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Cell Chem Biol ; 28(12): 1795-1806.e5, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1599513


Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found âˆ¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

Drug Design , Protein Kinase Inhibitors/chemistry , SARS-CoV-2/enzymology , Viral Matrix Proteins/antagonists & inhibitors , Acrylamide/chemistry , Acrylamide/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Computational Biology/methods , Databases, Protein , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
J Am Chem Soc ; 143(48): 20095-20108, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1531986


Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.

Agammaglobulinaemia Tyrosine Kinase/chemistry , Fluorescent Dyes/chemistry , Ligands , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Adenine/analogs & derivatives , Adenine/chemistry , Adenine/metabolism , Agammaglobulinaemia Tyrosine Kinase/metabolism , Catalytic Domain , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Half-Life , Humans , Piperidines/chemistry , Piperidines/metabolism , Proteolysis , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , SARS-CoV-2/enzymology
J Clin Invest ; 131(13)2021 07 01.
Article in English | MEDLINE | ID: covidwho-1236496


BACKGROUNDThe significant risks posed to mothers and fetuses by COVID-19 in pregnancy have sparked a worldwide debate surrounding the pros and cons of antenatal SARS-CoV-2 inoculation, as we lack sufficient evidence regarding vaccine effectiveness in pregnant women and their offspring. We aimed to provide substantial evidence for the effect of the BNT162b2 mRNA vaccine versus native infection on maternal humoral, as well as transplacentally acquired fetal immune response, potentially providing newborn protection.METHODSA multicenter study where parturients presenting for delivery were recruited at 8 medical centers across Israel and assigned to 3 study groups: vaccinated (n = 86); PCR-confirmed SARS-CoV-2 infected during pregnancy (n = 65), and unvaccinated noninfected controls (n = 62). Maternal and fetal blood samples were collected from parturients prior to delivery and from the umbilical cord following delivery, respectively. Sera IgG and IgM titers were measured using the Milliplex MAP SARS-CoV-2 Antigen Panel (for S1, S2, RBD, and N).RESULTSThe BNT162b2 mRNA vaccine elicits strong maternal humoral IgG response (anti-S and RBD) that crosses the placenta barrier and approaches maternal titers in the fetus within 15 days following the first dose. Maternal to neonatal anti-COVID-19 antibodies ratio did not differ when comparing sensitization (vaccine vs. infection). IgG transfer ratio at birth was significantly lower for third-trimester as compared with second trimester infection. Lastly, fetal IgM response was detected in 5 neonates, all in the infected group.CONCLUSIONAntenatal BNT162b2 mRNA vaccination induces a robust maternal humoral response that effectively transfers to the fetus, supporting the role of vaccination during pregnancy.FUNDINGIsrael Science Foundation and the Weizmann Institute Fondazione Henry Krenter.

Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Maternal-Fetal Exchange/immunology , SARS-CoV-2/immunology , Adult , Cohort Studies , Female , Fetal Blood/immunology , Humans , Immunization, Passive , Immunoglobulin G/blood , Infant, Newborn , Male , Pregnancy , Young Adult