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GSH Reactivity: An in Chemico Method and Overview of its Database

GSH Reactivity: An in Chemico Method and Overview of its Database. T. W. Schultz Presented at the Logan Workshop March 23-24, 2010. Topics. Background Method & Data Base Reactions & Coverage What We are Currently Doing. Reactions of Protein Binding.

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GSH Reactivity: An in Chemico Method and Overview of its Database

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  1. GSH Reactivity: An in Chemico Method and Overview of its Database T. W. Schultz Presented at the Logan Workshop March 23-24, 2010

  2. Topics • Background • Method & Data Base • Reactions & Coverage • What We are Currently Doing

  3. Reactions of Protein Binding • No consensus in number > 25 < 50 (40) • Vary in preferred target moiety • Typically45% -SH and 45% -NH2 • Vary in structural domain • Simple:isothiocyanate RN=C=S • Complicated:Michael addition • Complex:SNAr addition

  4. Nucleophilic Sitein Amino Acids • In order of increasing hardness include: • Thiol-group of cysteine • S-atom of methionine • Primary amino-group of lysine • Secondary amino-group of histidine

  5. Relative Reactivity Different nucleophiles can differ in their absolute reactivity towards a given electrophile, but relative reactivity is well correlated over a range of nucleophiles within the same mechanism. CH2=CH- k(thiol) K(amine) ______________________________________________________ C(=O)OMe 0.0110.00076 C#N 0.0027 0.00020 C(=O)NH2 0.00046 0.000026

  6. In Chemico Reactivity • Use in a similar context to in vitro or in silico • Quantitative, rapid, inexpensive experiments with model nucleophiles • Verify reaction-based rules of reactivity • Define the chemical space of a reaction • Provides a measure of relative potency • Useful in refining categories and modeling

  7. Ways of Measurements Reactivity • Full Kinetics- measured at several time intervals with several initial concentrations of electrophile (100 chemicals) • Partial Kinetics- measured at several time intervals with one initial concentrations of electrophile (O’Brien Assay) • Concentration giving 50% reaction in a fixed time- measured at one time with several initial concentrations of electrophile(1000 chemicals) • Extent of reaction after a fixed time- measured at one time with one initial concentrations of electrophile (Gerberick Assay)

  8. The UTK GSH Reactivity Method • Quantitative with kinetics-linked endpoint • Simple, rapid, repeatable, and inexpensive • Cysteine-based thiol target • Depletion-based (% free thiol) • Analyses by • Concentration-Response (RC50) • Full kinetic

  9. Log (RC50) versus log K

  10. + & - of GSH-BasedReactivity Assay • Readily available • Concentration can be analyzed by simple methods • Odorless, non-hazardous • Water soluble but NOT readily soluble in organic solvents • Does not lead itself to HPLC

  11. The RC50 Value • Initial concentration of electrophile that gives a half-life of 120 minutes • Good if electrophile is in excess • Adequate if concentrations of electrophile and GSH are similar • Poor if GSH is in excess; RC50 values are then extrapolated

  12. Overview of The Data Set • > 2,600 individual assays • ≈ 1,000 separate structure • > 25 different organic reactions (mechanisms) • > 300 not reactive because of structure • < 100 not reactive because of solubility • < 50 not reactive because of color interference

  13. Group 1 Thiol–Targeted Reactions • Highly relevant with multiple domains • 1) Michael Addition • 2) Nucleophilic substitution (N-sub) of haloaliphatics • 3) N-sub of haloaromatics (SNAr)

  14. Current Status of Testing on Group 1Thiol–Targeted Reactions • 1) Michael Addition > 250 compounds (cpds) • 2) Pre-Michael Addition > 50 cpds • 3) N-sub of Haloaliphatics > 150 cpds • 4) N-sub of Haloaromatics > 125 cpds

  15. Group 2 Thiol–Targeted Reactions • Highly relevant with simple domains • 1) disulfide exchange • 2) O-heterocyclic ring opening • 3) N-sub of alkyl sulfates & sulfonates • 4) nitroso- & N-oxides • 5) disulfide formation

  16. Current Status of Testing onGroup 2Thiol–Targeted Reactions • 1) disulfide exchange, >10 cpds • 2) O-heterocyclic ring opening, 20 cpds • 3) N-sub of alkyl sulfates, 5 cpds • 4) N-sub of alkyl sulfonates, >10 cpds • 5) nitroso-compounds, >5 cpds • 6) N-oxides, 10 cpds • All demonstrate GSH reactivity &are related to sensitization

  17. Group 3 Thiol–Related Reactions • Less relevant • 1) arenesulfinic acid substitution • 2) azomethyne addition • 3) thiocyanate addition • 4) mercury thiolate formation • 5) others

  18. Current Testing Program • Other chemical classes • 1) unsaturated alcohols • 2) secondary amines • 3) dialkyl acetals • 4) lactates • 5) anhydrides (hydrolysis) • 6) aldehydes (Schiff-base formers) • 7) diones (cycloaddition to diamines)

  19. Variations in RC50Values for Unsaturated Alcohols only ,-unsaturates are reactive Compounds RC50 (mM) _______________________________________________ C=CC(O)Cn 25 - 50 C#CC(O)Cn 1.0 – 3.0 OCC=CCn 5.0 - 10.0 OCC#CCn  3.0 - 5.0 1-pentyn-3-ol 1.3, 1.7 4-CH3-1-pentyn-3-ol 16, 13 3,4-CH3-1-pentyn-3-ol NR at 50mM

  20. N-sub Haloaliphatic Base Structure and Special Features R1C(X)YR2 Y = C6H5 > C#C > C=C, etc X = I > Br > Cl > F R1 = H > CnH(2n +1) R2 no effect

  21. N-sub Haloaromatic Base Structure and Special Features XC6H3Y2 Y = NO2, > in-ring-N > CHO > CN X = F > Cl > Br > I Position of leaving group in relationship to activity groups effects potency

  22. Thank you

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