Structure, Mechanism, and Inhibition of Aspergillus fumigatus Thioredoxin Reductase

Abstract

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K_i = 0.22 μM) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth.

Keywords: Aspergillus fumigatus; X-ray crystallography; antifungal; ebselen; thioredoxin reductase.

FIG 1

TrxR reaction mechanism and inhibition by EbSe. (a) TrxR catalyzes the NADPH-dependent reduction of Trx via a dithiol exchange reaction. (b) Proposed mechanism by which EbSe (bottom left) inhibits low-MW TrxR (32).

FIG 2

MS/MS data show that EbSe forms a covalent bond with Cys148 of AfTrxR. The MS/MS data for a peptide with m/z 1642.74 derived from AfTrxR(C145S) after incubation with excess EbSe are shown. A mass shift at Cys148 equal to the addition of one EbSe molecule (MW = 274.18) confirms the formation of a covalent bond between EbSe and Cys148. The peptide sequence modified at Cys148 is shown, with the C145S mutation marked by an asterisk. Full peptide annotation along with equivalent nonmodified peptides derived from AfTrxR wild-type, C145S, and C148S proteins is provided in Fig. S2 and S3 in the supplemental material.

FIG 3

Crystal structure of AfTrxR in flavin-oxidizing conformation at 3.2 Å. The NADPH molecule (cyan), the FAD molecule (magenta), and key residues are shown as sticks. The polypeptide backbone is shown in cartoon representation. A 2mFo-DFc composite omit electron density map for the bound dinucleotides and active-site cysteines (Cys145 and Cys148) is contoured at 1.0 (blue mesh) and 3.0 (pink mesh) RMSD (right panels only). Lengths of hydrogen bonds (dashes) are shown in angstroms. (a) The AfTrxR homodimer. α-Helices (cylinders) and β-stands (arrows) are labeled for one subunit. Each subunit (colored different shades of green) contains an NADPH domain connected to an FAD domain via a short two-stranded “hinge.” Each domain is composed of a central parallel β-sheet sandwiched between an antiparallel β-sheet and three α-helices (note that β1 and β6 contribute to both β-sheets of the FAD domain). (b) Cross-eyed stereo view of the adenosine of NADPH with key binding site residues. Electron density was insufficient to model the nicotinamide riboside. (c) Cross-eyed stereo view of the bound FAD cofactor and key residues. The flavin moiety is poised for electron transfer to the Sγ atom of Cys148.

FIG 4

Modeling of the EbSe-AfTrxR interaction. (a) The flavin-reducing (FR) conformation of AfTrxR was modeled by rotating the NADPH domain by ∼66°, exposing the active-site cysteines. Cys145 and Cys148 (yellow), FAD (magenta), and NADPH (cyan) are shown as sticks. (b) The reaction of EbSe with Cys148 was performed in silico using the FR conformation of AfTrxR. The results are shown in cross-eyed stereo view. Representative examples of group A (wheat carbons), group B (pink carbons), and group C (cyan carbons) poses are shown as sticks. Key interacting residues are also shown. A transparent molecular surface representation is shown for the protein, colored by electrostatic potential, −5 kT/e (red) to +5 kT/e (blue). The Sγ atom of Cys148 is colored yellow, and the Se atom of EbSe is orange. All hydrogen bonds (dashes) are 2.9 to 3.0 Å in length. The FAD cofactor (magenta) can be seen in the background (bottom of view).