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We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target., Competing Interests: The authors have declared that no competing interests exist.

Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone., (open access.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (M pro ) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective M pro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative M pro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as M pro inhibitors. In this manner, we aimed to complement enzymatic activity assays of M pro performed by other groups with information on ligand affinity. We have made the M pro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of M pro STD-NMR data, thereby accelerating ongoing drug design efforts.

An accurate centriolar structure is crucial for organelle function, necessitating the existence of molecular mechanisms for the tight control of centriole assembly. Formation of an initial scaffold, the cartwheel, assists the correct placement of centriolar proteins during assembly and templates key structural parameters of the organelle. Past work illustrated how cartwheel and centriolar symmetry are linked, and grounded organelle symmetry and diameter to the properties of the centriolar protein SAS-6. However, questions remained over how centriole polarity and length are controlled. Recent advances in resolving cartwheel structure and cell biology showed that these assemblies are polarized and that their length is under the control of a homeostatic mechanism. These cartwheel properties may, in turn, influence the centriolar polarity and length., (Copyright © 2020 Elsevier Ltd. All rights reserved.)