Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: Structural basis of their function.

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.

Structure of monkeypox virus poxin: implications for drug design.

Monkeypox, or mpox, is a disease that has recently resurfaced and spread across the globe. Despite the availability of an FDA-approved vaccine (JYNNEOS) and an effective drug (tecovirimat), concerns remain over the possible recurrence of a viral pandemic. Like any other virus, mpox virus must overcome the immune system to replicate. Viruses have evolved various strategies to overcome both innate and adaptive immunity. Poxviruses possess an unusual nuclease, poxin, which cleaves 2'-3'-cGAMP, a cyclic dinucleotide, which is an important second messenger in the cGAS-STING signaling pathway. Here, we present the crystal structure of mpox poxin. The structure reveals a conserved, predominantly ß-sheet fold and highlights the high conservation of the cGAMP binding site and of the catalytic residues His17, Tyr138, and Lys142. This research suggests that poxin inhibitors could be effective against multiple poxviruses.

Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure.

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

Realgar (As4S4) bioprecipitation in microcosm fed by a natural groundwater and organic matter.

Sequestration of arsenic to biogenic sulfide minerals is known from As-contaminated anoxic environments. Despite numerous successful laboratory experiments, the process remains difficult to predict in moderate arsenic conditions. We performed microcosm experiments using naturally contaminated groundwater (containing ca. 6 mg/L As) and natural organic matter (NOM) particles both collected from wetland soil. Macroscopic realgar precipitates, occasionally accompanied by bonazziite, a FeS phase, elementary S, calcite, and whewellite, appeared after 4 to 18 months. Realgar only precipitated in microcosms moderately poisoned by azide or antibiotics and those in which oxidation of hydrogen sulfide to sulfur took place. The biomineralization process was not affected by the presence of additional carbon sources or the diversity, community structure, and functional composition of the microbial community. Hydrogen sulfide concentration was greater in the realgar-free microcosms, suggesting that arsenic thiolation prevented precipitation of realgar. We compared our data to available microbial community data from soils with different rates of realgar precipitation, and found that the communities from realgar-encrusted NOM particles usually showed limited sulfate reduction and the presence of fermentative metabolisms, whereas communities from realgar-free NOM particles were strongly dominated by sulfate reducers. We argue that the limited sulfate supply and intensive fermentation amplify reducing conditions, which make arsenic sulfide precipitation plausible in high-sulfate, low-arsenic groundwaters.

Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Substantial Variability of Multiple Microbial Communities Collected at Similar Acidic Mine Water Outlets.

Deep sequencing of prokaryotic 16S rDNA regularly reveals thousands of microbial species thriving in many common habitats. It is still unknown how this huge microbial diversity, including many potentially competing organisms, may persist at a single site. One of plausible hypotheses is that a large number of spatially separated microcommunities exist within each complex habitat. Smaller subset of the species may exist in each microcommunity and actually interact with each other. We sampled two groups of microbial stalactites growing at a single acidic mine drainage outlet as a model of multiplicated, low-complexity microhabitat. Samples from six other sites were added for comparison. Both tRFLP and 16S rDNA pyrosequencing showed that microbial communities containing 6 to 51 species-level operational taxonomic units (OTU) inhabited all stalactites. Interestingly, most OTUs including the highly abundant ones unpredictably alternated regardless of physical and environmental distance of the stalactites. As a result, the communities clustered independently on sample site and other variables when using both phylogenetic dissimilarity and OTU abundance metrics. Interestingly, artificial communities generated by pooling the biota of several adjacent stalactites together clustered by the locality more strongly than when the stalactites were analyzed separately. The most probable interpretation is that each stalactite contains likely random selection from the pool of plausible species. Such degree of stochasticity in assembly of extremophilic microbial communities is significantly greater than commonly proposed and requires caution when interpreting microbial diversity.