Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Peripheral neuronal activation shapes the microbiome and alters gut physiology.

The gastrointestinal (GI) tract is innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Here, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic contractility and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally activated neurons differentially regulate the gut microbiome and GI physiology in mice without involvement of signals from the brain.

PROTAC-Mediated Selective Degradation of Cytosolic Soluble Epoxide Hydrolase Enhances ER Stress Reduction.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme responsible for lipid metabolism and is a promising drug target. Here, we report the first-in-class PROTAC small-molecule degraders of sEH. Our optimized PROTAC selectively targets the degradation of cytosolic but not peroxisomal sEH, resulting in exquisite spatiotemporal control. Remarkably, our sEH PROTAC molecule has higher potency in cellular assays compared to the parent sEH inhibitor as measured by the significantly reduced ER stress. Interestingly, our mechanistic data indicate that our PROTAC directs the degradation of cytosolic sEH via the lysosome, not through the proteasome. The molecules presented here are useful chemical probes to study the biology of sEH with the potential for therapeutic development. Broadly, our results represent a proof of concept for the superior cellular potency of sEH degradation over sEH enzymatic inhibition, as well as subcellular compartment-selective modulation of a protein by PROTACs.

Diversity oriented clicking delivers ß-substituted alkenyl sulfonyl fluorides as covalent human neutrophil elastase inhibitors.

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.

Sialic acid diversity in the human gut: Molecular impacts and tools for future discovery.

Sialic acids are a family of structurally related sugars that are prevalent in mucosal surfaces, including the human intestine. In the gut, sialic acids have diverse biological roles at the interface of the host epithelium and the microbiota. N-acetylneuraminic acid (Neu5Ac), the best studied sialic acid, is a nutrient source for bacteria and, when displayed on the cell surface, a binding site for host immune factors, viruses, and bacterial toxins. Neu5Ac is extensively modified by host and microbial enzymes, and the impacts of Neu5Ac derivatives on host-microbe interactions, and generally on human and microbial biology, remain underexplored. In this mini-review, we highlight recent reports describing how host and microbial proteins differentiate Neu5Ac and its derivatives, draw attention to gaps in knowledge related to sialic acid biology, and suggest cutting-edge methodologies that may expand our appreciation and understanding of Neu5Ac in health and disease.

The Development of the Bengamides as New Antibiotics against Drug-Resistant Bacteria.

The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.

Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis.

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified ("dark peptidome") by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers.

Elevated mitochondrial reactive oxygen species (mROS) and an increase in caspase-3 activity are established mechanisms that lead to skeletal muscle atrophy via the upregulation of protein degradation pathways. However, the mechanisms upstream of an increase in mROS and caspase-3 activity in conditions of muscle atrophy have not been identified. Based upon knowledge that an event known as mitochondrial permeability transition (MPT) causes an increase in mROS emission and the activation of caspase-3 via mitochondrial release of cytochrome c, as well as the circumstantial evidence for MPT in some muscle atrophy conditions, we tested MPT as a mechanism of atrophy. Briefly, treating cultured single mouse flexor digitorum brevis (FDB) fibers from adult mice with a chemical inducer of MPT (Bz423) for 24 h caused an increase in mROS and caspase-3 activity that was accompanied by a reduction in muscle fiber diameter that was able to be prevented by inhibitors of MPT, mROS, or caspase-3 (p < 0.05). Similarly, a four-day single fiber culture as a model of disuse caused atrophy that could be prevented by inhibitors of MPT, mROS, or activated caspase-3. As such, our results identify MPT as a novel mechanism of skeletal muscle atrophy that operates through mROS emission and caspase-3 activation.

Metabolomics activity screening of T cell-induced colitis reveals anti-inflammatory metabolites.

Untargeted metabolomics of disease-associated intestinal microbiota can detect quantitative changes in metabolite profiles and complement other methodologies to reveal the full effect of intestinal dysbiosis. Here, we used the T cell transfer mouse model of colitis to identify small-molecule metabolites with altered abundance due to intestinal inflammation. We applied untargeted metabolomics to detect metabolite signatures in cecal, colonic, and fecal samples from healthy and colitic mice and to uncover differences that would aid in the identification of colitis-associated metabolic processes. We provided an unbiased spatial survey of the GI tract for small molecules, and we identified the likely source of metabolites and biotransformations. Several prioritized metabolites that we detected as being altered in colitis were evaluated for their ability to induce inflammatory signaling in cultured macrophages, such as NF-κB signaling and the expression of cytokines and chemokines upon LPS stimulation. Multiple previously uncharacterized anti-inflammatory and inflammation-augmenting metabolites were thus identified, with phytosphingosine showing the most effective anti-inflammatory activity in vitro. We further demonstrated that oral administration of phytosphingosine decreased inflammation in a mouse model of colitis induced by the compound TNBS. The collection of distinct metabolites we identified and characterized, many of which have not been previously associated with colitis, may offer new biological insight into IBD-associated inflammation and disease pathogenesis.

Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions.

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.

Chemical Inhibition of ENL/AF9 YEATS Domains in Acute Leukemia.

Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.

A photoaffinity probe that targets folate-binding proteins.

Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.

Identification of an N-acetylneuraminic acid-presenting bacteria isolated from a human microbiome.

N-Acetylneuraminic acid is the most abundant sialic acid (SA) in humans and is expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacteria harvest and display host SA on their own surfaces to evade Siglec-mediated host immunity. While previous studies have identified bacterial enzymes associated with SA catabolism, no reported methods permit the selective labeling, tracking, and quantitation of SA-presenting microbes within complex multi-microbial systems. We combined metabolic labeling, click chemistry, 16S rRNA gene, and whole-genome sequencing to track and identify SA-presenting microbes from a cultured human fecal microbiome. We isolated a new strain of Escherichia coli that incorporates SA onto its own surface and encodes for the nanT, neuA, and neuS genes necessary for harvesting and presenting SA. Our method is applicable to the identification of SA-presenting bacteria from human, animal, and environmental microbiomes, as well as providing an entry point for the investigation of surface-expressed SA-associated structures.

Metaproteomics Analysis of SARS-CoV-2-Infected Patient Samples Reveals Presence of Potential Coinfecting Microorganisms.

In this Letter, we reanalyze published mass spectrometry data sets of clinical samples with a focus on determining the coinfection status of individuals infected with SARS-CoV-2 coronavirus. We demonstrate the use of ComPIL 2.0 software along with a metaproteomics workflow within the Galaxy platform to detect cohabitating potential pathogens in COVID-19 patients using mass spectrometry-based analysis. From a sample collected from gargling solutions, we detected Streptococcus pneumoniae (opportunistic and multidrug-resistant pathogen) and Lactobacillus rhamnosus (a probiotic component) along with SARS-Cov-2. We could also detect Pseudomonas sps. Bc-h from COVID-19 positive samples and Acinetobacter ursingii and Pseudomonas monteilii from COVID-19 negative samples collected from oro- and nasopharyngeal samples. We believe that the early detection and characterization of coinfections by using metaproteomics from COVID-19 patients will potentially impact the diagnosis and treatment of patients affected by SARS-CoV-2 infection.

Correction to "Synthetic Elaboration of Native DNA by RASS (SENDR)".

[This corrects the article DOI: 10.1021/acscentsci.0c00680.].

Synthetic Elaboration of Native DNA by RASS (SENDR).

Controlled site-specific bioconjugation through chemical methods to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this conjugation through the tactical combination of two recently developed technologies: one for the manipulation of DNA in organic media and another for the chemoselective labeling of alcohols. Reversible adsorption of solid support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent reaction with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3' or 5' alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs.

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen.

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes Cysteine Protease SpeB in Evasion of Host Complement Defenses.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 µM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.