SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.
© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.
Copyright © 2022. Published by Elsevier Inc.

Protein-protein interactions (PPIs) have emerged as promising yet challenging therapeutic targets. A robust bioassay is required for rapid PPI modulator discovery. Here, we present a time-resolved Förster's (fluorescence) resonance energy transfer assay protocol for PPI modulator screening in a 1536-well plate format. We use hypomorph SMAD4R361H-SMAD3 PPI as an example to illustrate the application of the protocol for screening of variant-directed PPI inducers. This platform can be readily adapted for the discovery of both small-molecule PPI inducers and inhibitors. For complete details on the use and execution of this protocol, please refer to Tang et al. (2020).
© 2021.

The transcription master regulator MYC plays an essential role in regulating major cellular programs and is a well-established therapeutic target in cancer. However, MYC targeting for drug discovery is challenging. New therapeutic approaches to control MYC-dependent malignancy are urgently needed. The mitogen-activated protein kinase kinase 3 (MKK3) binds and activates MYC in different cell types, and disruption of MKK3-MYC protein-protein interaction may provide a new strategy to target MYC-driven programs. However, there is no perturbagen available to interrogate and control this signaling arm. In this study, we assessed the drugability of the MKK3-MYC complex and discovered the first chemical tool to regulate MKK3-mediated MYC activation. We have designed a short 44-residue inhibitory peptide and developed a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay to discover the first small molecule MKK3-MYC PPI inhibitor. We have optimized and miniaturized the assay into an ultra-high-throughput screening (uHTS) 1536-well plate format. The pilot screen of ~6,000 compounds of a bioactive chemical library followed by multiple secondary and orthogonal assays revealed a quinoline derivative SGI-1027 as a potent inhibitor of MKK3-MYC PPI. We have shown that SGI-1027 disrupts the MKK3-MYC complex in cells and in vitro and inhibits MYC transcriptional activity in colon and breast cancer cells. In contrast, SGI-1027 does not inhibit MKK3 kinase activity and does not interfere with well-known MKK3-p38 and MYC-MAX complexes. Together, our studies demonstrate the drugability of MKK3-MYC PPI, provide the first chemical tool to interrogate its biological functions, and establish a new uHTS assay to enable future discovery of potent and selective inhibitors to regulate this oncogenic complex.
Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example. SMAD4, a central mediator of the TGF-β pathway, is recurrently mutated in many tumors. Here, we report the development of a TR-FRET technology that recapitulated the dynamic differential interaction of SMAD4 and SMAD4R361H with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGF-β-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small-molecule protein-protein interaction (PPI) molecular glues for biological insights and therapeutic discovery.
Copyright © 2020 Elsevier Ltd. All rights reserved.