Hirdesh Uppal

Identification of a Kinase Profile that Predicts Chromosome Damage Induced by Small Molecule Kinase Inhibitors

Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs) are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases), yielding a 113×290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT). Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM) for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity). This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing compounds, thereby providing a basis for rational drug design away from genotoxicity.

In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates.

The development of predictive toxicology assays is necessary to optimize the drug candidate selection process. The colony forming assay (CFA) is used routinely to assess bone marrow toxicity and represents a viable tool for the discovery toxicologist, but the assay is not widely accepted as a standard screening tool due to technical challenges. A higher throughput and standardized version of the assay recently was developed such that the proliferative capacity of a cell lineage is measured indirectly via ATP levels, replacing the cumbersome identification and enumeration of specific colonies. In this study, a high-throughput assay of bone marrow toxicity prediction using the granulocyte, erythrocyte, monocyte, and macrophage (GEMM) progenitor cell lineage was evaluated using a training set of 56 structurally diverse compounds with known in vivo bone marrow effects. In general, compounds identified as toxic in vivo had lower IC(50) values, whereas those identified as non-toxic had higher IC(50) values. Concordance (i.e., predictive accuracy) to in vivo bone marrow toxicity results was 82% when an in vitro toxicity threshold of 20 microM was used. Additional experiments in other hematopoietic lineages were conducted to determine if predictivity of several false positive and negative compounds in the GEMM lineage could be improved; however an increase in sensitivity or specificity was not observed. The high-throughput GEMM assay has good concordance to in vivo bone marrow toxicity results and, with the high-throughput and standardized format, can be incorporated readily into the pharmaceutical toxicological screening paradigm, aiding in the early identification of compounds that eventually may fail due to bone marrow toxicity.

Evaluation of the GreenScreen GADD45α-GFP indicator assay with non-proprietary and proprietary compounds

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.

Mechanism of subendocardial cell proliferation in the rat and relevance for understanding drug-induced valvular heart disease in humans

A number of drugs and drug candidates, including fenfluramine and ergot derivatives, are associated with valvulopathy in humans; however, these responses are poorly predicted from animal studies. In vitro and in vivo evidence suggests that these compounds exert their pathological effect through activation of serotonin 2B receptor (5HT2BR) signaling. However, the variable effect of fenfluramine and other 5HT2BR agonists in rodents has cast doubt on the relevance of animal findings to predicting human risk. Herein, a candidate compound, RO3013, induced subendocardial cell proliferation in the mitral and tricuspid valves in rats after only 3 days of daily dosing. Additionally, there was a treatment-related increase in immunostaining of the proliferation marker Ki67, and phosphorylated Smad3 in the heart indicative of TGFβ signaling co-localized with 5HT2BR expression. To substantiate the hypothesis that RO3013-induced valvular proliferation is secondary to 5HT2BR activation, the compound was evaluated in vitro and found to bind to the human 5HT2BR with a K(i) of 3.8μM; however, it was virtually devoid of agonist activity in a functional assay in human cells. By contrast, RO3013 bound to the rat 5HT2BR with a K(i) of 1.2μM and activated the receptor with an EC50 of 0.5μM. This agonist potency estimate is in good agreement with the free plasma concentrations of RO3013 at which valvular proliferation was observed. These results suggest that the rat may be susceptible to 5HT2BR-mediated valvular proliferation similar to humans; yet, the significant differences between binding and functional activities can be a possible explanation for the observed species-selective receptor responses.