De novo design of picomolar SARS-CoV-2 miniprotein inhibitors
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Abstract
We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC 50 values ranging from 35 pM to 35 nM; the most potent of these — 56 and 64 residue hyperstable proteins made using the second approach — are roughly six times more potent on a per mass basis (IC 50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.
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SciScore for 10.1101/2020.08.03.234914: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Plates were washed and sequentially incubated with 1 μg/mL of CR3022 (5) anti-S antibody and HRP-conjugated goat anti-human IgG in PBS supplemented to contain 0.1% saponin and 0.1% BSA. anti-Ssuggested: Noneanti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focus-forming assay on Vero E6 cells. Vero CCL81suggested: NoneRBD binder-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated for 1 h at 37°C. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources All libraries … SciScore for 10.1101/2020.08.03.234914: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Plates were washed and sequentially incubated with 1 μg/mL of CR3022 (5) anti-S antibody and HRP-conjugated goat anti-human IgG in PBS supplemented to contain 0.1% saponin and 0.1% BSA. anti-Ssuggested: Noneanti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focus-forming assay on Vero E6 cells. Vero CCL81suggested: NoneRBD binder-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated for 1 h at 37°C. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources All libraries were amplified using Kapa HiFi Polymerase (Kapa Biosystems) with a qPCR machine (BioRAD CFX96). BioRADsuggested: NoneData was processed using Prism software (GraphPad Prism 8.0). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking, and extraction were carried out using Warp. Warpsuggested: (Warp, RRID:SCR_018071)Local resolution estimation, filtering, and sharpening were carried out using CryoSPARC. CryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 26 and 28. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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