Applications of Reactive Cysteine Profiling

Backus, Keriann M.

doi:10.1007/82_2018_120

Cited by 35 publications

(30 citation statements)

References 283 publications

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“…Electrophile fragment screens were recently performed on a small scale, with libraries of up to ∼100 compounds in vitro against a recombinant target 38−43 or in a cellular phenotypic context. 44−46 Small-scale screens were also performed with reversible covalent fragments. 47,48 We hypothesized that significantly increasing the library size and screening it against a diverse panel of targets will allow robust discovery of covalent ligands.…”

Section: Introductionmentioning

confidence: 99%

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

et al. 2019

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Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

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Section: Introductionmentioning

confidence: 99%

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

et al. 2019

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“…Fig 1), on a subset of kinases from our database, showing an increase in the number of structures that can be covalentized. New covalent 'warheads', including reversible covalent warheads, such as cyanoacrylamides 60 , and clorofluoroacetamides 61 become available, both for cysteine residues 62 , but also for other amino acids [63][64][65] . These can be incorporated with little effort into the covalentizer pipeline.…”

Section: Discussionmentioning

confidence: 99%

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 M^proinhibitor

Zaidman

Gehrtz

Filep

et al. 2020

Preprint

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Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 μM. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

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“…3B) that different target cysteines show different potential for electrophilic labeling. Proteomic approaches for the identification of functional or reactive cysteines 42,44,[67][68][69] can identify potential target cysteines in a much larger scale than ever before. However, the throughput of the proteomics pipeline does not allow to assess the covalent ligandability.…”

Section: Discussionmentioning

confidence: 99%

“…Disulfides are not, however, suitable as cellular probes, and replacing them with a suitable electrophile is in general no less challenging than starting from a reversible ligand.A potential solution is to directly screen mild electrophile fragments. Electrophile fragment screens were recently performed in small scale, with libraries of up-to ~100 compounds in vitro against a recombinant target 37-41 or in a cellular phenotypic context [42][43][44] . Small scale screens were also performed with reversible covalent fragments 45,46 .…”

mentioning

confidence: 99%

Rapid covalent-probe discovery by electrophile fragment screening

Resnick

Bradley

Gan

et al. 2018

Preprint

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Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.Fragment based screening, which focuses on very low molecular-weight compounds, is a successful hit discovery approach for reversible inhibitors 28,29 , that has led to several drugs and chemical probes 29,30 . Compared to traditional HTS, fragment-based screening offers better coverage of chemical space and higher probability of binding due to lower complexity 31,32 . The major limitation in fragment-based screening is the weak binding affinity of fragment hits, which not only necessitates very sensitive biophysical detection methods, coupled with elaborate validation cascades to eliminate attendant artefacts, but additionally makes progressing hits to potency difficult and expensive. In particular, it requires large compound series with typically ambiguous structure-activity relationships, because no method to date can reliably rationalize which are the dominant interactions of the original fragment. Screening covalent fragments addresses both problems: covalent binders are easy to detect by mass spectrometry; and because the dominant interaction is unambiguous, namely the covalent bond, designing followup series is simplified, and the primary hits are already potent.A prominent covalent fragment screening approach is disulfide tethering 33,34 , which entails incubating a library of disulfide-containing fragments with the target. Disulfide exchange with the target cysteine selects for fragments that are reversibly stabilized in its vicinity. Disulfide tethering was successfully applied to a variety of targets containing both native and introduced cysteine residues 35 . Recently it led to the discovery of a promising K-Ras G12C inhibitor 36 . Disulfides are not, however, suitable as cellular probes, and replacing them with a suitable electrophile is in general no less challenging than starting from a reversible ligand.A potential solution is to directly screen mild electrophile fragments. Electrophile...

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Applications of Reactive Cysteine Profiling

Cited by 35 publications

References 283 publications

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 M^proinhibitor

Rapid covalent-probe discovery by electrophile fragment screening

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Applications of Reactive Cysteine Profiling

Cited by 35 publications

References 283 publications

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mproinhibitor

Rapid covalent-probe discovery by electrophile fragment screening

Contact Info

Product

Resources

About

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 M^proinhibitor