Design and characterization of a heterocyclic electrophilic fragment library for the discovery of cysteine-targeted covalent inhibitors

Keeley, Aaron; Ábrányi‐Balogh, Péter; Keserü, György M.

doi:10.1039/c8md00327k

Cited by 67 publications

(91 citation statements)

References 14 publications

(16 reference statements)

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“…Two covalent hits (2-cyanopyrimidine (Cov_HetLib 030 (×2097)) and 2-cyanoimidazole (Cov_HetLib 053 (×2119) came from a library of small heterocyclic electrophiles 47 . These are essentially covalent MiniFrags 48 , comprising five and six-membered nitrogen-containing heterocycles with electron-withdrawing character that activates small electrophilic substituents (halogens, ethylyl, vinyl and nitrile groups).…”

Section: Resultsmentioning

confidence: 99%

“…Fragments were soaked into crystals by adding dissolved compound directly to the crystallisation drops. The following libraries were screened: the DSipoised library (Enamine), a version of the poised library 25 ; a version of the MiniFrags library 48 assembled in-house; the FragLites library 45 ; a library of shape-diverse 3D fragments (“York3D”) 69 ; heterocyclic electrophiles 47 ; and the SpotFinder library. All fragments were in 100% DMSO at varying stock concentrations, detailed at https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/Fragment-Libraries.html ).…”

Section: Methodsmentioning

confidence: 99%

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Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

et al. 2020

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COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

et al. 2020

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“…A potential solution is to directly screen mild electrophile fragments. 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.…”

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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“…Formation of a thiohydroximate adduct in the reaction of a nitro group with a cysteine residue in the active site has been reported. 47 In fragment screening conducted prior to the COVID Moonshot project, 48 an electrophile library 49,50 oriented toward covalent bonding was used. As these electrophile libraries did not cover nitro groups, they evaluated a different chemical space from that examined in the present study.…”

Section: Discussionmentioning

confidence: 99%

Screening for inhibitors of main protease in SARS-CoV-2: in silico and in vitro approach avoiding secondary amides

Sekijima

Yamamoto

Yasuo

2021

Preprint

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In addition to vaccines, antiviral drugs are essential for suppressing COVID-19.Although several inhibitor candidates were reported for SARS-CoV-2 main protease, most are highly polar peptidomimetics with poor oral bioavailability and cell membrane permeability. Here, we conducted structure-based virtual screening and in vitro assays to obtain hit compounds belonging to a new chemical space excluding secondary amides. In total, 180 compounds were subjected to the primary assay at 20 µM, and nine compounds with inhibition rates higher than 5% were obtained. The IC 50 of six compounds was determined in dose-response experiments, with the values on the order of 10 -4 µM. Although nitro groups were enriched in the substructure of the hit compounds, they did not significantly contribute to the binding interaction in 1 the predicted docking poses. Physicochemical properties prediction showed good oral absorption. These new scaffolds are promising candidates for future optimization.

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Design and characterization of a heterocyclic electrophilic fragment library for the discovery of cysteine-targeted covalent inhibitors

Abstract: A fragment library of electrophilic small heterocycles was characterized through cysteine-reactivity and aqueous stability tests that suggested their potential as covalent warheads.

Cited by 67 publications

References 14 publications

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Screening for inhibitors of main protease in SARS-CoV-2: in silico and in vitro approach avoiding secondary amides

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