Notch Activation Complex Kinase (NACK) is a key player in Notch-mediated tumorigenesis and is an attractive novel target for the treatment of esophageal adenocarcinoma. NACK, annotated in the kinome as SgK223, is an atypical pseudokinase and an established Notch transcriptional co-activator. However, there is no known endogenous or exogenous ligand, no existing co-crystal structure and no reported biological data. To identify a scaffold for NACK inhibition, an artificial intelligence (AI) multitask classification approach was employed. Over 6 million commercially available compounds were screened against established kinase machine learning classifiers, and nearly 8000 were prioritized based on the predicted probability of being active. A homology structure model of the NACK kinase domain was generated and further optimized by all-atom explicit water molecular dynamics (MD) simulations, followed by virtual screening of prioritized compounds. Top-scoring compounds were purchased and screened in in-vitro and in-vivo assays. Commercially available compound Z271-0326 (iNACK) displayed the best inhibitory activity and was further validated in rodent models. A robust novel chemical synthesis for iNACK was accomplished in six steps with an overall yield of 26%. Current efforts are aimed towards optimizing iNACK into the first NACK molecular probe. We optimized our virtual NACK kinase domain structure model via MD simulations, and performed in-silico structure-activity relationship (SAR) studies to better understand putative binding interactions. A second SAR library was recently designed and synthesized with the goal of improving binding affinity and inhibitory activity. Preliminary assay results demonstrate that analogue UM-73 has a 4-fold increase in affinity with an IC50 of 330 nm in cells. We will continue to optimize iNACK through future SAR studies to further enhance inhibitory activity, while balancing affinity with favorable ADMET properties. Thus, arriving at a First-in-Class advanced preclinical NACK inhibitor.

Design and optimization of a first-in-class NACK inhibitor: A novel path to notch inhibition

2019 Spring National Meeting

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