Generation and Characterization of a CRISPR/Cas9 -Induced 3-mst Deficient Zebrafish.

3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H2S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced 3-mst mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The 3-mst zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H2S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H2S levels were reduced in 3-mst-/- zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following 3-mst deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in 3-mst deficient zebrafish. Overall, we provide evidence for the expression of 3-mst in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.

1H, 13C, 15N backbone and side-chain resonance assignment of the native form of UbcH7 (UBE2L3) through solution NMR spectroscopy.

Ubiquitination is a post-translational modification that regulates a plethora of processes in cells. Ubiquitination requires three type of enzyme: E1 ubiquitin (Ub) activating enzymes, E2 Ub conjugating enzymes and E3 ubiquitin ligases. The E2 enzymes perform a variety of functions, as Ub chain initiation, elongation and regulation of the topology and the process of chain formation. The E2 enzymes family is mainly characterized by a highly conserved ubiquitin conjugating domain (UBC), which comprises the binding region for the activated Ub, E1 and E3 enzymes. The E2 enzyme UbcH7 (UBE2L3) is a known interacting partner for different types of E3 Ub ligases such as HECT, RING and RBR. A structural analysis of the apo form of the native UbcH7 will provide the structural information to understand how this E2 enzyme is implicated in a wide range of diseases and how it interacts with its partners. In the present study we present the high yield expression of the native UbcH7 E2 enzyme and its preliminary analysis via solution NMR spectroscopy. The E2 enzyme is folded in solution and nearly a complete backbone assignment was achieved. Additionally, TALOS+ analysis was performed and the results indicated that UbcH7 adopts a αßßßßααα topology which is similar to that of the majority of E2 enzymes.

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

Cystathionine-ß-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 (IC50 of ∼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.

Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

D-Penicillamine modulates hydrogen sulfide (H2S) pathway through selective inhibition of cystathionine-γ-lyase.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2S) is a gasotransmitter produced from L-cysteine through the enzymatic action of cystathionine-γ-lyase (CSE) and/or cystathionine-ß-synthase. D-Penicillamine is the d isomer of a dimethylated cysteine and has been used for the treatment of rheumatoid arthritis. AsD-penicillamine is structurally very similar to cysteine, we have investigated whether D-penicillamine, as a cysteine analogue, has an effect on the H2 S pathway. EXPERIMENTAL APPROACH: We tested the effect of D-penicillamine (0.01-1 mM) in mouse aortic rings mounted in isolated organ baths and determined whether it could affect H2 S biosynthesis. In particular, we investigated any possible inhibitor or donor behaviour by using recombinant enzyme-based assays and an in vivo approach. KEY RESULTS: D-Penicillamine, per se, showed little or no vasodilator effect, and it cannot be metabolized as a substrate in place of l-cysteine. However, d-penicillamine significantly reduced L-cysteine-induced vasodilatation in a concentration-dependent manner through inhibition of H2 S biosynthesis, and this effect occurred at concentrations 10 times lower than those needed to induce the release of H2 S. In particular, D-penicillamine selectively inhibited CSE in a pyridoxal-5'-phosphate-dependent manner. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that D-penicillamine acts as a selective CSE inhibitor, leading to new perspectives in the design and use of specific pharmacological tools for H2 S research. In addition, the inhibitory effect of D-penicillamine on CSE could account for its beneficial action in rheumatoid arthritis patients, where H2 S has been shown to have a detrimental effect.

Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-ß-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

Recent data show that colon cancer cells selectively overexpress cystathionine-ß-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects.

Selectivity of commonly used pharmacological inhibitors for cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE).

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ß-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 µM vs. 40 ± 8 µM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 µM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 µM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Oxidative stress suppresses the cellular bioenergetic effect of the 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide pathway.

Recent data show that lower concentrations of hydrogen sulfide (H2S), as well as endogenous, intramitochondrial production of H2S by the 3-mercaptopyruvate (3-MP)/3-mercaptopyruvate sulfurtransferase (3-MST) pathway serves as an electron donor and inorganic source of energy to support mitochondrial electron transport and ATP generation in mammalian cells by donating electrons to Complex II. The aim of our study was to investigate the role of oxidative stress on the activity of the 3-MP/3-MST/H2S pathway in vitro. Hydrogen peroxide (H2O2, 100-500 µM) caused a concentration-dependent decrease in the activity of recombinant mouse 3-MST enzyme. In mitochondria isolated from murine hepatoma cells, H2O2 (50-500 µM) caused a concentration-dependent decrease in production of H2S from 3-MP. In cultured murine hepatoma cells H2O2, (3-100 µM), did not result in overall cytotoxicity, but caused a partial decrease in basal oxygen consumption and respiratory reserve rapacity. The positive bioenergetic effect of 3-MP (100-300 nM) was completely abolished by pre-treatment of the cells with H2O2 (50 µM). The current findings demonstrate that oxidative stress inhibits 3-MST activity and interferes with the positive bioenergetic role of the 3-MP/3-MST/H2S pathway. These findings may have implications for the pathophysiology of various conditions associated with increased oxidative stress, such as various forms of critical illness, cardiovascular diseases, diabetes or physiological aging.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation.

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.