Structural Basis of Small-Molecule Aggregate Induced Inhibition of a Protein–Protein Interaction

Blevitt, Jonathan M.; Hack, Michael D.; Herman, Krystal; Jackson, Paul F.; Krawczuk, Paul J.; Lebsack, Alec D.; Liu, Annie X.; Mirzadegan, Taraneh; Nelen, Marina I.; Patrick, Aaron; Steinbacher, Stefan; Milla, Marcos E.; Lumb, Kevin J.

doi:10.1021/acs.jmedchem.6b01836

Cited by 60 publications

(56 citation statements)

References 33 publications

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“…Molecules with greasy and polar groups at opposite ends (so-called “amphiphilic” molecules) are particularly prone to self-aggregation. Unsurprisingly, this phenomenon frequently leads to non-specific effects on multiple proteins and hence misleading observations unrelated to the pharmacology of the component small molecule (Blevitt et al., 2017, Irwin et al., 2015, Leeson and Springthorpe, 2007). Similarly, hydrophobic interactions between ligand and protein contribute significantly to favorable binding energies and, as a result, lipophilic molecules benefit from a greater driving force to leave the aqueous environment and enter into complementary lipid/protein environments, thereby resulting in multiple weak off-target interactions.…”

Section: Main Textmentioning

confidence: 99%

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

2017

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Small-molecule chemical probes or tools have become progressively more important in recent years as valuable reagents to investigate fundamental biological mechanisms and processes causing disease, including cancer. Chemical probes have also achieved greater prominence alongside complementary biological reagents for target validation in drug discovery. However, there is evidence of widespread continuing misuse and promulgation of poor-quality and insufficiently selective chemical probes, perpetuating a worrisome and misleading pollution of the scientific literature. We discuss current challenges with the selection and use of chemical probes, and suggest how biologists can and should be more discriminating in the probes they employ.

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Section: Main Textmentioning

confidence: 99%

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

2017

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“…Current Protocols in Chemical Biology of aggregate formation is provided by the biophysical methods of resonant waveguide grating (RWG; Blevitt et al, 2017;Chan et al, 2009) and by dynamic light scattering (DLS; Coan & Shoichet, 2008;Coan, Maltby, Burlingame, & Shoichet, 2009;Irwin et al, 2015;McGovern et al, 2003;Pohjala & Tammela, 2012;Seidler, McGovern, Doman, & Shoichet, 2003). Here we present high-throughput microplate-based RWG and DLS protocols for monitoring small-molecule aggregation.…”

Section: Of 15mentioning

confidence: 99%

Detection of Small‐Molecule Aggregation with High‐Throughput Microplate Biophysical Methods

Allen¹,

Dower²,

Liu³

et al. 2020

CP Chemical Biology

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Small‐molecule drug discovery can be hindered by the formation of aggregates that act as non‐selective inhibitors of drug targets. Such aggregates appear as false positives in high‐throughput screening campaigns and can bedevil structure‐activity relationships during compound optimization. Protocols are described for resonant waveguide grating (RWG) and dynamic light scattering (DLS) as microplate‐based high‐throughput approaches to identify compound aggregation. Resonant waveguide grating and dynamic light scattering give equivalent results for the compound test set, as assessed with Bland‐Altman analysis. © 2019 The Authors. Basic Protocol 1: Resonant waveguide grating (RWG) in 384‐well or 1536‐well plate format to detect compound aggregation Basic Protocol 2: Dynamic light scattering (DLS) in 384‐well plate format to detect compound aggregation

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“…In seminal work (9), Shoichet and colleagues proposed that the colloidal aggregates exert their effects by enzyme sequestration, thereby blocking and inhibiting their activities in unexpected ways. The lists of aggregators are not just limited to synthetic small molecules, and are also found among natural products (14) and marketed drugs (15); these issues are widespread.Besides enzymes, targets involved in protein-protein interactions are affected as well (16,17).Importantly, these artefacts are difficult to discern if the readout from the assay is reproducible and dose-dependent. One tool for addressing this requires the addition of detergents into the biochemical assay such that the apparent activities of colloidal aggregates can be alleviated or even completely abrogated (18).Macrocyclic peptides represent an exciting chemical modality with the potential to therapeutically address intracellular protein-protein interactionsthese represent targets that are most often intractable with a small-molecule modality (19,20).…”

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De-risking drug discovery of intracellular targeting peptides: screening strategies to eliminate false-positive hits

Juang²,

Chandramohan³

et al. 2019

Preprint

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AbstractDiscovery of false-positive target binding, due to assay interference or aggregation, presents a significant problem for drug discovery programs. These issues may often be unrealized and could lead researchers astray if not subject to independent verification of reproducibility and/or on-target mechanism of action. Although well-documented for small molecules, this issue has not been widely explored for peptide modality. As a case study, we demonstrate that two purported KRas inhibitors, stapled peptide SAH-SOS1A and macrocyclic peptide cyclorasin 9A5, exemplify false-positive molecules – both in terms of their sub-micromolar KRas binding affinities and their on-target cellular activities. We observed that the apparent binding of fluorescein-labeled SAH-SOS1A given by a fluorescence polarization assay is sensitive to detergent. False-positive readouts can arise from peptide adsorption to the surface of microplates. Hence, we used surface plasmon resonance and isothermal titration calorimetry to unambiguously show that both SAH-SOS1A and cyclorasin 9A5 are non-binders for KRas. Thermal shift assay and hydrogen-deuterium exchange mass spectrometry further demonstrate that both peptides destabilize KRas and induce unfolding of the protein. Furthermore, both peptides caused significant release of intracellular lactate dehydrogenase, suggesting that membrane rupture rather than on-target activity is accountable for their reported cytotoxicity. Finally, both peptides exhibited off-target activities by inhibiting the proliferation of U-2 OS and A549 cells, despite their independency of the KRas signaling pathway. Our findings demonstrate the critical need to employ orthogonal binding assays and cellular counter-screens to de-risk false-positive molecules. More rigorous workflows should lead to improved data and help obviate inadvertent scientific conclusions.Significance statementFalse positive molecule hits occur frequently in high-throughput screens and can contaminate the scientific literature. This has become an increasingly serious issue in small molecule drug discovery and chemical probe development and it is not surprising that peptides may be similarly prone to assay interference. Using KRas as a target and two known macrocyclic peptide inhibitors as a case study, we clearly show that reporter-free biophysical assays and cellular counter-screens offer the solution to detect and de-risk the potential of false-positive compounds. We further discuss the advantages, limitations and overall strategic importance of such methods.

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Structural Basis of Small-Molecule Aggregate Induced Inhibition of a Protein–Protein Interaction

Cited by 60 publications

References 33 publications

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

Detection of Small‐Molecule Aggregation with High‐Throughput Microplate Biophysical Methods

De-risking drug discovery of intracellular targeting peptides: screening strategies to eliminate false-positive hits

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