Craig W. Lindsley

Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinsons disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100μM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.

Abstract 2534: High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The protein-protein interaction (PPI) between menin and Mixed Lineage Leukemia (MLL) plays a critical role in acute leukemias with translocations of MLL gene, and inhibition of this interaction with small molecules represents a new potential therapeutic strategy for the MLL leukemia patients. We identified novel small molecule inhibitors of the menin-MLL interaction with distinct molecular scaffolds by performing a High Throughput Screening (HTS) of over 300,000 compounds. Extensive medicinal chemistry efforts performed for two lead classes to improve inhibitory activity of these compounds resulted in menin-MLL inhibitors with low nanomolar binding affinities. The menin-inhibitor co-crystal structures revealed that these compounds directly bind to menin and closely mimic the key interactions of MLL with menin, resulting in their high binding affinity. Interestingly, the hydroxymethylpiperidine class of the menin-MLL inhibitors extends beyond the MLL binding region on menin, providing additional opportunity for their optimization. We combined extensive crystallography studies with structure-based design to perform rational optimization and scaffold modification to rapidly improve activity and modulate physicochemical properties of the menin-MLL inhibitors. Treatment of MLL leukemia cells with the most potent menin-MLL inhibitors we developed resulted in very effective and selective inhibition of cell proliferation, induced apoptosis and differentiation of these cells. These effects were associated with downregulation of Hoxa9 and Meis1 expression, the downstream targets of MLL fusion proteins required for their leukemogenicity, demonstrating a very specific mechanism of action for these newly developed menin-MLL inhibitors. In vivo studies are currently undergoing to assess the effect of these compounds on leukemia progression in animal models of MLL leukemia. Our studies provide a novel and very attractive scaffolds for further development as a new potential therapeutic approach for the MLL leukemia patients. Citation Format: Jolanta E. Grembecka, Shihan He, Timothy J. Senter, Dmitry Borkin, Jonathan Pollock, Changho Han, Sunil Kumar Upadhyay, Trupta Purohit, Hongzhi Miao, Rocco D. Gogliotti D. Gogliotti, Craig W. Lindsley, Tomasz Cierpicki, Shaun R. Stauffer. High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2014-2534