title: 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: ∼60μM), tannic acid (IC50: ∼40μM) and benserazide (IC50: ∼30μM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3μM) and NSC67078 (IC50: ∼1μM), while aurintricarboxylic acid (IC50: ∼3μM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1μM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50of ∼6μM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6μM), tannic acid (IC50: ∼20μM), benserazide (IC50: ∼20μM), and NSC67078 (IC50: ∼0.3μM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300μM). 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 300μM. 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 300μM, 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 100μM, 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 (50mg/kg/days.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.
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Nadiya
authors: N
authors: Druzhyna
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Bartosz
authors: B
authors: Szczesny
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Gabor
authors: G
authors: Olah
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA; Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Katalin
authors: K
authors: Módis
authors: Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece; Center of Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation of the Academy of Athens, Greece.
authors: Antonia
authors: A
authors: Asimakopoulou
authors: National and Kapodistrian University of Athens, School of Pharmacy, Athens, Greece.
authors: Athanasia
authors: A
authors: Pavlidou
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Petra
authors: P
authors: Szoleczky
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Domokos
authors: D
authors: Gerö
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Kazunori
authors: K
authors: Yanagi
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Gabor
authors: G
authors: Törö
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Isabel
authors: I
authors: López-García
authors: National and Kapodistrian University of Athens, School of Pharmacy, Athens, Greece.
authors: Vassilios
authors: V
authors: Myrianthopoulos
authors: National and Kapodistrian University of Athens, School of Pharmacy, Athens, Greece.
authors: Emmanuel
authors: E
authors: Mikros
authors: Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA.
authors: John R
authors: JR
authors: Zatarain
authors: Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA.
authors: Celia
authors: C
authors: Chao
authors: Center of Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation of the Academy of Athens, Greece; National and Kapodistrian University of Athens, School of Pharmacy, Athens, Greece.
authors: Andreas
authors: A
authors: Papapetropoulos
authors: Department of Surgery, The University of Texas Medical Branch, Galveston, TX, USA; CBS Therapeutics Inc., Galveston, TX, USA.
authors: Mark R
authors: MR
authors: Hellmich
authors: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, USA; CBS Therapeutics Inc., Galveston, TX, USA. Electronic address: szabocsaba@aol.com.
authors: Csaba
authors: C
authors: Szabo
mesh_terms: D000818:Animals; D001545:Benserazide / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy*; D003374:Coumarins / Q000494:pharmacology; D003541:Cystathionine beta-Synthase / Q000037:antagonists & inhibitors*; D058492:Drug Repositioning / Q000379:methods; D004734:Energy Metabolism / Q000187:drug effects; D005260:Female; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D006862:Hydrogen Sulfide / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000187:drug effects; D009025:Morpholines / Q000494:pharmacology; D009946:Organothiophosphorus Compounds / Q000494:pharmacology; D035703:Therapies, Investigational / Q000379:methods
chemical_list: C097886:7-azido-4-methylcoumarin; D003374:Coumarins; C529376:GYY 4137; D006834:Hydrazines; D009025:Morpholines; D009946:Organothiophosphorus Compounds; C006307:2,3,4-trihydroxybenzylhydrazine; D001545:Benserazide; D003541:Cystathionine beta-Synthase; D006862:Hydrogen Sulfide
keywords: Aminooxyacetic acid (PubChem CID:286); Aurintricarboxylic acid (PubChem CID:2259); Benserazide (PubChem CID:26964); Bioenergetics; Cancer; Cell proliferation; Hexachlorophene (PubChem CID: 3598); Hydrogen sulfide; NSC67078 (PubChem CID:66541); Nitric oxide; Tannic acid (PubChem CID:16129778)