Brief Guide: Experimental Strategies for High-Quality Hit Selection from Small-Molecule Screening Campaigns

Abstract

Small-molecule screening is a powerful approach to identify modulators of either specific biological targets or cellular pathways with phenotypic endpoints. A myriad of assay technologies are available to assess the activity of enzymes, monitor protein–protein interactions, measure transcription factor activity in reporter assays, or detect cellular features and activities using high-content imaging. A common challenge during small-molecule screening is, however, the presence of hit compounds generating assay interference, thereby producing false-positive hits. Thus, efforts are needed to uncover such interferences to prioritize high-quality hits for further analysis. This process encompasses (1) computational approaches to flag undesirable compounds, and (2) the use of experimental approaches like counter, orthogonal, and cellular fitness screens to identify and eliminate artifacts. In this brief guide, we provide an overview for first-time users, highlighting experimental screening strategies to prioritize high-quality bioactive hits from high-throughput screening/high-content screening (HTS/HCS) campaigns.

Methods to Detect Small Molecule Inhibition of RING E3 Ligase Activity

“…Chemoinformatics filters can help identify these frequent hitters (Baell & Holloway, 2010; Brenke et al., 2016; Schorpp et al., 2014). Hit evaluation in orthogonal and counter screens is mandatory to confirm activity and specificity (Rothenaigner & Hadian, 2021; Weber et al., 2017).…”

“…A genuine TRAF6-Ubc13 inhibitory compound should diminish signals of different tag combinations with the same PPI of interest (here TRAF6 and Ubc13). However, it should not reduce AlphaScreen signals with other PPI pairs or in assays using the linear fusion constructs (Rothenaigner & Hadian, 2021). A study by Brenke et al (2016) used a GST-His construct to verify GST frequent hitters.…”

Tyrosine kinase inhibitor-loaded biomimetic nanoparticles as a treatment for osteosarcoma

“…As shown in Additional file 1: Fig. S1, we assessed proof of principle single-concentration drug discovery screenings of up to 114 compounds and we identified several drugs with the ability to drastically reduce the viability and stem-like properties of bone sarcoma, including several chemotherapies such as doxorubicin and etoposide (Rothenaigner et al 2021;Al Shihabi et al 2021). We also identified Pon as being highly effective at targeting OS spheroids formation using the metastatic human 143B OS cell line (Additional file 1: Fig.…”

Discovery of Clinical Candidate AZD0095, a Selective Inhibitor of Monocarboxylate Transporter 4 (MCT4) for Oncology

“…This screen used a lactate efflux assay in SKBr3 cells, with counter-screening in a lactate influx fluorometric imaging plate reader (FLIPR) assay in NCI-H358 cells to remove false positives. 14,15 These acidic hit compounds were developed into in vivo probe compounds such as AZ1422 (Figure 1), although they carried risks over human PK prediction and secondary pharmacology, specifically inhibition of bile salt export pump (BSEP) 16 and multidrug resistance-associated protein 2 (MRP2) 17,18 transporters, as well as a perceived risk of idiosyncratic toxicity from formation of an acyl glucuronide metabolite. 19,20 Zwitterionic analogues were subsequently developed and successfully optimized, although the PK properties and designing a compound with a low predicted dose to man remained a challenge.…”

“…Initially, the AstraZeneca compound collection was screened for acidic hits, using the rationale that natural substrates for the monocarboxylate transporters are low-molecular-weight carboxylic acids such as lactic acid and pyruvic acid. This screen used a lactate efflux assay in SKBr3 cells, with counter-screening in a lactate influx fluorometric imaging plate reader (FLIPR) assay in NCI-H358 cells to remove false positives. , These acidic hit compounds were developed into in vivo probe compounds such as AZ1422 (Figure ), although they carried risks over human PK prediction and secondary pharmacology, specifically inhibition of bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) , transporters, as well as a perceived risk of idiosyncratic toxicity from formation of an acyl glucuronide metabolite. , Zwitterionic analogues were subsequently developed and successfully optimized, although the PK properties and designing a compound with a low predicted dose to man remained a challenge. In addition, it was subsequently found that it was difficult to establish a sufficiently high tolerated dose in nude mice to explore preclinical efficacy, and although the mechanistic reason for this lack of tolerability was not well understood, a chemically distinct series that could be optimized toward a candidate drug for clinical studies was viewed as desirable.…”