SA for Protein Binding

SA for Protein Binding

T. W. Schultz Presented at the Logan Workshop March 23-24, 2010 Background Method & Data Base Reactions & Coverage What We are Currently Doing No consensus in number > 25 < 50 (40)

Vary in preferred target moiety Typically 45% -SH and 45% -NH2 Vary in structural domain Simple: isothiocyanate RN=C=S Complicated: Michael addition Complex: SNAr addition

In order of increasing hardness include: Thiol-group of cysteine S-atom of methionine Primary amino-group of lysine Secondary amino-group of histidine 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 0.011

0.00076 C#N 0.0027 0.00020 C(=O)NH2

0.00046 0.000026 ______________________________________________________ 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 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 (OBrien 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) Quantitative Simple, with kinetics-linked endpoint

rapid, repeatable, and inexpensive Cysteine-based thiol target Depletion-based Analyses (% free thiol)

by Concentration-Response (RC50) Full kinetic 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 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 > 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 Highly relevant with multiple domains

1) Michael Addition 2) Nucleophilic substitution (N-sub) of haloaliphatics 3) N-sub of haloaromatics (SNAr) 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

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 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 Less relevant 1) arenesulfinic acid substitution 2) azomethyne addition

3) thiocyanate addition 4) mercury thiolate formation 5) others 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) 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

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) 2

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

Thank you

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