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Covalent docking of large libraries for the discovery of chemical probes.

London N, Miller RM, Krishnan S, Uchida K, Irwin JJ, Eidam O, Gibold L, Cimermančič P, Bonnet R, Shoichet BK, Taunton J - Nat. Chem. Biol. (2014)

Bottom Line: Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns.Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules.Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, California, USA.

ABSTRACT
Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).

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Cyanoacrylamide inhibitors of RSK2 and MSK1 predicted by covalent dockinga and b. Blind docking predictions of two cyanoacrylamide fragments covalently bound to RSK2 (magenta) recapitulate their crystallographic poses (yellow, PDB: 4JG7,4JG6). c. Chemical structures of cyanoacrylamide fragments selected for synthesis and testing. d. Docking prediction for the most potent fragment 24 corresponds well to the experimental structure. e. Docking prediction of the binding mode of compound 21. f. Compounds 24 and 21 inhibit autophosphorylation of RSK2 and MSK1 in PMA-stimulated cells. Neither compound inhibits the cysteine to valine mutant of MSK1 at concentrations up to 20 μM. Western blots are representative of duplicate biological measurements. g. Dose-response curves comparing pyrrolopyrimidine 27 and 21 vs. WT RSK2. 27 was designed based on the docked structure of 21 (See e.). Data are plotted as the mean of duplicate measurements ± the range. h. Docked pose of 27. i. Compound 27 inhibits MSK1 autophosphorylation in PMA-stimulated cells. All western blots are representative of duplicate experiments. Full gel images can be found in Supplementary Fig. 15.
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Figure 3: Cyanoacrylamide inhibitors of RSK2 and MSK1 predicted by covalent dockinga and b. Blind docking predictions of two cyanoacrylamide fragments covalently bound to RSK2 (magenta) recapitulate their crystallographic poses (yellow, PDB: 4JG7,4JG6). c. Chemical structures of cyanoacrylamide fragments selected for synthesis and testing. d. Docking prediction for the most potent fragment 24 corresponds well to the experimental structure. e. Docking prediction of the binding mode of compound 21. f. Compounds 24 and 21 inhibit autophosphorylation of RSK2 and MSK1 in PMA-stimulated cells. Neither compound inhibits the cysteine to valine mutant of MSK1 at concentrations up to 20 μM. Western blots are representative of duplicate biological measurements. g. Dose-response curves comparing pyrrolopyrimidine 27 and 21 vs. WT RSK2. 27 was designed based on the docked structure of 21 (See e.). Data are plotted as the mean of duplicate measurements ± the range. h. Docked pose of 27. i. Compound 27 inhibits MSK1 autophosphorylation in PMA-stimulated cells. All western blots are representative of duplicate experiments. Full gel images can be found in Supplementary Fig. 15.

Mentions: As an initial blind test, we used the method to predict the poses of two cyanoacrylamide fragments bound to RSK2, prior to determining the crystal structures. The predicted binding modes anticipated the experimental structures to 1.93 Å and 1.56 Å RMSD (Fig. 3a and 3b). Retrospective docking of two larger cyanoacrylamides also recapitulated their crystal structures (0.66 Å and 1.52 Å RMSD; Supplementary Fig. 8a–b). In each prediction, the scaffold portion of the molecule, which forms critical non-covalent interactions with RSK2, closely matched the x-ray structures (0.91–1.36 Å RMSD).


Covalent docking of large libraries for the discovery of chemical probes.

London N, Miller RM, Krishnan S, Uchida K, Irwin JJ, Eidam O, Gibold L, Cimermančič P, Bonnet R, Shoichet BK, Taunton J - Nat. Chem. Biol. (2014)

Cyanoacrylamide inhibitors of RSK2 and MSK1 predicted by covalent dockinga and b. Blind docking predictions of two cyanoacrylamide fragments covalently bound to RSK2 (magenta) recapitulate their crystallographic poses (yellow, PDB: 4JG7,4JG6). c. Chemical structures of cyanoacrylamide fragments selected for synthesis and testing. d. Docking prediction for the most potent fragment 24 corresponds well to the experimental structure. e. Docking prediction of the binding mode of compound 21. f. Compounds 24 and 21 inhibit autophosphorylation of RSK2 and MSK1 in PMA-stimulated cells. Neither compound inhibits the cysteine to valine mutant of MSK1 at concentrations up to 20 μM. Western blots are representative of duplicate biological measurements. g. Dose-response curves comparing pyrrolopyrimidine 27 and 21 vs. WT RSK2. 27 was designed based on the docked structure of 21 (See e.). Data are plotted as the mean of duplicate measurements ± the range. h. Docked pose of 27. i. Compound 27 inhibits MSK1 autophosphorylation in PMA-stimulated cells. All western blots are representative of duplicate experiments. Full gel images can be found in Supplementary Fig. 15.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232467&req=5

Figure 3: Cyanoacrylamide inhibitors of RSK2 and MSK1 predicted by covalent dockinga and b. Blind docking predictions of two cyanoacrylamide fragments covalently bound to RSK2 (magenta) recapitulate their crystallographic poses (yellow, PDB: 4JG7,4JG6). c. Chemical structures of cyanoacrylamide fragments selected for synthesis and testing. d. Docking prediction for the most potent fragment 24 corresponds well to the experimental structure. e. Docking prediction of the binding mode of compound 21. f. Compounds 24 and 21 inhibit autophosphorylation of RSK2 and MSK1 in PMA-stimulated cells. Neither compound inhibits the cysteine to valine mutant of MSK1 at concentrations up to 20 μM. Western blots are representative of duplicate biological measurements. g. Dose-response curves comparing pyrrolopyrimidine 27 and 21 vs. WT RSK2. 27 was designed based on the docked structure of 21 (See e.). Data are plotted as the mean of duplicate measurements ± the range. h. Docked pose of 27. i. Compound 27 inhibits MSK1 autophosphorylation in PMA-stimulated cells. All western blots are representative of duplicate experiments. Full gel images can be found in Supplementary Fig. 15.
Mentions: As an initial blind test, we used the method to predict the poses of two cyanoacrylamide fragments bound to RSK2, prior to determining the crystal structures. The predicted binding modes anticipated the experimental structures to 1.93 Å and 1.56 Å RMSD (Fig. 3a and 3b). Retrospective docking of two larger cyanoacrylamides also recapitulated their crystal structures (0.66 Å and 1.52 Å RMSD; Supplementary Fig. 8a–b). In each prediction, the scaffold portion of the molecule, which forms critical non-covalent interactions with RSK2, closely matched the x-ray structures (0.91–1.36 Å RMSD).

Bottom Line: Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns.Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules.Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, California, USA.

ABSTRACT
Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC β-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).

Show MeSH
Related in: MedlinePlus