The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

In humans both the levels and activity of the ␣-subunit of the hypoxia-inducible transcription factor (HIF-␣) are regulated by its post-translation hydroxylation as catalyzed by iron-and 2-oxoglutarate (2OG)-dependent prolyl and asparaginyl hydroxylases (PHD1-3 and factor-inhibiting HIF (FIH), respectively). One consequence of hypoxia is the accumulation of tricarboxylic acid cycle intermediates (TCAIs). In vitro assays were used to assess non-2OG TCAIs as inhibitors of purified PHD2 and FIH. Under the assay conditions, no significant FIH inhibition was observed by the TCAIs or pyruvate, but fumarate, succinate, and isocitrate inhibited PHD2. Mass spectrometric analyses under nondenaturing conditions were used to investigate the binding of TCAIs to PHD2 and supported the solution studies. X-ray crystal structures of FIH in complex with Fe(II) and fumarate or succinate revealed similar binding modes for each in the 2OG co-substrate binding site. The in vitro results suggest that the cellular inhibition of PHD2, but probably not FIH, by fumarate and succinate may play a role in the Warburg effect providing that appropriate relative concentrations of the components are achieved under physiological conditions.In humans and other mammals, the ␣␤ heterodimeric hypoxia-inducible transcription factor (HIF) 4 (1) regulates the oxygen-dependent transcription of an array of genes, including those encoding for enzymes involved in glycolysis and those proteins involved in oxygen delivery. The HIF-␤ subunit (HIF-␤) is identical to aryl hydrocarbon nuclear translocator, and its levels are oxygen-independent. Both the levels and transcriptional activity of the ␣-subunit (HIF-␣) are oxygen-regulated, and hydroxylation of human HIF-␣ at one of two prolyl residues (Pro-402 or Pro-564 in human HIF-1␣) (2, 3) within the oxygen-dependent degradation domain enables binding of HIF-␣ to the von Hippel-Lindau protein, the targeting element of an ubiquitin-protein isopeptide ligase complex. Subsequent ubiquitylation leads to proteasomal degradation of HIF-␣ (for recent reviews see Refs. 4 -7). In humans this mechanism is augmented by hydroxylation of an asparaginyl residue in the C-terminal transcriptional activation domain of HIF-1␣ (8); this modification blocks the interaction of HIF-1␣ with the transcriptional co-activator CBP/p300 so disabling HIF-mediated transcription. During hypoxia, the reduction or ablation of HIF hydroxylase activity leads to accumulation of HIF-1␣, dimerization with HIF-1␤, and consequent transcription of genes involved in the hypoxic response. Hydroxylation of human HIF-1␣ is catalyzed by four Fe(II)-and 2-oxoglutarate (2OG)-dependent dioxygenases (9, 10), which use molecular oxygen and the tricarboxylic acid cycle intermediate (TCAI) 2OG as co-substrates to produce CO 2 and the TCAI succinate as co-products (Fig. 1). Of the HIF hydroxylases, i.e. three prolyl hydroxylases (PHD1, -2, and -3) (11-13) and one asparaginyl hydroxylase (factor-inhibiting HIF (FIH)) (8, 14), PHD2 is proposed to be the most important...

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.There are three human trypsins encoded by different genes; cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) are located at proximal loci on chromosome 7q35, while mesotrypsinogen (PRSS3) is found on chromosome 9p13 (1). All three isoforms are secreted as digestive zymogens in the pancreas and activated by enteropeptidase in the duodenum (2). A differentially spliced form of mesotrypsinogen termed trypsinogen 4, transcribed from an alternative promoter (3) and utilizing an unconventional CUG translation initiation codon (4), is highly expressed in brain tissue (5) and in some epithelial cell lines (6) and tumors (7). The two zymogen forms differ only at the N terminus, and processing of either form by removal of the prodomain results in active mesotrypsin of identical amino acid sequence (3). Trypsinogen 4 lacks a recognizable signal sequence and it is not known whether or how the enzyme might be secreted, though there is some evidence for processing of the prodomain and deposition of activated mesotrypsin in the extracellular neuronal matrix (8).The most striking characteristic of mesotrypsin is its unique resistance to polypeptide trypsin inhibitors (9, 10). Canonical trypsin inhibitors feature characteristic binding loops that bind to the trypsin active site extremely tightly, mimicking a substrate, yet are cleaved extremely slowly (11-13). Mesot...

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