Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer

Abstract

Cystathionine-β-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the `Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ~60 μM), tannic acid (IC50: ~40 μM) and benserazide (IC50: ~30 μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ~3 μM) and NSC67078 (IC50: ~1 μM), while aurintricarboxylic acid (IC50: ~3 μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ~1 μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ~6 μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ~6 μM), tannic acid (IC50: ~20 μM), benserazide (IC50: ~20 μM), and NSC67078 (IC50: ~0.3 μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ~300 μM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300 μM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300 μM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100 μM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50 mg/kg/day s.q.) prevented tumor growth. In silico docking simulations...

Role of 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation, Migration, and Bioenergetics in Murine Colon Cancer Cells

“…In particular in human colon cancer, the most significant upregulation in tumor tissue (compared to surrounding tissue) was noted in CBS, with a trend for increase for 3-MST [26]. Likewise, in many human colon cancer cell lines, CBS was found to be highly expressed, and genetic silencing or pharmacological inhibition of CBS exerted antitumor effects in vitro and in vivo [24][25][26][37][38][39][40][41] while forced expression of CBS into non-transformed human epithelial cells increased their growth in situ (but did not render them metastatic) [42]. In contrast, in the current, murine colon cancer cell line, CBS (mRNA or protein) was undetectable (and, accordingly, the CBS inhibitor AOAA was without significant biological effects), while 3-MST was highly expressed.…”

International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors

“…When benserazide was tested against the other two H 2 S-producing enzymes, it was found to only weakly inhibit CSE and 3-MST activity (16% and 35% at 100 mM, respectively). Moreover, the major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity (Druzhyna et al, 2016). By using in silico docking simulations, we proposed that the mechanism of action of benserazide results from binding in the active site of the enzyme and reacting with the PLP cofactor, leading to the formation of a Schiff base-like adduct with the formyl moiety of pyridoxal (Druzhyna et al, 2016).…”

“…It should be noted, however, that NSC67078 appears to have several additional pharmacological targets in addition to CBS; moreover, in the fluorescent assay used to estimate its potency, it also inhibits the H 2 S H 2 S Donors and H 2 S Biosynthesis Inhibitors signal elicited by GYY4137, indicative of additional pharmacological actions beyond inhibition of the catalytic activity of CBS (e.g., H 2 S scavenging and potent inhibition of the b-catenin pathway (as discussed in Druzhyna et al, 2016). The most recently published effort to identify new CBS inhibitors through highthroughput screening used a natural compound library, yielding 11 hits with IC 50 below 20 mΜ.…”

“…We used a pool of 8871 well-annotated pharmacological compounds and clinically used drugs that included the LOPAC Library, the Food and Drug Administration Approved Drug Library, the National Institutes of Health Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the "Killer Plates" collection, and a small custom compilation of PLP-dependent enzyme inhibitors (Druzhyna et al, 2016). After using two counterscreens, the hit list was narrowed down to four compounds, hexachlorophene, tannic acid, aurin tricarboxylic acid, and benserazide, all of which were less potent than AOAA.…”

Cbsoverdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically withDyrk1a

“…A few studies have reported the identification of CBS inhibitors (39-44) but most of them were based on in vitro assays using a recombinant CBS enzyme as a drug target and led to the isolation of inhibitors with relatively low potency and limited selectivity, hence leading to the idea that CBS may be an undruggable enzyme. Therefore we oriented toward an in cellulo phenotype-based assay that would allow screening drugs that interfere with the phenotypical consequences of CBS overexpression and thereby that do not necessarily directly target the CBS enzyme.…”