{"id":6,"date":"2013-09-10T12:21:20","date_gmt":"2013-09-10T12:21:20","guid":{"rendered":"https:\/\/sites.krieger.jhu.edu\/template-research\/?page_id=6"},"modified":"2026-05-13T18:16:39","modified_gmt":"2026-05-13T22:16:39","slug":"research","status":"publish","type":"page","link":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/research\/","title":{"rendered":"Research"},"content":{"rendered":"\n
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Development of Novel Hydropersulfide Precursors<\/strong><\/p>\n\n\n\n

\"RSSH<\/figure>\n\n\n\n

Many of the signaling functions and cytoprotective effects of hydrogen sulfide (H2<\/sub>S) are thought to occur via the transient generation of hydropersulfides (RSSH) and “reactive sulfur species” from biological thiols. Because of their inherent instability, RSSH must be generated in situ<\/em> using precursor molecules for study. We have developed four classes of RSSH prodrugs: S<\/em>-substituted thioisothioureas<\/a> (TIU<\/strong>), N<\/em>-alkylamine perthiocarbamates<\/a> (APT<\/strong>), alkylsulfenyl thiocarbonates<\/a> (AST<\/strong>), and arylsulfonothioates<\/a> (AT<\/strong>). All four classes demonstrate efficient and tunable release of RSSH under physiological conditions and in cardiomyocyte cell models.<\/p>\n\n\n\n

In the presence of thiol, release of RSSH from APT- and AST-class precursors dominates over release of carbonyl sulfide (COS), a precursor to H2<\/sub>S, while these compounds are insensitive to amines at pH 7.4. Arylsulfonothioates are thiol-activated slow-release sulfane sulfur donors superior to sodium thiosulfate, with RSSH release influenced by substituents on the aryl ring.<\/p>\n\n\n\n

We have extended initial studies into APT- and AST-class RSSH precursors towards developing more controllable systems for RSSH release. Targeted, enzyme-activated [1<\/strong><\/a>,2<\/strong><\/a>], and photocleavable<\/a> precursors are being developed and evaluated in our group.<\/p>\n\n\n\n

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Chemical Biology and Therapeutic Applications of Hydropersulfide Precursors<\/strong><\/p>\n\n\n\n

Hydropersulfides are excellent single-electron reductants and hydrogen-atom donors compared to the corresponding thiols, and they are naturally produced intracellularly under oxidative stress conditions. They are thought to induce antioxidant response signaling and protect protein Cys residues from overoxidation. We are broadly interested in the biochemistry and pharmacology of RSSH, mechanisms of reactive\/sulfane sulfur species homeostasis, and cellular responses to RSSH elevation.<\/p>\n\n\n\n

In collaboration with the Pratt group at the University of Ottawa, we have applied our persulfide donors to the inhibition of ferroptosis<\/a>, a cell death pathway characterized by excessive lipid peroxidation driven in part by iron-mediated Fenton chemistry. Using the fluorescence-enabled inhibited autoxidation (FENIX) assay, alkyl hydropersulfides were found to have outstanding dose-dependent lipid peroxyl trapping kinetics in liposomes. The faster-releasing APT-class precursors proved to be the most potent. These results were validated in cells through rescue experiments under GPX4-knockout-induced ferroptosis.<\/p>\n\n\n\n

\"Dox<\/figure>\n\n\n\n

Following Langendorff studies<\/a> conducted with APT 7b<\/strong> (see above section) showing that treatment is cardioprotective against myocardial ischemic\/reperfusion injury, we examined the efficacy of persulfide precursors against doxorubicin-induced cardiotoxicity (DIC). This is an adverse effect thought to occur via direct ROS generation from single-electron chemistry, aberrant iron accumulation and other mechanisms. <\/p>\n\n\n\n

The slow-releasing AST-class precursor was found to provide superior dose-dependent protection against DIC-associated ROS<\/a> in cardiomyocytes via Nrf2 and PGC-1\u03b1 signaling. However, in cancer cells, our precursors are synergistic with doxorubicin: AST treatment increased the sensitivity of cancer cells to doxorubicin by inducing reductive stress due to elevated basal levels of reducing equivalents in cancer cells.<\/p>\n\n\n\n

\"Sulfane<\/figure>\n\n\n\n

Using AT-class precursors<\/a> as mimics of gradual intracellular RSSH biogenesis, we have demonstrated that cysteine persulfide (CysSSH) export through xCT, the cystine-glutamate antiporter, responds dynamically to the redox state of H9c2 cardiomyocytes. AT treatment resulted in xCT-dependent elevation of extracellular CysSSH, while challenge with oxidative stress significantly decreased CysSSH export. We believe that modulation of RSSH export buffers cells against reductive stress under basal conditions while retaining RSSH when needed for antioxidant response.<\/p>\n\n\n\n

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Analytical Chemistry of Nitroxyl (HNO) and Reactive Sulfur Species<\/strong><\/p>\n\n\n\n

Using precursor molecules, we aim to understand the fundamental reactivity of gaseous signaling molecules and their short-lived intermediates. Much of this chemistry is biologically relevant, and our work provides insight into the elusive mechanisms by which gaseous signaling molecules exert their biological functions.<\/p>\n\n\n\n

\"Sulfur<\/figure>\n\n\n\n

We have studied the reactivity of nitroxyl (HNO) with H2<\/sub>S and RSSH<\/a>. The reaction of HNO with H2<\/sub>S produces either hydrogen polysulfides (H2<\/sub>Sn<\/sub>) or S8<\/sub> depending on their relative concentrations. Both S8 <\/sub>and H2<\/sub>Sn<\/sub> are believed to be involved in cellular sulfane sulfur homeostasis. In this study, we also confirmed that persulfides exhibit enhanced nucleophilicity compared to thiols, which is the result of more than just differences in pK<\/em>a<\/sub>. This difference in reactivity provides a possible explanation for the specificity of HNO signaling. <\/p>\n\n\n\n

Follow-up work examining the reactivity of RSSH (using APT 7b<\/strong>) with S<\/em>-nitrosothiols<\/a> (RSNO) demonstrated that transnitrosation is the major reaction pathway, reducing RSNO to the corresponding thiols while generating a S<\/em>-nitrosopersulfide (RSSNO) intermediate. RSSNO readily homolyze, ultimately generating nitric oxide (NO) and tetrasulfides. S<\/em>-thiolation to generate a new trisulfide and release HNO also occurs as a secondary pathway. RSSH may serve as a physiological mechanism to liberate NO directly from RSNO as well as counter pathological elevation of S<\/em>-nitrosylation under oxidizing conditions.<\/p>\n\n\n\n

\"HNO<\/figure>\n\n\n\n

Membrane inlet (or introduction) mass spectrometry (MIMS) allows for the detection of hydrophobic gasses such as NO, HNO and H2<\/sub>S dissolved in aqueous solutions. We developed a MIMS method for detection of nitroxyl<\/a> in aqueous solution for the first time, using the canonical donors Angeli’s salt (AS) and Piloty’s acid (PA) along with 2-bromo-Piloty’s acid (2BrPA), an efficient HNO donor developed by our lab. We have demonstrated that HNO can be produced from N<\/em>-hydroxy-L<\/em>-arginine<\/a> by the physiologically relevant oxidant hypochlorous acid (HOCl) as a potential endogenous mechanism for HNO generation. This chemistry also applies to hydroxylamine, hydroxyurea, and acetohydroxamic acid.<\/p>\n\n\n\n

\"HNO<\/figure>\n\n\n\n

HNO, a potential heart failure therapeutic, is known to uniquely post-translationally modify cysteine residues to sulfinamides [RS(O)NH2<\/sub>], which can alter protein structure and function. At physiological pH and temperature, sulfinamides may be reduced to free thiols in the presence of excess thiol and hydrolyzed to sulfinic acids [RS(O)OH]. We have used 15<\/sup>N-edited\u00a01<\/sup>H-NMR techniques to examine the reactivity of sulfinamides<\/a> and the modification of protein C-terminal cysteines<\/a> by HNO. HNO modification of the cardiac protein phospholamban (PLN)<\/a> was shown to enhance cardiac sarcoplasmic reticulum Ca2+<\/sup>\u00a0cycling independent of the \u03b2-adrenergic pathway.<\/p>\n\n\n\n

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New Physiologically Useful HNO Precursors<\/strong><\/p>\n\n\n\n

\"Combined<\/figure>\n\n\n\n

Due to its inherent reactivity, HNO must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N<\/em>-substituted hydroxylamines with carbon-based leaving groups have been developed to generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. Previous work has focused on pyrazolone [1<\/a><\/strong>,2<\/a><\/strong>,3<\/a><\/strong>] and N<\/em>-hydroxy-N<\/em>-acylsulfonamide<\/a> derivatives.
This research, in collaboration with Dr. Nazareno Paolocci and the David Kass lab at the JHU School of Medicine, was continued by the start-up Cardioxyl Pharmaceuticals<\/a>, eventually leading to the discovery of cimlanod<\/a> as a potential therapeutic for heart failure.<\/p>\n\n\n\n

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Past and present funding sources:<\/strong><\/p>\n\n\n\n

\"National<\/figure>\n\n\n\n
\"NIH<\/figure>\n\n\n\n

<\/p>\n","protected":false},"excerpt":{"rendered":"

Development of Novel Hydropersulfide Precursors Many of the signaling functions and cytoprotective effects of hydrogen sulfide (H2S) are thought to occur via the transient generation of hydropersulfides (RSSH) and “reactive sulfur species” from biological thiols. Because of their inherent instability, RSSH must be generated in situ using precursor molecules for study. We have developed four […]<\/p>\n","protected":false},"author":40,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-6","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/pages\/6","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/users\/40"}],"replies":[{"embeddable":true,"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/comments?post=6"}],"version-history":[{"count":4,"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/pages\/6\/revisions"}],"predecessor-version":[{"id":508,"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/pages\/6\/revisions\/508"}],"wp:attachment":[{"href":"https:\/\/sites.krieger.jhu.edu\/toscano-lab\/wp-json\/wp\/v2\/media?parent=6"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}