Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility.

Aquatic bacteria frequently are divided into lifestyle categories oligotroph or copiotroph. Oligotrophs have proportionately fewer transcriptional regulatory genes than copiotrophs and are generally non-motile/chemotactic. We hypothesized that the absence of chemotaxis/motility in oligotrophs prevents them from occupying nutrient patches long enough to benefit from transcriptional regulation. We first confirmed that marine oligotrophs are generally reduced in genes for transcriptional regulation and motility/chemotaxis. Next, using a non-motile oligotroph (Ca. Pelagibacter st. HTCC7211), a motile copiotroph (Alteromonas macleodii st. HOT1A3), and [14 C]l-alanine, we confirmed that l-alanine catabolism is not transcriptionally regulated in HTCC7211 but is in HOT1A3. We then found that HOT1A3 took 2.5-4 min to initiate l-alanine oxidation at patch l-alanine concentrations, compared to <30 s for HTCC7211. By modelling cell trajectories, we predicted that, in most scenarios, non-motile cells spend <2 min in patches, compared to >4 min for chemotactic/motile cells. Thus, the time necessary for transcriptional regulation to initiate prevents transcriptional regulation from being beneficial for non-motile oligotrophs. This is supported by a mechanistic model we developed, which predicted that HTCC7211 cells with transcriptional regulation of l-alanine metabolism would produce 12% of their standing ATP stock upon encountering an l-alanine patch, compared to 880% in HTCC7211 cells without transcriptional regulation.

Acoustic Ejection Mass Spectrometry for High-Throughput Analysis.

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.

SAR11 bacteria have a high affinity and multifunctional glycine betaine transporter.

Marine bacterioplankton face stiff competition for limited nutrient resources. SAR11, a ubiquitous clade of very small and highly abundant Alphaproteobacteria, are known to devote much of their energy to synthesizing ATP-binding cassette periplasmic proteins that bind substrates. We hypothesized that their small size and relatively large periplasmic space might enable them to outcompete other bacterioplankton for nutrients. Using uptake experiments with 14 C-glycine betaine, we discovered that two strains of SAR11, Candidatus Pelagibacter sp. HTCC7211 and Cand. P. ubique HTCC1062, have extraordinarily high affinity for glycine betaine (GBT), with half-saturation (K s ) values around 1 nM and specific affinity values between 8 and 14 L mg cell-1 h-1 . Competitive inhibition studies indicated that the GBT transporters in these strains are multifunctional, transporting multiple substrates in addition to GBT. Both strains could use most of the transported compounds for metabolism and ATP production. Our findings indicate that Pelagibacter cells are primarily responsible for the high affinity and multifunctional GBT uptake systems observed in seawater. Maximization of whole-cell affinities may enable these organisms to compete effectively for nutrients during periods when the gross transport capacity of the heterotrophic plankton community exceeds the supply, depressing ambient concentrations.

Complete genome sequence of a winter season Vibrio facilitates discovery of a novel subclade of cold-adapted species in the albus clade.

In temperate marine climate zones, seasonal changes in water temperature contribute to distinct populations of warm- and cold-water vibrios. We report here the complete genome sequence (BUSCO score=94.8) of the novel strain Vibrio sp. VB16 isolated in late winter from the intertidal zone near Virginia Beach, Virginia, USA with the ability to form colonies at 4 °C. The 5.2 Mbp genome is composed of a large (3.6 Mbp) and small (1.6 Mbp) chromosome. Based on paired average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH), V. sp. VB16 is the same species as V. sp. UBA2437 from a North Sea tidal flat and is closely related to V. sp. DW001 from Antarctic sea ice. Our phylogenomic and bioinformatic analyses placed VB16, UBA2437 and DW001 into a cold-tolerant subclade within the albus clade, along with two non-cold-tolerant subclades. Orthovenn analysis indicated that VB16 and its other albus clade members shared 1544 gene orthologue clusters, including clusters for biosynthesis of polar flagella and tight adhesion pili that predict multiple lifestyles, either free-living or as an opportunistic pathogen within a marine eukaryotic host. The cold-tolerant subclade shared 552 orthologue proteins, including genes known to promote survival in cold or freezing temperatures, such as the eicosapentaenoic acid biosynthetic gene cluster, syp exopolysaccharide gene cluster and novel giant proteins with ice-binding domains. This subclade represents a group of psychrotolerant or 'moderate psychrophile' winter season Vibrio species. The discovery of this subclade opens the door for experimental work on the physiological features, virulence potential and ecological importance of this subclade.

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

A Reduction of Transcriptional Regulation in Aquatic Oligotrophic Microorganisms Enhances Fitness in Nutrient-Poor Environments.

In this review, we consider the regulatory strategies of aquatic oligotrophs, microbial cells that are adapted to thrive under low-nutrient concentrations in oceans, lakes, and other aquatic ecosystems. Many reports have concluded that oligotrophs use less transcriptional regulation than copiotrophic cells, which are adapted to high nutrient concentrations and are far more common subjects for laboratory investigations of regulation. It is theorized that oligotrophs have retained alternate mechanisms of regulation, such as riboswitches, that provide shorter response times and smaller amplitude responses and require fewer cellular resources. We examine the accumulated evidence for distinctive regulatory strategies in oligotrophs. We explore differences in the selective pressures copiotrophs and oligotrophs encounter and ask why, although evolutionary history gives copiotrophs and oligotrophs access to the same regulatory mechanisms, they might exhibit distinctly different patterns in how these mechanisms are used. We discuss the implications of these findings for understanding broad patterns in the evolution of microbial regulatory networks and their relationships to environmental niche and life history strategy. We ask whether these observations, which have emerged from a decade of increased investigation of the cell biology of oligotrophs, might be relevant to recent discoveries of many microbial cell lineages in nature that share with oligotrophs the property of reduced genome size.

Structural basis of the acyl-transfer mechanism of human GPAT1.

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.

Unique Geothermal Chemistry Shapes Microbial Communities on Mt. Erebus, Antarctica.

Mt. Erebus, Antarctica, is the world's southernmost active volcano and is unique in its isolation from other major active volcanic systems and its distinctive geothermal systems. Using 16S rRNA gene amplicon sequencing and physicochemical analyses, we compared samples collected at two contrasting high-temperature (50°C-65°C) sites on Mt. Erebus: Tramway Ridge, a weather-protected high biomass site, and Western Crater, an extremely exposed low biomass site. Samples were collected along three thermal gradients, one from Western Crater and two within Tramway Ridge, which allowed an examination of the heterogeneity present at Tramway Ridge. We found distinct soil compositions between the two sites, and to a lesser extent within Tramway Ridge, correlated with disparate microbial communities. Notably, pH, not temperature, showed the strongest correlation with these differences. The abundance profiles of several microbial groups were different between the two sites; class Nitrososphaeria amplicon sequence variants (ASVs) dominated the community profiles at Tramway Ridge, whereas Acidobacteriotal ASVs were only found at Western Crater. A co-occurrence network, paired with physicochemical analyses, allowed for finer scale analysis of parameters correlated with differential abundance profiles, with various parameters (total carbon, total nitrogen, soil moisture, soil conductivity, sulfur, phosphorous, and iron) showing significant correlations. ASVs assigned to Chloroflexi classes Ktedonobacteria and Chloroflexia were detected at both sites. Based on the known metabolic capabilities of previously studied members of these groups, we predict that chemolithotrophy is a common strategy in this system. These analyses highlight the importance of conducting broader-scale metagenomics and cultivation efforts at Mt. Erebus to better understand this unique environment.

SAR11 Cells Rely on Enzyme Multifunctionality To Metabolize a Range of Polyamine Compounds.

In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations at seawater pH produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells uptake and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, "Candidatus Pelagibacter" strain HTCC7211 and "Candidatus Pelagibacter ubique" strain HTCC1062. Both strains took up all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but polyamines did not fully substitute for their requirements of glycine (or related compounds) or pyruvate (or related compounds). Our data suggest that potABCD transports all five polyamines and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they metabolize. IMPORTANCE Genome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically, they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality, referring to enzymes that are adapted to have broader substrate and catalytic range than canonically defined, is hypothesized to be an adaptation that increases the range of organic compounds metabolized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells take up and metabolize multiple polyamine compounds and propose that a small set of multifunctional enzymes catalyze this metabolism. We report that polyamine uptake rates can exceed metabolic rates, resulting in both high intracellular concentrations of these nitrogen-rich compounds (in comparison to native polyamine levels) and an increase in cell size.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Draft Genome Sequence of Janthinobacterium sp. Strain PC23-8, Isolated from a Freshwater Stream Impacted by Acid Mine Drainage.

We present the draft genome sequence of Janthinobacterium sp. strain PC23-8, a bacterium isolated from freshwater stream sediment downstream from acid mine drainage. The 6.4-Mb genome sequence of this strain contains several secondary metabolite gene clusters, including one similar to the cyclic peptide jagaricin, synthesized by Janthinobacterium agaricidamnosum.

A Parasitic Arsenic Cycle That Shuttles Energy from Phytoplankton to Heterotrophic Bacterioplankton.

In many regions of the world oceans, phytoplankton face the problem of discriminating between phosphate, an essential nutrient, and arsenate, a toxic analogue. Many phytoplankton, including the most abundant phytoplankton group known, Prochlorococcus, detoxify arsenate (AsV) by reduction to arsenite (AsIII), followed by methylation and excretion of the methylated arsenic products. We synthesized [14C]dimethyl arsenate (DMA) and used it to show that cultured Pelagibacter strain HTCC7211 (SAR11) cells oxidize the methyl group carbons of DMA, producing 14CO2 and ATP. We measured [14C]DMA oxidation rates in the P-depleted surface waters of the Sargasso Sea, a subtropical ocean gyre. [14C]DMA was oxidized to 14CO2 by Sargasso Sea plankton communities at a rate that would cause turnover of the estimated DMA standing stock every 8.1 days. SAR11 strain HTCC7211, which was isolated from the Sargasso Sea, has a pair of arsenate resistance genes and was resistant to arsenate, showing no growth inhibition at As/P ratios of >65:1. Across the global oceans, there was a strong inverse relationship between the frequency of the arsenate reductase (LMWPc_ArsC) in Pelagibacter genomes and phosphate concentrations. We propose that the demethylation of methylated arsenic compounds by Pelagibacter and possibly other bacterioplankton, coupled with arsenate resistance, results in the transfer of energy from phytoplankton to bacteria. We dub this a parasitic cycle because the release of arsenate by Pelagibacter in principle creates a positive-feedback loop that forces phytoplankton to continually regenerate arsenate detoxification products, producing a flow of energy to P-limited ocean regions.IMPORTANCE In vast, warm regions of the oceans, phytoplankton face the problem of arsenic poisoning. Arsenate is toxic because it is chemically similar to phosphate, a scarce nutrient that phytoplankton cells need for growth. Many phytoplankton, including the commonest phytoplankton type in warm oceans, Prochlorococcus, detoxify arsenate by adding methyl groups. Here we show that the most abundant non-photosynthetic plankton in the oceans, SAR11 bacteria, remove the methyl groups, releasing poisonous forms of arsenic back into the water. We postulate that the methylation and demethylation of arsenic compounds creates a cycle in which the phytoplankton can never get ahead and must continually transfer energy to the SAR11 bacteria. We dub this a parasitic process and suggest that it might help explain why SAR11 bacteria are so successful, surpassing all other plankton in their numbers. Field experiments were done in the Sargasso Sea, a subtropical ocean gyre that is sometimes called an ocean desert because, throughout much of the year, there is not enough phosphorous in the water to support large blooms of phytoplankton. Ocean deserts are expanding as the oceans absorb heat and grow warmer.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA).

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.

Tool compounds robustly increase turnover of an artificial substrate by glucocerebrosidase in human brain lysates.

Mutations in glucocerebrosidase (GBA1) cause Gaucher disease and also represent a common risk factor for Parkinson's disease and Dementia with Lewy bodies. Recently, new tool molecules were described which can increase turnover of an artificial substrate 4MUG when incubated with mutant N370S GBA1 from human spleen. Here we show that these compounds exert a similar effect on the wild-type enzyme in a cell-free system. In addition, these tool compounds robustly increase turnover of 4MUG by GBA1 derived from human cortex, despite substantially lower glycosylation of GBA1 in human brain, suggesting that the degree of glycosylation is not important for compound binding. Surprisingly, these tool compounds failed to robustly alter GBA1 turnover of 4MUG in the mouse brain homogenate. Our data raise the possibility that in vivo models with humanized glucocerebrosidase may be needed for efficacy assessments of such small molecules.