes interaction energies. Utilizing these methods we were able to elucidate the previously unknown substrate binding modes of a series of hydroxyurea analogs to hemoglobin and the concomitant structural changes of the enzyme. We identify a backbone carbonyl that forms a hydrogen bond with bound substrates. Our results are consistent with kinetic and EPR measurements of hydroxyurea-hemoglobin reactions and a full mechanism is proposed that offers new insights into possibly improving substrate binding and/or reactivity.

How Does Catalase Release Nitric Oxide? A Computational Structure–Activity Relationship Study

“…Analogs 1, 5, 6 , and 8 produce NO due to the presence of an –NHOH group and its adjacent H N′ which stabilizes the radical intermediates via a hydrogen bond to Gly25. 18 These binding and reaction mechanisms differ from the present case of catalase CpdI where HUAs must first form C-nitroso formamide intermediates before final NO production. The absence of 3D structures further complicates the elucidation of structure-reaction details of HUAs with CpdI.…”

“…17 Our previous flexible docking studies, determined that 7 ’s inability to form NO was attributed to the absence of the H N′ atom. 18 However, this analog may interact with catalase CpdI to produce NO as it contains the essential –NHOH moiety required to form the C-nitroso formamide intermediate. IFD resulted in 22 binding conformations, however, neither pose A nor AB had any representative conformations.…”

“…Previous work in our lab has focused on correlating experimental SAR studies of the hemoglobin catalyzed production of NO to structural and mechanistic information. 18 This was the first step in our long-term goal of elucidating the binding and mechanistic detail of enzymes primarily responsible for in vivo conversion of HU → NO. Catalase CpdI is such an enzyme; known to produce NO upon interacting with HU and lacking characterization of its binding and reaction mechanism.…”

Elucidating a chemical defense mechanism of Antarctic sponges: A computational study

“…Previous work by Weihua Li et al has shown that refining homology models via MD simulation can be a necessary step in accurately predicting ligand binding modes in P450 proteins (e.g., CYP2J2). 46 From our own past experience with flexible docking studies, 47,48,49,50,51 we have also seen that it is advisable to utilize protein structures with receptor conformations that are predisposed to ligand binding (i.e.,"productive" conformations). Past research has shown that apo P450 binding sites in particular can be very malleable, 52,53 therefore "holo-like" structures were desirable, i.e., protein structures similar to those with a bound ligand, to ensure we had productive receptor conformations for docking.…”

Accessing and applying molecular history

“…However, in a pinch, doctors prescribe non-infants hydroxyurea. For reasons still being studied [63], hydroxyurea enhances production of gamma-hemoglobin in adults and children, thereby improving sickle-cell anemia symptoms.…”