Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Molnupiravir maintains antiviral activity against SARS-CoV-2 variants and exhibits a high barrier to the development of resistance.

Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1-3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.

Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch.

A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 µM), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.

Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry.

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

Deposition of artificial radionuclides in sediments of Loch Etive, Scotland.

The nuclear fuel reprocessing plants on the Sellafield site (UK) have released low-level effluents into the Irish Sea under authorisation since 1952. This has led to the labelling of nearby offshore sediments with a range of artificial radionuclides. In turn, these sediments act as a long-term secondary source of both soluble and particle-associated radionuclides to coastal areas. These radionuclides are of interest both in assessing possible environmental impacts and as tracers for marine processes. Here we present results from a study of the geochemistry of natural (234, 238U) and artificial (137Cs, 241Am, 238Pu, 239+240Pu, and 236U) radionuclides and their accumulation in sediments from Loch Etive, Scotland. The data are interpreted in the context of the historical radioactive discharges to the Irish Sea and biogeochemical processes in marine sediments. Loch Etive is divided into two basins; a lower, seaward basin where the sedimentation rate (∼0.6 cm/yr) is about twice that of the more isolated upper basin (∼0.3 cm/yr). These accumulation rates are consistent with the broad distribution of 137Cs in the sediment profiles which can be related to the maximum Sellafield discharges of 137Cs in the mid-1970s and suggest that 137Cs was mainly transported in solution to Loch Etive during that period. Enrichments of Mn, Fe, and Mo in sediment and porewater from both Loch Etive basins result from contemporary biogeochemical redox processes. Enrichments of 238U and 234U in the lower basin may be a result of the cycling of natural U. By contrast, the Sellafield-derived artificial isotope 236U does not seem to be affected by the redox-driven reactions in the lower basin. The 238Pu/239,240Pu ratios suggest contributions from both historical Sellafield discharges and global fallout Pu. The uniform sediment distributions of Pu and Am, which do not reflect Sellafield historical discharges, suggest the existence of a homogenous secondary source. This could be the offshore 'mud patch' in the vicinity of Sellafield from which the supply of radionuclides reflects time-integrated Sellafield discharges. This source could also account for the continuing supply of Cs to Loch Etive, even after substantial reductions in discharge from the Sellafield site.

Nuclear reprocessing-related radiocarbon (14C) uptake into UK marine mammals.

To evaluate the transfer of Sellafield-derived radiocarbon (14C) to top predators in the UK marine environment, 14C activities were examined in stranded marine mammals. All samples of harbour porpoise (Phocoena phocoena) obtained from the Irish Sea showed 14C enrichment above background. Mammal samples obtained from the West of Scotland, including harbour porpoise, grey seals (Halichoerus grypus) and harbour seals (Phoca vitulina) showed 14C enrichment but to a lesser extent. This study demonstrates, for the first time, enriched 14C is transferred through the marine food web to apex predators as a consequence of ongoing nuclear reprocessing activities at Sellafield. Total Sellafield 14C discharge activity 24months prior to stranding and, in particular, distance of animal stranding site from Sellafield are significant variables affecting individual 14C activity. 14C activities of West of Scotland harbour porpoises suggest they did not forage in the Irish Sea prior to stranding, indicating a high foraging fidelity.

Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting.

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Ecosystem uptake and transfer of Sellafield-derived radiocarbon (14C). Part 1. The Irish Sea.

Ecosystem uptake and transfer processes of Sellafield-derived radiocarbon (14C) within the Irish Sea were examined. Highly variable activities in sediment, seawater and biota indicate complex 14C dispersal and uptake dynamics. All east basin biota exhibited 14C enrichments above ambient background while most west basin biota had 14C activities close to background, although four organisms including two slow-moving species were significantly enriched. The western Irish Sea gyre is a suggested pathway for transfer of 14C to the west basin and retention therein. Despite ongoing Sellafield 14C discharges, organic sediments near Sellafield were significantly less enriched than associated benthic organisms. Rapid scavenging of labile, 14C-enriched organic material by organisms and mixing to depth of 14C-enriched detritus arriving at the sediment/water interface are proposed mechanisms to explain this. All commercially important fish, crustaceans and molluscs showed 14C enrichments above background; however, the radiation dose from their consumption is extremely low and radiologically insignificant.

Ecosystem uptake and transfer of Sellafield-derived radiocarbon (14C) part 2: The West of Scotland.

Ecosystem uptake and transfer of Sellafield-derived radiocarbon (14C) were examined within the West of Scotland marine environment. The dissolved inorganic carbon component of seawater, enriched in 14C, is transported to the West of Scotland where it is transferred through the marine food web. Benthic and pelagic biota with variable life-spans living in the North Channel and Clyde Sea show comparable 14C activities. This suggests that mixing of 14C within the Irish Sea results in a relatively constant northwards dispersal of activity. Benthic species in the Firth of Lorn have similar 14C enrichments, demonstrating that Irish Sea residual water is the dominant source to this area. Measured 14C activities in biota show some similarity to western Irish Sea activities, indicating that dispersion to the West of Scotland is significant with respect to the fate of Sellafield 14C releases. Activities measured in commercially important species do not pose any significant radiological risk.

Atomic resolution mechanistic studies of ribocil: A highly selective unnatural ligand mimic of the E. coli FMN riboswitch.

Bacterial riboswitches are non-coding RNA structural elements that direct gene expression in numerous metabolic pathways. The key regulatory roles of riboswitches, and the urgent need for new classes of antibiotics to treat multi-drug resistant bacteria, has led to efforts to develop small-molecules that mimic natural riboswitch ligands to inhibit metabolic pathways and bacterial growth. Recently, we reported the results of a phenotypic screen targeting the riboflavin biosynthesis pathway in the Gram-negative bacteria Escherichia coli that led to the identification of ribocil, a small molecule inhibitor of the flavin mononucleotide (FMN) riboswitch controlling expression of this biosynthetic pathway. Although ribocil is structurally distinct from FMN, ribocil functions as a potent and highly selective synthetic mimic of the natural ligand to repress riboswitch-mediated ribB gene expression and inhibit bacterial growth both in vitro and in vivo. Herein, we expand our analysis of ribocil; including mode of binding in the FMN binding pocket of the riboswitch, mechanisms of resistance and structure-activity relationship guided efforts to generate more potent analogs.

Latest Quaternary palaeoceanographic change in the eastern North Atlantic based upon a dinoflagellate cyst event ecostratigraphy.

The analyses of dinoflagellate cyst records, from the latest Quaternary sediments recovered from DSDP Core 610A taken on the Feni Ridge in the southern Rockall Trough, and part of core MD01-2461 on the continental margin of the Porcupine Seabight in the eastern North Atlantic Ocean, has provided evidence for significant oceanographic change encompassing the Last Glacial Maximum (LGM) and part of the Holocene. This together with other published records has led to a regional evaluation of oceanographic change in the eastern North Atlantic over the past 68 ka, based upon a distinctive dinoflagellate event ecostratigraphy. These changes reflect changes in the surface waters of the North Atlantic Current (NAC), and perhaps the deeper thermohaline Atlantic Meridional Overturning Circulation (AMOC), driving fundamental regime changes within the phytoplanktonic communities. Three distinctive dinoflagellate cyst associations based upon both factor and cluster analyses have been recognised. Associations characterised by Bitectatodinium tepikiense (between 61.1 ± 6.2 to 13.4 ± 1.1 ka BP), Nematosphaeropsis labyrinthus (between 10.5 ± 0.3 and 11.45 ± 0.8 ka. BP), and the cyst of Protoceratium reticulatum (between 8.5 ± 0.9 and 5.2 ± 1.3 ka. BP) indicate major change within the eastern North Atlantic oceanography. The transitions between these changes occur over a relatively short time span (c.1.5 ka), given our sampling resolution, and have the potential to be incorporated into an event stratigraphy through the latest Quaternary as recommended by the INTIMATE (INTegrating Ice core, MArine and TErrestrial records) group. The inclusion of a dinoflagellate cyst event stratigraphy would highlight changes within the phytoplankton of the North Atlantic Ocean as a fully glacial world changed to our present interglacial.