Hydroxyurea (HU) is the only FDA approved treatment of sickle cell disease (SCD). It is believed the primary mechanism of action is associated with the pharmacological elevation of nitric oxide (NO), and subsequent induction of fetal hemoglobin (HbF) in the blood. Although HU therapy has been used for over 15 years in treating SCD, the in vivo mechanism of NO production from HU has yet to be fully elucidated. HU interacts with oxy hemoglobin (oxyHb) and deoxy hemoglobin (deoxyHb) resulting in slow NO production rates. However, this did not correlate with the observed increase of NO concentrations in patients undergoing HU therapy. The discrepancy can be attributed to the interaction of HU competing with other heme-based enzymes such as catalase and peroxidases.  The primary goal of our current work is the full characterization of biomedically relevant enzymes involved in the in vivo conversion of HU to NO, including oxyHb, metHb, and catalase compound I (Cpd I). Further, we intend to propose new compounds for enhanced NO generation / inhibition, and identify new biological targets for these reactions. We investigate the atomic level details of this mutliscale process using a combination of flexible-ligand / flexible-receptor virtual screening studies, sequence based bioinformatics, classical molecular dynamics simulations, DFT based hybrid quantum mechanical / molecular mechanical (QM/MM) methods, and QM/MM charge perturbation analysis (CPA). From this study, we were able to determine the previously unknown substrate binding modes of a series of HUAs to human oxyHbA, metHbA, catalase Cpd I, and the concomitant structural changes of the enzymes. We are currently undertaking reaction path studies to identify the underlying mechanisms for the aforementioned enzymes. Following the successful generation of reaction profiles, the key components of transition state (TS) stabilization (e.g. energetic contribution of acids/bases/waters) were determined using QM/MM charge perturbation analysis (CPA). Results from these calculations and analyses identified the key structural feature of the ligand and protein (i.e. active site residues) that are most responsible for stabilizing the binding of substrates as compared to their reactive transition states. Our studies were able to shed light onto the experimentally reported reaction rates and explain the proposed radical stabilizations of several HU analogs interacting with hemoglobin. Our results are consistent with kinetic and EPR measurements with a full mechanism identified using the QM/MM Replica Path method; subsequently, results offer new insights into possibly improving substrate binding and/or reactivity. Results from computational reaction path studies (i.e. TS structures) will feed directly into rational design of both new NO donors and inhibitors.

Examining binding and reaction mechanisms of hydroxyurea as a biological nitric oxide donor using QM/MM reaction path methods

247th National Meeting (2014)

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