Predictive Models for Acute Oral Systemic Toxicity: A Workshop to Bridge the Gap from Research to Regulation.

In early 2018, the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) published the "Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States" (ICCVAM 2018). Cross-agency federal workgroups have been established to implement this roadmap for various toxicological testing endpoints, with an initial focus on acute toxicity testing. The ICCVAM acute toxicity workgroup (ATWG) helped organize a global collaboration to build predictive in silico models for acute oral systemic toxicity, based on a large dataset of rodent studies and targeted towards regulatory needs identified across federal agencies. Thirty-two international groups across government, industry, and academia participated in the project, culminating in a workshop in April 2018 held at the National Institutes of Health (NIH). At the workshop, computational modelers and regulatory decision makers met to discuss the feasibility of using predictive model outputs for regulatory use in lieu of acute oral systemic toxicity testing. The models were combined to yield consensus predictions which demonstrated excellent performance when compared to the animal data, and workshop outcomes and follow-up activities to make these tools available and put them into practice are discussed here.

Is skin penetration a determining factor in skin sensitization potential and potency? Refuting the notion of a LogKow threshold for skin sensitization.

It is widely accepted that substances that cannot penetrate through the skin will not be sensitizers. LogKow and molecular weight (MW) have been used to set thresholds for sensitization potential. Highly hydrophilic substances e.g. LogKow ≤ 1 are expected not to penetrate effectively to induce sensitization. To investigate whether LogKow >1 is a true requirement for sensitization, a large dataset of substances that had been evaluated for their skin sensitization potential under Registration, Evaluation, Authorisation and restriction of CHemicals (REACH), together with available measured LogKow values was compiled using the OECD eChemPortal. The incidence of sensitizers relative to non-sensitizers above and below a LogKow of 1 was explored. Reaction chemistry principles were used to explain the sensitization observed for the subset of substances with a LogKow ≤0. 1482 substances were identified with skin sensitization data and measured LogKow values. 525 substances had a measured LogKow ≤ 1, 100 of those were sensitizers. There was no significant difference in the incidence of sensitizers above and below a LogKow of 1. Reaction chemistry principles that had been established for lower MW and more hydrophobic substances were found to be still valid in rationalizing the skin sensitizers with a LogKow ≤ 0. The LogKow threshold arises from the widespread misconception that the ability to efficiently penetrate the stratum corneum is a key determinant of sensitization potential and potency. Copyright © 2016 John Wiley & Sons, Ltd.

What determines skin sensitization potency: Myths, maybes and realities. The 500 molecular weight cut-off: An updated analysis.

It is widely accepted that substances must have a molecular weight (MW) < 500 to penetrate effectively through the skin to induce sensitization. Roberts et al. (2012. Contact Dermatitis 68: 32-41) evaluated a data set of 699 substances taken from the TIMES-SS expert system and identified that of the 13 substances with a MW > 500, five were sensitizers. This provided good evidence to refute such a MW 500 threshold. While Roberts et al. (2012) made a convincing case that the MW > 500 cut-off was not a true requirement for sensitization, the number of counter examples identified were too few to draw any statistical conclusions. This updated analysis systematically interrogated a large repository of sensitization information collected under the EU REACH regulation. A data set of 2904 substances that had been tested for skin sensitization, using guinea pigs and/or mice were collected. The data set contained 197 substances with a MW > 500; 33 of these were skin sensitizers. Metal containing complexes, reaction products and mixtures were excluded from further consideration. The final set of 14 sensitizers substantiated the original findings. The study also assessed whether the same reaction chemistry principles established for low MW sensitizers applied to chemicals with a MW > 500. The existing reaction chemistry considerations were found appropriate to rationalize the sensitization behaviour of the 14 sensitizers with a MW > 500. The existence of the MW 500 threshold, based on the widespread misconception that the ability to penetrate efficiently the stratum corneum is a key determinant of skin sensitization potential and potency, was refuted. Copyright © 2016 John Wiley & Sons, Ltd.

Current and Future Perspectives on the Development, Evaluation, and Application of in Silico Approaches for Predicting Toxicity.

Exploiting non-testing approaches to predict toxicity early in the drug discovery development cycle is a helpful component in minimizing expensive drug failures due to toxicity being identified in late development or even during clinical trials. Changes in regulations in the industrial chemicals and cosmetics sectors in recent years have prompted a significant number of advances in the development, application, and assessment of non-testing approaches, such as (Q)SARs. Many efforts have also been undertaken to establish guiding principles for performing read-across within category and analogue approaches. This review offers a perspective, as taken from these sectors, of the current status of non-testing approaches, their evolution in light of the advances in high-throughput approaches and constructs such as adverse outcome pathways, and their potential relevance for drug discovery. It also proposes a workflow for how non-testing approaches could be practically integrated within testing and assessment strategies.

Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

Small molecule inhibition of the Na(+)/H(+) exchange regulatory factor 1 and parathyroid hormone 1 receptor interaction.

We have identified a series of small molecules that bind to the canonical peptide binding groove of the PDZ1 domain of NHERF1 and effectively compete with the association of the C-terminus of the parathyroid hormone 1 receptor (PTH1R). Employing nuclear magnetic resonance and molecular modeling, we characterize the mode of binding that involves the GYGF loop important for the association of the C-terminus of PTH1R. We demonstrate that the common core of the small molecules binds to the PDZ1 domain of NHERF1 and displaces a (15)N-labeled peptide corresponding to the C-terminus of PTH1R. The small size (molecular weight of 192) of this core scaffold makes it an excellent candidate for further elaboration in the development of an inhibitor for this important protein-protein interaction.