Abstract C078: Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia

Abstract

Abstract AMP-Activated protein kinase (AMPK) acts as a central metabolic sensor at the interface of metabolic and signaling networks. Activated AMPK promotes multiple catabolic processes to generate ATP, such as glucose uptake, glycolysis, fatty acid uptake and oxidation, and mitochondrial biogenesis. In addition, AMPK activation suppresses the cell cycle, and anabolic processes such as the mammalian Target of Rapamycin Complex 1 (mTORC1)-dependent protein synthesis and fatty acid biosynthesis via inactivating phosphorylation of cytosolic acetyl-CoA carboxylase 1 (ACC1) and mitochondrial ACC2. The activation of AMPK under conditions of energetic stress is modulated by an allosteric mechanism where the depletion of ATP promotes AMP binding at the γ subunit of AMPK with a subsequent conformational change in the catalytic α subunit. In the context of cancer AMPK has been reported to promote tumor survival and progression under hypoxia and the maintenance of cancer stem cells (CSCs). A major challenge to interrogating the therapeutic potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity, but has broad-spectrum activity across the kinome. Although sunitinib targets multiple kinases, its nanomolar potency for AMPK inhibition and large scope for chemical substitution on the core oxindole ring make it an attractive lead for AMPK inhibitor development. We have used a computational model of sunitinib docked into the ATP-binding site of the α subunit of AMPK to design and synthesize several series of oxindoles to examine structural modifications to improve AMPK inhibition and selectivity. These candidate inhibitors were evaluated against the activity of the α1 and α2 isoforms of AMPK using the TR-FRET assay and cell engagement was determined by ELISA, measuring p-ACC(Ser79), in the chronic myeloid leukemia K562 cell line. Our assays identified two novel, potent oxindole-based AMPK inhibitors that hadsignificantly reduced binding affinity when compared with sunitinib against members of the receptor tyrosine kinase (RTK) family, including VEGFR1, VEGFR2 and CSF1R, as well as BTK, a member of the B cell receptor (BCR) signaling pathway. Interestingly, cellular AMPK inhibition did not impact cell viability or result in cytotoxicity in K562 cells and may only sensitize these cells to chemotherapy. AMPK inhibition may be a more effective strategy to eliminate cancer cells in hypoxic microenvironments or against certain cell types, such as CSCs, that may be more reliant on AMPK for survival. Our current studies are designed to examine our AMPK inhibitors as single agents and in combination therapy in cancer cells grown under hypoxia and in CSC populations in order to eliminate these drug-resistant cancer cell populations with an aim to prevent cancer recurrence. In summary, we have developed two novel, potent AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of the α subunit of AMPK that are undergoing evaluation in advanced models of leukemia. Citation Format: Christopher J Matheson, Kimberly A Casalvieri, Donald S Backos, Craig T Jordan, Philip Reigan. Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C078. doi:10.1158/1535-7163.TARG-19-C078