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

Nadiya Druzhyna, Bartosz Szczesny, Gabor Olah, Katalin Modis, Antonia Asimakopoulou, Athanasia Pavlidou, Petra Szoleczky, Domokos Gerö, Kazunori Yanagi, Gabor Törö, Isabel López-García, Vassilios Myrianthopoulos, Emmanuel Mikros, John R. Zatarain, Celia Chao, Andreas Papapetropoulos, Mark Hellmich, Csaba Szabo

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Original languageEnglish (US)
Pages (from-to)18-37
Number of pages20
JournalPharmacological Research
Volume113
DOIs
StatePublished - Nov 1 2016

Fingerprint

Benserazide
Cystathionine
Investigational Therapies
Colonic Neoplasms
Inhibitory Concentration 50
Libraries
Pharmacology
Pharmaceutical Preparations
Aurintricarboxylic Acid
Hexachlorophene
Cell Proliferation
Tannins
HCT116 Cells
Copper
Fluorescence
Pyridoxal
Ions
Small Molecule Libraries
Neoplasms
Hydrogen Sulfide

Keywords

  • Bioenergetics
  • Cancer
  • Cell proliferation
  • Hydrogen sulfide
  • Nitric oxide

ASJC Scopus subject areas

  • Pharmacology

Cite this

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. / Druzhyna, Nadiya; Szczesny, Bartosz; Olah, Gabor; Modis, Katalin; Asimakopoulou, Antonia; Pavlidou, Athanasia; Szoleczky, Petra; Gerö, Domokos; Yanagi, Kazunori; Törö, Gabor; López-García, Isabel; Myrianthopoulos, Vassilios; Mikros, Emmanuel; Zatarain, John R.; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark; Szabo, Csaba.

In: Pharmacological Research, Vol. 113, 01.11.2016, p. 18-37.

Research output: Contribution to journalArticle

Druzhyna, Nadiya ; Szczesny, Bartosz ; Olah, Gabor ; Modis, Katalin ; Asimakopoulou, Antonia ; Pavlidou, Athanasia ; Szoleczky, Petra ; Gerö, Domokos ; Yanagi, Kazunori ; Törö, Gabor ; López-García, Isabel ; Myrianthopoulos, Vassilios ; Mikros, Emmanuel ; Zatarain, John R. ; Chao, Celia ; Papapetropoulos, Andreas ; Hellmich, Mark ; Szabo, Csaba. / 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. In: Pharmacological Research. 2016 ; Vol. 113. pp. 18-37.
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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 showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.",
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T1 - 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

AU - Druzhyna, Nadiya

AU - Szczesny, Bartosz

AU - Olah, Gabor

AU - Modis, Katalin

AU - Asimakopoulou, Antonia

AU - Pavlidou, Athanasia

AU - Szoleczky, Petra

AU - Gerö, Domokos

AU - Yanagi, Kazunori

AU - Törö, Gabor

AU - López-García, Isabel

AU - Myrianthopoulos, Vassilios

AU - Mikros, Emmanuel

AU - Zatarain, John R.

AU - Chao, Celia

AU - Papapetropoulos, Andreas

AU - Hellmich, Mark

AU - Szabo, Csaba

PY - 2016/11/1

Y1 - 2016/11/1

N2 - 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 showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

AB - 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 showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

KW - Bioenergetics

KW - Cancer

KW - Cell proliferation

KW - Hydrogen sulfide

KW - Nitric oxide

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