Mechanistic understanding of molecular initiating events (MIEs) using NMR spectroscopy

Abstract

Toxicological risk assessments in the 21 century are increasingly being driven by the Adverse Outcome Pathways (AOP) conceptual framework in which the Molecular Initiating Event (MIE) is of fundamental importance to pathway progression. For those MIEs that involve covalent chemical reactions, such as protein haptenation, determination of relative rates and mechanisms of reactions is a prerequisite for their understanding. The utility of NMR spectroscopy as an experimental technique for effectively providing reaction rate and mechanistic information for early assessment of likely MIE(s) has been demonstrated. To demonstrate the concept, model systems exemplifying common chemical reactions involved in the covalent modification of proteins were utilized; these involved chemical reactions of electrophilic species (representing different mechanistic classes) with simple amine and thiol nucleophiles acting as surrogates for the reactive groups of lysine and cysteine protein side chains respectively. Such molecular interactions are recognized as critical mechanisms in a variety of chemical and drug toxicities, including respiratory and skin sensitization and liver toxicity as well as being the key mechanism of action for a number of therapeutic agents.

Relationship Between Adverse Outcome Pathways and Chemistry-BasedIn SilicoModels to Predict Toxicity

“…Information from the Molecular Initiating Event (MIE) is one of the key drivers for in silico models derived from an AOP. [25][26][27][28] Before computational modelling approaches are considered, it should be recognised that there are different types of MIEs and each may require a different modelling approach. These have been summarised in general terms in Table 1, along with examples and the types of models that can be derived from the different types of MIE.…”

“…At the outset it is well acknowledged that AOPs can support computational modelling. 21 In addition, the use of AOPs to support computational modelling deriving structural alerts [25][26][27][28] for toxicity prediction or as part of a grouping strategy leading to read-across, and for QSAR development, is well established. 29 Other computational approaches, beyond the ab initio risk assessment consideration, that utilise and extend the AOP framework are the development of Integrated Assessment and Testing Approaches (IATAs).…”

Reaction Chemistry to Characterize the Molecular Initiating Event in Skin Sensitization: A Journey to Be Continued

“…Distinguishing the two processes by a kinetic analysis of DPRA samples by LC/MS over time may help to identify the dominant reaction in questionable cases. Another approach very recently proposed used simple nucleophiles (butylamine and butylthiol) and measured kinetics of adduct formation and test chemical depletion by NMR . Reaction conditions were adapted for each chemical in order for the reaction to proceed significantly over a 1 h experiment.…”

Predictivity of the kinetic direct peptide reactivity assay (kDPRA) for sensitizer potency assessment and subclassification

“…As shown in detailed case studies (Natsch et al, 2018), an additional evaluation of adduct formation can further advance risk assessment for specific chemicals. From a conceptual point of view, the approach proposed by Sanderson et al (2016) would be most preferable, as it directly measures rate constants of adduct formation by real-time NMR analysis. However, we currently do not see how that approach could be adapted to screen a large library of chemicals and to arrive at an OECD test guideline.…”