A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

Sulfur(lone-pair)…π interactions with FAD in flavoenzymes

The interactions of π-systems with lone-pairs of electrons are known and have been described in biological systems, involving lone-pairs derived from metals, metalloids, sulfur, oxygen and nitrogen. This study describes a bibliographic survey of the disulfide-bound sulfur(lone-pair) interactions with π-systems residing in the flavin adenine dinucleotide (FAD) cofactor of oxidoreductase enzymes (flavoenzymes). Thus, of the 172 oxidoreductase enzymes evaluated for gamma-S(lone-pair)…π(FAD) interactions, 96 proteins (56%) exhibited these interactions corresponding; 61% of 350 the constituent monomers featured at least one gamma-S(lone-pair)…π(FAD) interaction. Two main points of association between the S(lone-pair) and the isoalloxazine moiety of FAD were identified, namely at the centroid of the bond linking the uracil and pyrazine rings (60%), and the centroid of the uracil ring (37%). Reflecting the nature of the secondary structure in three prominent classes of oxidoreductase enzymes: glutathione disulfide reductases (GR; 21 proteins), trypanothione disulfide reductases (TR, 14) and sulfhydryl oxidases (SOX, 22), the approach of the gamma-S(lone-pair) to the FAD residue was to the si-face of the isoalloxazine ring system, i.e. to the opposite side as the carbonyl residue, for all GR and TR examples, and to the re-face for all SOX examples. Finally, the attractive nature of the gamma-S(lone-pair)…π(FAD) interactions was confirmed qualitatively by an examination of the non-covalent interaction plots.

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium falciparum

ABSTRACT Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M−1 s−1), thioredoxins (k = 8.5 to 26 M−1 s−1), dihydrolipoamide (k = 53 M−1 s−1), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the k cat values ranging from 0.03 s−1 to 10 s−1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts.