Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability

Abstract

NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC50 = ∼275 μM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC50 = 10.81 μM) and ZINC39395747 (IC50 = 9.14 μM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development.

Keywords: computational biology; cytochrome b5 reductase 3; hemoglobin; nitric oxide; oxidation-reduction (redox); vascular.

FIGURE 1.

Flow chart representing the approach taken to identify new small molecule inhibitors for CYB5R3. a, molecular structure of CYB5R3 consisting of two domains: the FAD-binding domain (Thr-30–Ser-145, blue) and a NADH binding domain (Ser-173–Phe-300, red). These domains are connected by a linker (hinge) at the bottom (Gly-146–Lys-172, purple). b shows the NADH and FAD binding domain of CYB5R3. c illustrates the proposed binding of PTU in the NADH binding pocket. d shows the structure of PTU and an example of a carbon tail modification. e represents an example (ZINC 39395747) of a modification to that carbon tail that is predicted to increase the binding affinity to the NADH pocket. f shows a list of ZINC compounds identified through thiouracil-biased pharmacophore screening. Structures show modified carbon tail and side chain modifications to the parent thiouracil compound.

FIGURE 2.

Primary screening, secondary screening, and IC₅₀ values of identified compounds. a shows the primary screen using recombinant human CYB5R3 incubated with each compound (500 μm) followed by activity measurements with an NADH-ferricyanide reductase assay. b represents the secondary screen with 50 μm concentrations of each compound. c shows the calculated IC₅₀ values of PTU, ZINC39395747, and ZINC05626394. d shows the binding constants potential of PTU, ZINC39395747, and ZINC05626394 with CYB5R3 using ITC. e shows the time course of CYB5R3 inhibition by ZINC39395747 using an in vitro ferricyanide reduction assay. All error bars represent the S.E.

FIGURE 3.

List of ZINC compounds with modified pyrimidine rings and their inhibitory effect. a shows the derivatives of ZINC05626394, and b shows the ZINC39395747 derivative with modified pyrimidine rings. c illustrates the primary screen using recombinant human CYB5R3 incubated with each compound (500 μm) followed by activity measurements with an NADH-ferricyanide reductase assay. d represents a secondary screen with 50 μm concentrations. All error bars represent the S.E.

FIGURE 4.

Effects of ZINC05626394 and ZINC39395747 on cellular CYB5R3 activity. a, CYB5R3 knockdown efficiency in HEK cells using shRNA. b, CYB5R3 activity inhibition in HEK CYB5R3 knockdown cells determined by ferricyanide reduction assay. CYB5R3 activity inhibition by ZINC39395747 and ZINC05626394 in HEK cells (c) and rat renal endothelial cells (d) was determined by ferricyanide reduction assay. e, CYB5R3 protein levels in rat renal endothelial cells treated with ZINC39395747 and ZINC05626394. f, reduction rate of met α globin by CYB5R3 in the presence of ZINC39395747 and ZINC05626394. g, met α globin formation in isolated mouse RBCs treated with ZINC39395747 for 36–48 h. AU, absorption units. h, a representative image of met α globin formed in RBCs treated with ZINC39395747 or vehicle. * indicates significant difference of p < 0.05. All error bars represent S.E.

FIGURE 5.

Renal smooth muscle cells and renal endothelial cells co-culture assays show the effect of ZINC39395747 on nitric oxide bioavailability. a, Western blot showing protein level of tubulin, CYB5R3, and Hb α in renal endothelial cells, myoendothelial junctions, and preglomerular vascular smooth muscle cells (SMC). b shows schematic of the vascular cell co-culture transwell assay with pretreatment of ZINC39395747followed by stimulation with DEA NONOate. The graph shows the cGMP response in smooth muscle cells in response to DEA NONOate pretreated with vehicle or ZINC39395747. All error bars represent S.E. *, significant difference at p < 0.05.

FIGURE 6.

Effect of ZINC39395747 on renal blood flow in vivo. Shown are MABP, RBF, and RVR responses to AngII (0.1–1.0 μg) (a), NE (1.0–10.0 μg) (b), and NPY_1–36 (1.0–10.0 μg) (c). The asterisk indicates a significant difference of p < 0.05. All error bars represent S.E.