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S4-2 Development of a H2S fluorescent probe and its application to inhibitor screening for H2S producing enzyme
Abstracts / Nitric Oxide 39 (2014) S1–S14
S5
S4-2 Development of a H2S fluorescent probe and its application to inhibitor screening for H2S producing enzyme
data on signalling, pharmacological profiles and biological activities in the context of cardiovascular disease of different classes of H2Sgenerating compounds will be analysed.
Kenjiro Hanaoka Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
http://dx.doi.org/10.1016/j.niox.2014.03.018
For detailed studies of the physiological functions of H2S, a new method to measure H2S concentration in biological samples is required, because the existing methylene blue method and the sulfide ion-selective electrode method are destructive, requiring homogenization of samples. In this presentation, we report the design and synthesis of a novel fluorescence probe for H2S, HSip-1 (Hydrogen Sulfide Imaging Probe-1), utilizing macroazacyclic complex chemistry with copper ion (II) (J. Am. Chem. Soc. 2011 (33), 18003). HSip-1 is more sensitive and selective for H2S than previously reported H2S probes. Further, HSip-1 showed a large and immediate fluorescence increment (by 50-fold) upon addition of 10 lM H2S, whereas almost no fluorescence increment was observed upon addition of 10 mM GSH. HSip-1 also showed high selectivity over other biothiols, ROS, and RNS. We applied HSip-1 to the detection of enzymatic activity to produce H2S. We successfully monitored the time-dependent H2S production by 3-mercaptopyruvate sulfurtransferase (3MST) or lysate of 3MST-expressing HEK293 cells, and cystathionine gamma-lyase (CSE), i.e., H2S-producing enzymes. We then applied HSip-1 to the inhibitor high-throughput screening (HTS) of a chemical library containing 160,000 compounds from The University of Tokyo, Open Innovation Center for Drug Discovery, and found selective inhibitors for 3MST and CSE. http://dx.doi.org/10.1016/j.niox.2014.03.017
S4-3 Novel pharmacological tools for the H2S pathway Andreas Papapetropoulos School of Health Sciences, Faculty of Pharmacy, University of Athens, Greece Hydrogen sulfide (H2S) regulates smooth muscle tone, cell metabolism and growth, apoptosis and migration. It has, thus, been implicated in a plethora of physiological and pathophysiological processes including angiogenesis, cardioprotection, atherosclerosis, inflammation and cancer. H2S is produced by cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST). Aminoethoxyvinylglycine (AVG), b-cyano-L-alanine (BCA) and propargylglycine (PAG) selectively inhibit CSE over CBS and 3-MST. All of them, exhibit limited potency and block other PLP-dependent enzymes, too. It should be emphasized that no selective CBS or 3-MST inhibitors are currently available. During this presentation data obtained from a screen of chemical libraries against H2S-synthesizing enzymes will be presented. The available donors can be divided based on their chemical nature and rate of release to inorganic vs organic compounds and fast vs slow releasers. The most widely used inorganic compounds to generate H2S are NaSH and Na2S. These compounds dissociate rapidly in solution leading to immediate H2S formation. The organic donors available can be divided according to their source of origin into naturally occurring and synthetic. Contrary to inorganic salts, the organic donors release H2S more slowly; the rate of H2S liberation may differ substantially depending on the absence or presence of biological material. Mitochondrial-targeted donors and hybrid molecules between known drugs and H2S-releasing moieties are special subclasses of H2S donors. During the second part of the presentation
S4-4 Photo-inducible hydrogen sulfide releaser using ketoprofenate photocages Hidehiko Nakagawa a, Naoki Fukushima a, Naoya Ieda a, Takayoshi Suzuki b, Naoki Miyata a a Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan b Graduate School of Medical Scieces, Kyoto Prefectural University, Kyoto, Japan Hydrogen sulfide (H2S) has been recognized as one of the important gaseous cellular signaling molecules. H2S is considered to be involved in vascular relaxation, neurotransmission, and inflammation. To investigate the physiological and pharmacological actions of H2S, inorganic sulfide salts such as NaSH have been commonly used as H2S donors. However, these approaches suffer from an inability to precisely control the release rate and dosage. Although several H2S releasers have been reported, they are still incontrollable for H2S release except one example. To overcome these shortcomings, we focused on photochemical H2S generation. Controlled H2S release with photoirradiation has the potential to provide a high degree of control over release location, timing, and dosage. We envisaged that modification of H2S with suitable photolabile protecting groups would afford an H2S donor directly controllable with light, which would release H2S upon rapid photodissociation of the two protecting groups. A ketoprofenate photocage offers many advantages including good photochemical properties. In this study, we adopted ketoprofenate photocages for our new H2S photo-releaser, and developed a novel photocontrollable H2S releaser, which releases H2S proportionally to the photo-irradiation time and intensity. Photocontrolled H2S release from this compound was also demonstrated in biological bovine serum systems. http://dx.doi.org/10.1016/j.niox.2014.03.019
S4-5 New chemistry and chemical tools for hydrogen sulfide research Ming Xian Department of Chemistry, Washington State University, Pullman, WA 99164, USA Hydrogen sulfide (H2S) is a newly recognized signaling molecule with very potent cytoprotective actions. The fields of H2S physiology and pharmacology have been rapidly growing in recent years, but a number of fundamental issues must be addressed to advance our understanding of the biology and clinical potential of H2S in the future. In this regard hydrogen sulfide releasing agents (i.e. H2S donors) and H2S sensors are important research tools. The research in our laboratory focuses on the development of such chemical tools. In this presentation, I will report our recent progress in controllable H2S donors and reaction-based H2S fluorescent probes. Some works related to the detection and reactions of sulfane sulfur species will also be discussed. http://dx.doi.org/10.1016/j.niox.2014.03.020
S5
S4-2 Development of a H2S fluorescent probe and its application to inhibitor screening for H2S producing enzyme
data on signalling, pharmacological profiles and biological activities in the context of cardiovascular disease of different classes of H2Sgenerating compounds will be analysed.
Kenjiro Hanaoka Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
http://dx.doi.org/10.1016/j.niox.2014.03.018
For detailed studies of the physiological functions of H2S, a new method to measure H2S concentration in biological samples is required, because the existing methylene blue method and the sulfide ion-selective electrode method are destructive, requiring homogenization of samples. In this presentation, we report the design and synthesis of a novel fluorescence probe for H2S, HSip-1 (Hydrogen Sulfide Imaging Probe-1), utilizing macroazacyclic complex chemistry with copper ion (II) (J. Am. Chem. Soc. 2011 (33), 18003). HSip-1 is more sensitive and selective for H2S than previously reported H2S probes. Further, HSip-1 showed a large and immediate fluorescence increment (by 50-fold) upon addition of 10 lM H2S, whereas almost no fluorescence increment was observed upon addition of 10 mM GSH. HSip-1 also showed high selectivity over other biothiols, ROS, and RNS. We applied HSip-1 to the detection of enzymatic activity to produce H2S. We successfully monitored the time-dependent H2S production by 3-mercaptopyruvate sulfurtransferase (3MST) or lysate of 3MST-expressing HEK293 cells, and cystathionine gamma-lyase (CSE), i.e., H2S-producing enzymes. We then applied HSip-1 to the inhibitor high-throughput screening (HTS) of a chemical library containing 160,000 compounds from The University of Tokyo, Open Innovation Center for Drug Discovery, and found selective inhibitors for 3MST and CSE. http://dx.doi.org/10.1016/j.niox.2014.03.017
S4-3 Novel pharmacological tools for the H2S pathway Andreas Papapetropoulos School of Health Sciences, Faculty of Pharmacy, University of Athens, Greece Hydrogen sulfide (H2S) regulates smooth muscle tone, cell metabolism and growth, apoptosis and migration. It has, thus, been implicated in a plethora of physiological and pathophysiological processes including angiogenesis, cardioprotection, atherosclerosis, inflammation and cancer. H2S is produced by cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST). Aminoethoxyvinylglycine (AVG), b-cyano-L-alanine (BCA) and propargylglycine (PAG) selectively inhibit CSE over CBS and 3-MST. All of them, exhibit limited potency and block other PLP-dependent enzymes, too. It should be emphasized that no selective CBS or 3-MST inhibitors are currently available. During this presentation data obtained from a screen of chemical libraries against H2S-synthesizing enzymes will be presented. The available donors can be divided based on their chemical nature and rate of release to inorganic vs organic compounds and fast vs slow releasers. The most widely used inorganic compounds to generate H2S are NaSH and Na2S. These compounds dissociate rapidly in solution leading to immediate H2S formation. The organic donors available can be divided according to their source of origin into naturally occurring and synthetic. Contrary to inorganic salts, the organic donors release H2S more slowly; the rate of H2S liberation may differ substantially depending on the absence or presence of biological material. Mitochondrial-targeted donors and hybrid molecules between known drugs and H2S-releasing moieties are special subclasses of H2S donors. During the second part of the presentation
S4-4 Photo-inducible hydrogen sulfide releaser using ketoprofenate photocages Hidehiko Nakagawa a, Naoki Fukushima a, Naoya Ieda a, Takayoshi Suzuki b, Naoki Miyata a a Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan b Graduate School of Medical Scieces, Kyoto Prefectural University, Kyoto, Japan Hydrogen sulfide (H2S) has been recognized as one of the important gaseous cellular signaling molecules. H2S is considered to be involved in vascular relaxation, neurotransmission, and inflammation. To investigate the physiological and pharmacological actions of H2S, inorganic sulfide salts such as NaSH have been commonly used as H2S donors. However, these approaches suffer from an inability to precisely control the release rate and dosage. Although several H2S releasers have been reported, they are still incontrollable for H2S release except one example. To overcome these shortcomings, we focused on photochemical H2S generation. Controlled H2S release with photoirradiation has the potential to provide a high degree of control over release location, timing, and dosage. We envisaged that modification of H2S with suitable photolabile protecting groups would afford an H2S donor directly controllable with light, which would release H2S upon rapid photodissociation of the two protecting groups. A ketoprofenate photocage offers many advantages including good photochemical properties. In this study, we adopted ketoprofenate photocages for our new H2S photo-releaser, and developed a novel photocontrollable H2S releaser, which releases H2S proportionally to the photo-irradiation time and intensity. Photocontrolled H2S release from this compound was also demonstrated in biological bovine serum systems. http://dx.doi.org/10.1016/j.niox.2014.03.019
S4-5 New chemistry and chemical tools for hydrogen sulfide research Ming Xian Department of Chemistry, Washington State University, Pullman, WA 99164, USA Hydrogen sulfide (H2S) is a newly recognized signaling molecule with very potent cytoprotective actions. The fields of H2S physiology and pharmacology have been rapidly growing in recent years, but a number of fundamental issues must be addressed to advance our understanding of the biology and clinical potential of H2S in the future. In this regard hydrogen sulfide releasing agents (i.e. H2S donors) and H2S sensors are important research tools. The research in our laboratory focuses on the development of such chemical tools. In this presentation, I will report our recent progress in controllable H2S donors and reaction-based H2S fluorescent probes. Some works related to the detection and reactions of sulfane sulfur species will also be discussed. http://dx.doi.org/10.1016/j.niox.2014.03.020