Abstract
Protein-protein interactions (PPIs) represent important and promising therapeutic targets that are associated with the regulation of various molecular pathways, particularly in cancer. Although they were once considered “undruggable,” the recent advances in screening strategies, structure-based design, and elucidating the nature of hot spots on PPI interfaces, have led to the discovery and development of successful small-molecule inhibitors. In this report, we are describing an integrated high-throughput and computational screening approach to enable the discovery of small-molecule PPI inhibitors of the anti-apoptotic protein, Mcl-1. Applying this strategy, followed by biochemical, biophysical, and biological characterization, nineteen new chemical scaffolds were discovered and validated as Mcl-1 inhibitors. A novel series of Mcl-1 inhibitors was designed and synthesized based on the identified difuryl-triazine core scaffold and structure-activity studies were undertaken to improve the binding affinity to Mcl-1. Compounds with improved in vitro binding potency demonstrated on-target activity in cell-based studies. The obtained results demonstrate that structure-based analysis complements the experimental high-throughput screening in identifying novel PPI inhibitor scaffolds and guides follow-up medicinal chemistry efforts. Furthermore, our work provides an example that can be applied to the analysis of available screening data against numerous targets in the PubChem BioAssay Database, leading to the identification of promising lead compounds, fuelling drug discovery pipelines.
Highlights
Identification and development of novel bioactive small molecules play a key role in basic biological studies, along with drug discovery efforts, by providing chemical tools that lead to new therapeutic targets and potential drugs[1]
The Bcl-2 family of proteins, consisting of anti- (Bcl-2, Bcl-xL, Bcl-w, myeloid cell leukaemia-1 (Mcl-1), Bfl-1, Bcl-B) and pro- (Bax, Bak, Bim, Bad, Bmf, tBid, Noxa, Bik, Puma, Hrk) apoptotic proteins, are major regulators of the intrinsic programmed cell death pathway, and their activity is governed through a network of protein-protein interactions (PPIs) between pro- and anti- apoptotic members[9,10]
We have reported the development and optimization of a dual-readout high-throughput screening (HTS) assay that combines two assay technologies into one system, time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP), for the discovery of Mcl-1 protein inhibitors[46]
Summary
Identification and development of novel bioactive small molecules play a key role in basic biological studies, along with drug discovery efforts, by providing chemical tools that lead to new therapeutic targets and potential drugs[1]. PPIs have been traditionally characterized as “undruggable” targets, due to the major challenge of developing small molecules that potently bind to the relatively flat and large interfaces[7]. Many of these protein interfaces are hydrophobic in nature, so the developed drug leads usually display hydrophobicity themselves, with high molecular weight, low solubility, and poor oral bioavailability, representing unfavourable physicochemical properties. Our lab described selective Mcl-1 inhibitors (UMI-77 and compound 21) with hydroxynaphthalen-arylsulphonamide core scaffolds and demonstrated their on-target cellular activity[34] and efficacy in pancreatic cancer[35,36] and acute myeloid leukaemia cells[37]. A potent Mcl-1 inhibitor, S63845, developed by Servier was recently reported with a thienopyrimidine scaffold and displayed potent in vitro and in vivo activity[44]
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