In silico and crystallographic studies identify key structural features of biliverdin IXβ reductase inhibitors having nanomolar potency

Abstract

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXβ (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

Repositioning Food and Drug Administration-Approved Drugs for Inhibiting Biliverdin IXβ Reductase B as a Novel Thrombocytopenia Therapeutic Target

“…Erythrosin B and phloxine B (Figure A) are recognized as the two most potent inhibitors of BLVRB . Because the inhibition mechanism of these two compounds was investigated through in silico and crystallographic studies, we set out to investigate the inhibition mechanism in a physiological environment by NMR.…”

“…Multimerization of BLVRB Induced by Xanthene-Based Drug Candidates, Erythrosin B and Phloxine B. Erythrosin B and phloxine B (Figure 1A) are recognized as the two most potent inhibitors of BLVRB. 22 Because the inhibition mechanism of these two compounds was investigated through in silico and crystallographic studies, we set out to investigate the inhibition mechanism in a physiological environment by NMR. As expected from the previous studies, the two compounds induced chemical shift changes of NMR resonances of BLVRB observed by two-dimensional (2D) 1 H- 15 N heteronuclear single quantum correlation (HSQC), clearly indicating inhibitor binding to BLVRB.…”

“…These two compounds share structural similarity to a natural BLVRB substrate, flavin mononucleotide (FMN). Among these xanthene dyes, erythrosin B and phloxine B were found to be the two most potent inhibitors. , Molecular modeling and X-ray crystallographic studies showed that erythrosin B and phloxine B bind to the active site of BLVRB . In addition, the hydrogen bond network in the active site of BLVRB and the critical role of the S111 residue for the catalytic activity were also elucidated. − Interestingly, Lineweaver–Burk plot analyses showed that these compounds inhibit the activity in a noncompetitive manner, although they bind similarly but not as deeply in the binding pocket of BLVRB as the natural substrate, FMN, engages. ,, …”

“…Among these xanthene dyes, erythrosin B and phloxine B were found to be the two most potent inhibitors. 21,22 Molecular modeling and X-ray crystallographic studies showed that erythrosin B and phloxine B bind to the active site of BLVRB. 22 In addition, the hydrogen bond network in the active site of BLVRB and the critical role of the S111 residue for the catalytic activity were also elucidated.…”

“…23−25 Interestingly, Lineweaver−Burk plot analyses showed that these compounds inhibit the activity in a noncompetitive manner, although they bind similarly but not as deeply in the binding pocket of BLVRB as the natural substrate, FMN, engages. 22,25,26 Both erythrosin B and phloxine B have been used for coloring food, and the antimicrobial activity of phloxine B was also reported for various Gram-positive bacteria. 27 However, chronic administration of erythrosin B was shown to promote thyroid tumors in rats.…”