HKI-357

Dravet Syndrome is a severe childhood drug-resistant epilepsy. The predominant etiology of this condition is related to de novo mutations within the SCN1A gene, which codes for the alpha subunit of the NaV1.1 sodium channels. This dysfunction leads to hypoexcitability of GABAergic interneurons. In turn, the loss of electrical excitability in GABAergic interneurons leads to an imbalance of excitation over inhibition in many neural circuits. Notably, exacerbation of symptoms is observed when non-selective sodium channel blockers are administered to patients with Dravet. Recent studies in animal models of Dravet have highlighted the potential of highly specific sodium channel blockers capable of blocking other sodium channel subtypes without inhibiting NaV1.1 current and selective activators of NaV1.1 as viable therapeutic strategies for alleviating Dravet Syndrome symptoms. Here, we describe the development and validation of ligand-based machine learning models to identify ligands with inhibitory effects on sodium channel isoforms NaV1.1 and NaV1.2. These models were built based on in-house open-source routines and Mordred molecular descriptors. First, linear classifiers were inferred using a combination of feature-bagging and Forward Stepwise selection. Secondly, ensemble learning was applied to build meta-classifiers with improved predictive ability, whose performance was tested in retrospective screening experiments. After in silico validation, the models were applied to screen for drug repurposing opportunities in the DrugBank and Drug Repurposing Hub databases, to identify selective blocking agents of NaV1.2 devoid of NaV1.1 blocking activity, as potential compounds for the treatment of Dravet Syndrome. Forty in silico hits were later identified in a prospective screening experiment. Four of them were acquired and submitted to experimental confirmation via patch clamp: three of these candidates, Eltrombopag, Sufugolix, and Glesatinib, showed blocking effects on NaV1.2 currents, although no subtype selectivity was observed. The different predictive abilities of the NaV1.1 and NaV1.2 models may be attributed to the different sizes of the datasets used to train and validate the respective models.

Non-small cell lung cancers (NSCLCs) with activating mutations in the kinase domain of the epidermal growth factor receptor (EGFR) demonstrate dramatic, but transient, responses to the reversible tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Some recurrent tumors have a common secondary mutation in the EGFR kinase domain, T790M, conferring drug resistance, but in other cases the mechanism underlying acquired resistance is unknown. In studying multiple sites of recurrent NSCLCs, we detected T790M in only a small percentage of tumor cells. To identify additional mechanisms of acquired resistance to gefitinib, we used NSCLC cells harboring an activating EGFR mutation to generate multiple resistant clonesin vitro. These drug-resistant cells demonstrate continued dependence on EGFR and ERBB2 signaling for their viability and have not acquired secondary EGFR mutations. However, they display increased internalization of ligand-activated EGFR, consistent with altered receptor trafficking. Although gefitinib-resistant clones are cross-resistant to related anilinoquinazolines, they demonstrate sensitivity to a class of irreversible inhibitors of EGFR. These inhibitors also show effective inhibition of signaling by T790M-mutant EGFR and killing of NSCLC cells with the T790M mutation. Both mechanisms of gefitinib resistance are therefore circumvented by irreversible tyrosine kinase inhibitors. Our findings suggest that one of these, HKI-272, may prove highly effective in the treatment of EGFR-mutant NSCLCs, including tumors that have become resistant to gefitinib or erlotinib.