Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide

This is the peer reviewed version of the following article: Sarma, D., & Hanzlik, R. P. (2011). Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide. Journal of Labelled Compounds & Radiopharmaceuticals, 54(13), 795–798. http://doi.org/10.1002/jlcr.1933, which has been published in final form at http://doi.org/10.1002/jlcr.1933. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH3C(NH)SO2H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification we required both radio-labeled and stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C-14, C-13 and/or deuterium. The methods are straightforward, utilize readily available precursors and are amenable to small scale

Cyclopropylamine inactivation of cytochromes P450: Role of metabolic intermediate complexes

NOTICE: this is the author’s version of a work that was accepted for publication in Archives of Biochemistry and Biophysics. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Archives of Biochemistry and Biophysics, Volume 436, Issue 2, 15 April 2005 doi:10.1016/j.abb.2005.02.020The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5′ (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to α-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1

Cobalt complexes: correlation of redox potential with effectivity as oxygenation catalysts

The redox potential of a series of cobalt(II) chelates correlates with their properties as either oxygen carriers or phosphine oxidation catalysts

Metal-Catalyzed Hydration of 2-Pyridyloxirane

In the presence of CuII the hydration of 2-pyridyloxiran is accelerated 18,000-fold, and its reaction with Cl–, Br–, and MeO– becomes 100% regiospecific for β-attack

Reactive Metabolite Target Protein Database (TPDB)

This record contains a Narrative that explains the history of the Reactive Metabolite Target Protein Database (TPDB) developed by Robert Hanzlik and Yakov Koen. All of the appendices are zipped into a single file for easier downloading.The biotransformation of drugs and xenobiotic chemicals in living cells sometimes generates chemically reactive metabolites (CRMs) that diffuse through the cell and react covalently with cellular constituents, especially proteins. In some cases this covalent binding may impair the function of the protein(s) leading to cellular injury and even cell death. The elucidation of the chemistry involved in the bioactivation, the details of the ensuing protein modification, and the connection between protein binding and toxicity, became a major pursuit in a number of laboratories starting in the early 1970s. An attempt to collect all the relevant data about the proteins targeted by CRMs led to the construction of the Reactive Metabolite Target Protein Database (TPDB) at the University of Kansas. This curated, searchable database became freely available online in January 2006, and was maintained until July 2020, when its maintenance ceased. This Narrative describes the nature and extent of the data that was compiled and the searches that could be implemented within the TPDB. The results of analyses of that data have been published elsewhere. All of the data originally compiled, including a list of all pertinent literature references, is now available in the Narrative and the associated Appendices at http://hdl.handle.net/1808/30592. List of Appendices: 1. Home page.pdf 2. Help page.pdf 3. Distribution of studies.xlsx 4. Chemical structures and abbreviations.pdf 5. List of hits and proteins.xlsx 6. List of protein "orthologs" (≥90% similar).xlsx 7. Commonality matrix, orthologs excluded.xlsx 8. Commonality matrix, orthologs included.xlsx 9. Targets proteins ranked by hit frequency.xlsx 10. Chemicals ranked by number of targets.xlsx 11. References cited in TPDB.xls

Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

<p>Abstract</p> <p>Background</p> <p>Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity.</p> <p>Results</p> <p>Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein). Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine) content and protein instability index (i.e., rate of turnover in vivo) were among the features most important to determining susceptibility.</p> <p>Conclusion</p> <p>As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.</p

Absorption and elimination of formate following oral administration of calcium formate in female human subjects

Final publisher version is available online openly at: http://dmd.aspetjournals.org/content/33/2/282.full.pdf. Author accepted manuscript is available in compliance with NIH requirements at, http://dmd.aspetjournals.org/content/early/2004/11/16/dmd.104.001289. Abstract is posted in fulfillment of the KU faculty's Open Access policy.Published abstract: Calcium formate is a water-soluble salt of an essential mineral nutrient with potential for use as a dietary calcium supplement. Formate ion is a product of endogenous and xenobiotic metabolism, but sustained high plasma formate concentrations (such as occur in cases of methanol poisoning) are toxic to the retina and optic nerve. Humans and primates have reduced capacity for formate oxidation compared with rodents and dogs and are thus more sensitive to methanol (and formate) intoxication. To assess the potential for accumulation of formate ion upon repeated administration of calcium formate as a potential dietary calcium supplement, we measured plasma concentrations of formate in 14 adult human subjects before and after oral administration of a single large dose of calcium formate (3900 mg; ca. 3–6 times the anticipated dose for calcium supplementation). Plasma formate concentrations increased briskly from 0.024 ± 0.008 mM (endogenous formate) to reach Cmax (0.50 ± 0.04 mM) at 60 min postdose and then declined with a half-life of 59 ± 7 min. By 225 min postdose, plasma formate concentration had returned to baseline. With such a short half-life, repeated use of calcium formate as a dietary supplement, even three times daily, should not lead to progressive accumulation of formate. These findings are discussed in light of the production of formate by endogenous and xenobiotic metabolism and the kinetics of formate during methanol poisoning

Relative bioavailability of calcium from calcium formate, calcium citrate, and calcium carbonate

Final publisher version is available online openly at: http://jpet.aspetjournals.org/content/313/3/1217.full.pdf Author accepted manuscript is available in compliance with NIH requirements at, http://jpet.aspetjournals.org/content/early/2005/02/25/jpet.104.081893/. Abstract is posted in fulfillment of the KU faculty's Open Access policy.Published Abstract: Calcium is an essential nutrient required in substantial amounts, but many diets are deficient in calcium making supplementation necessary or desirable. The objective of this study was to compare the oral bioavailability of calcium from calcium formate, a new experimental dietary calcium supplement, to that of calcium citrate and calcium carbonate. In a four-way crossover study, either a placebo or 1200 mg of calcium as calcium carbonate, calcium citrate, or calcium formate were administered orally to 14 healthy adult female volunteers who had fasted overnight. After calcium carbonate, the maximum rise in serum calcium (~4%) and the fall in serum intact para-thyroid hormone 1–84 (iPTH) (~20–40%) did not differ significantly from placebo. After calcium citrate, the changes were modestly but significantly (p < 0.05) greater, but only at 135 to 270 min after ingestion. In contrast, within 60 min after calcium formate serum calcium rose by approximately 15% and serum iPTH fell by 70%. The mean increment in area under the plasma concentration-time curve (0–270 min) for serum calcium after calcium formate (378 mg∙min/dl) was double that for calcium citrate (178 mg∙min/dl; p < 0.01), whereas the latter was only modestly greater than either placebo (107; p < 0.05) or calcium carbonate (91; p < 0.05). In this study, calcium formate was clearly superior to both calcium carbonate and calcium citrate in ability to deliver calcium to the bloodstream after oral administration. Calcium formate may offer significant advantages as a dietary calcium supplement

Human oncoprotein Musashi-2 N-terminal RNA recognition motif backbone assignment and identification of RNA-binding pocket

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors

Ocular and Systemic Safety Evaluation of Calcium Formate as a Dietary Supplement

Purpose: The objective of this study was to perform a preliminary evaluation of the ocular and systemic safety of calcium formate, a dietary calcium supplement for prevention and management of osteoporosis. Although formate is an endogenous product of metabolism, high concentrations are associated with toxicity during methanol overdose. Methods: In this prospective clinical trial, 12 healthy women ingested calcium formate (1,300 mg) three times a day for 14 days. Study evaluations included physical and ocular examination, extensive laboratory testing, serum calcium and formate levels, Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, color vision, visual fields, visual evoked potential (VEP), and full-field, pattern, and multifocal electroretinograms (MERG). Results: The mean baseline serum level of formate was 0.572 ± 0.06 mM. Peak serum levels and final serum formate did not differ significantly from baseline. The final concentration was 0.582 ± 0.091 mM. Accumulation of serum formate did not occur. There was also no evidence of toxicity with calcium formate ingestion. All examinations and tests remained normal, including optic nerve and retinal function. Three subjects had mild transient symptoms attributable to any calcium formulation. Conclusions: Calcium formate is highly bioavailable and well-tolerated. Serum formate remained at basal levels and did not accumulate with repeated dosing. Systemic and ocular safety was demonstrated by objective testing. Given its high oral bioavailability, calcium formate may be a good choice for calcium supplementation in the prevention and management of osteoporosis. Further study will be needed to evaluate its long-term safety in a larger group of subjects representing more varied age, health, dietary, and nutritional status