High Throughput Kinetic Profiling Approach for Covalent Binding to Peptides: Application to Skin Sensitization Potency of Michael Acceptor Electrophiles

Abstract

Research aimed at nonanimal approaches to provide the relevant information needed for the effective assessment of skin sensitization, for both hazard characterization and risk assessment purposes, is currently an area of high activity, stimulated by regulatory initiatives related to chemicals used in consumer products. The ability of a chemical to react covalently with protein or peptide nucleophiles in the skin is recognized as the key determinant in determining sensitization potency, and initiatives to develop peptide reactivity assays to replace animal testing have been undertaken recently. This paper describes a high throughput kinetic profiling (HTKP) approach, developed as an extension of a published standard assay, with the aim of providing a quantitatively robust end point in the form of a kinetic profile from which reactivity to a model peptide can be quantified in the form of second order rate constants. The approach allows solubility issues to be identified and overcome; these are frequently encountered, but can often go undetected, in aqueous reactivity assays with organic compounds of interest in the skin sensitization context. Using rate constants determined by the HTKP approach we have obtained a quantitative mechanistic model for the Michael acceptor reaction mechanistic domain, relating the sensitization potency in the murine local lymph node assay to the rate constant. The observation that the correlation is not improved by incorporation of a hydrophobicity term has implications regarding the nature and location of the skin nucleophile whose reaction leads to sensitization by Michael acceptor electrophiles.