Direct Peptide Reactivity Assays

The circumventricular organs (CVO) of the brain are phylogenetically old aggregates of specialised nervous tissue that mediate exclusively direct feedback actions of circulating peptide hormones on neuronal signalling. The CVOs are highly vascularised and fenestrated, giving them an anatomic relationship with both the systemic and cerebrospinal fluid circulations. This unique property has led to the concept of them as receptors and mediators of circulating chemical sensitisation.

The kinetic direct peptide reactivity assay (kDPRA), which replaced the original white rabbit test in 2021, is an alternative to animal testing for assessing skin sensitisation hazards of substances using non-animal methods. In the kDPRA test the reaction kinetics of a chemical with a synthetic cysteine-containing peptide are evaluated by stopping the reaction with monobromobimane and measuring the relative amount of non-depleted peptide remaining. kDPRA has been shown to have good predictive performance for assigning chemicals into UN GHS sub-categories 1A and 1B, although there remain issues with the current kDPRA data analysis protocol which cannot with confidence be assumed to represent true rate constants. The kDPRA-cys and DPRA protocols also do not measure the reactivity of Schiff base electrophiles, acyl transfer agents and unreactive chemicals that sensitise via metabolic or abiotic activation.

Phthalic and trimellitic anhydrides react rapidly with the thiol groups of peptide cysteine residues, producing a thioester that is readily hydrolysed with regeneration of the sulphur nucleophile. As such, both anhydrides give a high level of peptide depletion in a kDPRA assay. However, the peptide thioester is not a protein and therefore does not contribute to the formation of interchain or intrachain disulfide bonds within proteins. Consequently, differences in DP values in the 90-100% range cannot with confidence be treated as meaningful and should not be used to make predictions about the potency of chemicals. direct peptides

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