Non-covalent SARS-CoV-2 Mpro inhibitors developed from in silico screen hits.

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.

Reversal of the DNA-binding-induced loop L1 conformational switch in an engineered human p53 protein.

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.

Crystal structure of a multidomain human p53 tetramer bound to the natural CDKN1A (p21) p53-response element.

The p53 tumor suppressor protein is a sequence-specific DNA-binding transcription factor. Structures of p53 bound to DNA have been described, but, so far, no structure has been determined of p53 bound to a natural p53-response element. We describe here the structure of a human p53 homotetramer encompassing both the DNA-binding and homo-oligomerization domains in complex with the natural p53-response element present upstream of the promoter of the CDKN1A (p21) gene. Similar to our previously described structures of human p53 tetramers bound to an artificial consensus DNA site, p53 DNA binding proceeds via an induced fit mechanism with loops L1 of two subunits adopting recessed conformations. Interestingly, the conformational change involving loop L1 is even more extreme than the one previously observed with the artificial consensus DNA site. In fact, the previously determined loop L1 conformation seems to be a transition intermediate between the non-DNA-bound and CDKN1A-bound states. Thus, the new structure further supports our model that recognition of specific DNA by p53 is associated with conformational changes within the DNA-binding domain of p53.

Lymphocytes in Peyer patches regulate clinical tolerance in a murine model of food allergy.

BACKGROUND: Food allergy can lead to severe, potentially life-threatening anaphylactic reactions. To generate efficient strategies aimed at an active cure, a better understanding of the pathogenic mechanisms is strongly needed. OBJECTIVE: To investigate T-cell-related mechanisms of food allergy and tolerance to beta-lactoglobulin (BLG) in gut-associated lymphoid structures. METHODS: Beta-lactoglobulin-specific IgG1, IgG2a, and IgE in serum from mice anaphylactic to BLG were analyzed by ELISA and compared with those obtained in mice actively tolerized to BLG. The number of Ab-secreting cells in the spleen and in Peyer patches was determined by ELISPOT. The numbers of cytokine-producing cells after antigen-specific activation were measured by the same method. Furthermore, mesenteric lymph node cells and Peyer patches cells were transferred to naive mice, and Ab production as well as Ab-secreting cells were measured. RESULTS: Serum IgG1 and IgE Ab titers as well as IL-4-producing cell numbers were strongly increased in anaphylactic mice. IL-10 was found in Peyer patch cells from tolerant mice after BLG activation but not in anaphylactic mice. Peyer patch cells, in contrast to mesenteric lymph node cells, proliferated weakly in anaphylactic mice after antigen activation, and activation of Peyer patches was partially inhibited by tolerization. CONCLUSIONS: Our data suggest a specific role for lymphocytes in Peyer patches in tolerance to BLG. Low IL-10 production in Peyer patches may favor symptoms of food allergy.