Inhibition of the FAD containing ER oxidoreductin 1 (Ero1) protein by EN-460 as a strategy for treatment of multiple myeloma

Abstract

Multiple myeloma (MM) cells demonstrate high basal endoplasmic reticulum (ER) stress and are typically exquisitely sensitive to agents such as proteasome inhibitors that activate the unfolded protein response. The flavin adenosine dinucleotide (FAD) containing endoplasmic reticulum oxidoreductin enzyme (Ero1L) catalyzes de-novo disulfide bridge formation of ER resident proteins and contributes to proper protein folding. Here we show that increased Ero1L expression is prognostic of poor outcomes for MM patients relapsing on therapy. We propose that targeting protein folding via inhibition of Ero1L may represent a novel therapeutic strategy for the treatment of MM. In this report we show that treatment of MM cells with EN-460, a known inhibitor of ERO1L, was sufficient to inhibit cell proliferation and induce apoptosis. Furthermore, we show that cell death correlated in part with induction of ER stress. We also show that EN460 inhibited the enzyme activity of Ero1L, with an IC50 of 22.13 μM, consistent with previous reports. However, EN-460 was also found to inhibit other FAD-containing enzymes including MAO-A (IC₅₀ = 7.91 μM), MAO-B (IC₅₀ = 30.59 μM) and LSD1 (IC₅₀ = 4.16 μM), suggesting overlap in inhibitor activity and the potential need to develop more specific inhibitors to enable pharmacological validation of ERO1L as a target for the treatment of MM. We additionally prepared and characterized azide-tagged derivatives of EN-460 as possible functional probe compounds (e.g., for photo-affinity labeling) for future target-engagement studies and further development of structure-activity relationships.

Keywords: Flavin adenosine dinucleotide; Oxidative stress; Unfolded protein response.

Figure 1:. High Ero1L expression levels are poor prognostic indicator of survival in relapsed MM patients.

(A) GSE9782, all patients were compared for differences in outcome related to ERO1L expression levels. Kaplan-Meier estimates were calculated for overall survival in the high (>median ERO1L expression) and low (<median ERO1L expression) groups. A log-rank test was used to assess significant difference in outcome. (B) Patients receiving Dexamethasone (n=76). (C) Patients receiving bortezomib (n=188).

Figure 2:. ERO1L inhibitor, EN460, induces apoptosis and ER stress in MM cells.

(A) U266 cells were treated with DMSO (vehicle control) or 25 μM EN460 for 18 h and then subjected to Annexin V-FITC and propidium iodide (PI) staining followed by flow cytometry analysis of apoptosis. Data are reported as means ± SD for three independent experiments. (B) U266 cells were treated with DMSO (vehicle control) and EN460 for 2, 4 and 8 h. Markers for the induction of ER stress include an increase in phosphorylation of eIf2 alpha, increased ATF4 levels and cleavage of ATF6. As such we measured the induction of these ER stress proteins following treatment of EN-460. Following exposure to EN-460 we observed a robust increase in p-eIF2α and a reduction of full length AYF6. The ATF6 antibody used does not recognize the cleaved product but as shown EN-460 treatment of U266 cells resulted in reduced levels of full length ATF6 consistent with cleavage of the protein. Surprisingly only a modest increase in ATF4 was detected following treatment with EN-460 in U266 cells. Equal protein loading is demonstrated by beta actin (n=3 independent experiments).

Figure 3:. Protein-protein interaction (PPI) of Ero1L and PDI.

The docked pose showing the orientation of the two proteins docked to allow for the redox cycling and electron transfer. Ero1L with the full sequence modeled onto 3AHQ, is shown as silver, and PDI as blue. The crystal structure of Ero1L the missing sequence is shown in green. The FAD ligand is shown in atomic colors.

Figure 4 .. Azide modification of EN460 increased efficacy.

(A) Structural formulas of EN460 and PB-EN-10 (an azide derivative of EN460). (B) PB-EN-10 shifts first derivatives, dF/dT of melt curve compared to vehicle (DMSO). Graph of first derivatives, dF/dT, of fluorescence intensities of Sypro Orange combined with Ero1α (0.25 mg/mL) and PB-EN-10 (20 μM) or DMSO. Melting temperatures (Tm) were defined by inflection point in dF/dT (dashed lines). (C) Bar graph of melting temperatures (Tm) of Ero1α with PB-En-10 (20 μM), EN460 (20 μM), or DMSO. Ero1α/PB-EN-10 interaction increased Tm and stability of Ero1α compared to EN460. (D) PB-EN-10 improved inhibition of hEro1α activity compared to parent compound, EN460. Purified human Ero1α (0.0625 mg/mL) incubated with increasing concentrations of EN460 or PB-EN-10 with Amplex Red Assay. (E) Graph of first derivatives, dF/dT of melt curves and (F) Melting temperatures (Tm) of PDI (0.25 mg.mL) were not altered with the addition of 20 μM of PB-EN-10 or EN460 suggesting the inhibition of Ero1α was not mediated by PDI. Graphs are represented images of two experiments performed in triplicate.

Figure 5:. Inhibition of FAD containing enzymes activity with EN460.

(A) Ero1L (B) MAO-A (C) MAO-B (D) LSD-1. Each data point is avg. and standard error of fit, with N = 2.

Figure 6.. Enzyme kinetics of EN460 inhibition of Ero1α oxidase.

Ero1α activity was monitored by using an Amplex Red assay. (A) The elevated hydrogen peroxide levels (intensifying fluorescence) observed with increasing concentrations of purified human PDI protein were progressively suppressed in the presence of escalating amounts of EN460. (B) Lineweaver–Burk plot of results in (A) shows EN460 functions as a competitive inhibitor of Ero1α, under these conditions.

Figure 7:. Docking of EN460 in FAD binding pocket of Ero1L.

(A) Hyperactive form of Ero1α (DOI: 10.2210/pdb3AHQ/pdb) was used to determine possible binding of EN460 in FAD binding pocket with PyMol (Schrödinger). (B)

Figure 8. Docking of EN460 in binding pockets of other FAD containing enzymes

(A) Monoamine oxidase A (MAO-A) (B) Monoamine oxidase B (MAO-B).

Figure 9:. Docking of EN460 in FAD binding pocket of LSD1.

(A) ligand interaction diagram showing interactions with Val764 and Ala539; (B) docked pose of EN460 in LSD1.

Scheme 1:. Synthesis of EN460 derivatives.

Pyrazolone (4.2) was synthesized as reported previously. (REF: Org. Lett. 2014, 16, 5060−5063) Substituted furfuraldehyes (3.1 and 3.2) were synthesized by Suzuki coupling of aryl iodides (2.1(b) and 2.2 (b)) (REF: Org. Lett. 2010, 12, 4217–4219) with furfural-boronic acid (3.1 (b)). (REF: Eur. J. Med. Chem. 2017, 126, 929–936) Knoevenagel condensation of the substituted furfuraldehyes (3.1 and 3.2) with pyrazolone (4.2) using catalytic sodium acetate in acetic acid provided EN460 derivatives PB-10 and PB-11 in 93% and 53% yields, respectively.