Inhibition of Radix Scutellariae flavones on carboxylesterase mediated activations of prodrugs.

AIMS: Carboxylesterase (CES) plays an essential role in the hydrolysis of ester prodrugs. Our study explored the inhibitions of Radix Scutellariae flavones, including baicalein (B), baicalin (BG), wogonin (W), wogonoside (WG), oroxylin A (OXA) and oroxylin A-7-O-glucuronide (OAG), on CES-mediated hydrolysis of seven prodrugs (capecitabine, clopidogrel, mycophenolate mofetil, dabigatran etexilate, acetylsalicylic acid, prasugrel and irinotecan). MAIN METHODS: In vitro screenings were developed by incubating the flavones with prodrugs in rat plasma, intestine S9 and liver S9. Docking simulations were conducted using AMDock v1.5.2. In vivo evaluations were performed in rats co-administered with the selected flavone and prodrug via oral gavage/intravenous administration for five consecutive days. KEY FINDINGS: The in vitro investigation showed that B and OXA demonstrated strongest inhibitions on the hydrolysis of irinotecan followed by dabigatran in rat plasma, intestine S9 and liver S9. Consistent results showed in the molecular docking analyses. Additionally, in rats receiving irinotecan, B/OXA intravenous and oral pre-treatments both led to reduction trends on the active metabolite SN-38 formation in plasma. Besides, significant decreases of SN-38/irinotecan plasma concentration ratios were found in the B/OXA oral pre-treatment group with quicker and stronger inhibition potential in OXA pre-treatment than that from B pre-treatment. OXA oral pre-treatment was also found to be able to significantly inhibit intestinal CES2 activities at 0.5 h and 5 h after irinotecan administration. SIGNIFICANCE: Our current findings for the first time alert on potential CES-mediated HDIs between RS flavones and prodrugs, which provide a constructive information referring to rational drug combinations in clinical practice.

Optochemical Control of mTOR Signaling and mTOR-Dependent Autophagy.

As an important regulator of cell metabolism, proliferation, and survival, mTOR (mammalian target of rapamycin) signaling provides both a potential target for cancer treatment and a research tool for investigation of cell metabolism. One inhibitor for both mTORC1 and mTORC2 pathways, OSI-027, exhibited robust anticancer efficacy but induced side effects. Herein, we designed a photoactivatable OSI-027 prodrug, which allowed the release of OSI-027 after light irradiation to inhibit the mTOR signaling pathway, triggering autophagy and leading to cell death. This photoactivatable prodrug can provide novel strategies for mTOR-targeting cancer therapy and act as a new tool for investigating mTOR signaling and its related biological processes.

Loss of TSC1/TSC2 sensitizes immune checkpoint blockade in non-small cell lung cancer.

Tuberous sclerosis complex subunit 1 (TSC1) and 2 (TSC2) are frequently mutated in non-small cell lung cancer (NSCLC), however, their effects on antitumor immunity remained unexplored. A CRISPR screening in murine KrasG12D/Trp53-/- (KP) model identified Tsc1 and Tsc2 as potent regulators of programmed cell death ligand 1 (Pd-l1) expression in vitro and sensitivity to anti-programmed cell death receptor 1 (PD-1) treatment in vivo. TSC1 or TSC2 knockout (KO) promoted the transcriptional and membrane expression of PD-L1 in cell lines. TSC2-deficient tumors manifested an inflamed microenvironment in patient samples and The Cancer Genome Atlas dataset. In syngeneic murine models, KP-Tsc2-KO tumors showed notable response to anti-PD-1 antibody treatment, but Tsc2-wild-type tumors did not. Patients with TSC1/TSC2-mutant NSCLC receiving immune checkpoint blockade (ICB) had increased durable clinical benefit and survival. Collectively, TSC1/TSC2 loss defines a distinct subtype of NSCLC characterized as inflamed tumor microenvironment and superior sensitivity to ICB.

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF2· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF2· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong "red-shifting" of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV-visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical "HC(O)·" equivalents enables first examples of C-C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally "edited" into biology.

Nucleic Acid-Based Technologies Targeting Coronaviruses.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.

Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain.

The molecular signatures of cells in the brain have been revealed in unprecedented detail, yet the ageing-associated genome-wide expression changes that may contribute to neurovascular dysfunction in neurodegenerative diseases remain elusive. Here, we report zonation-dependent transcriptomic changes in aged mouse brain endothelial cells (ECs), which prominently implicate altered immune/cytokine signaling in ECs of all vascular segments, and functional changes impacting the blood-brain barrier (BBB) and glucose/energy metabolism especially in capillary ECs (capECs). An overrepresentation of Alzheimer disease (AD) GWAS genes is evident among the human orthologs of the differentially expressed genes of aged capECs, while comparative analysis revealed a subset of concordantly downregulated, functionally important genes in human AD brains. Treatment with exenatide, a glucagon-like peptide-1 receptor agonist, strongly reverses aged mouse brain EC transcriptomic changes and BBB leakage, with associated attenuation of microglial priming. We thus revealed transcriptomic alterations underlying brain EC ageing that are complex yet pharmacologically reversible.

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Identification of the in vivo relevant dissolution media for the three active components in EGb 761 tablet for better correlation with their pharmacokinetics in healthy subjects.

Although EGb 761, the standardized dry extract of Ginkgo biloba leaves, exhibited numerous pharmacological activities and widely used in Asia, European and North America, the quality control of its dosage forms such as tablet mainly relies on monitoring the contents of the active marker components, namely quercetin, kaempferol, isorhamnetin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C. So far, the in vitro dissolution profiles of EGb761 tablet were barely used to monitor its quality and how these dissolution profiles correlate with their in vivo pharmacokinetics was not known. Thus, the present study was proposed aiming to 1) develop the in vitro-in vivo correlations (IVIVCs) for the marker components in EGb 761 tablet; 2) identify the in vivo relevant dissolution media for the marker components in EGb 761 tablet based on the established IVIVCs. The content analyses of the marker components in EGb 761 tablet was first carried out. Then, the dissolution profiles were further obtained using paddle method of United States Pharmacopeia for bilobalide, ginkgolides A, and ginkgolide B, that have previously reported human plasma pharmacokinetics after EGb 761 tablet oral administrations. About seven different media including 0.1 M hydrochloric acid (HCl), acetate buffer, H2O, fasted state simulated gastric fluid (FaSSGF), fasted state simulated intestinal fluid version 2 (FaSSIF-V2), fed state simulated intestinal fluid version 2 (FeSSIF-V2), and sequential medium (0.1 M HCl for 2 h with pH adjusted to 7 for another 2 h) were tested in the current investigation. The obtained in vitro dissolution profiles of bilobalide, ginkgolides A and ginkgolide B from EGb 761 tablet were first fitted with four dissolution models, namely Weibull, Double Weibull, Hill and Makoid-Banakar, to obtain the best-fit model for each component in each medium. The human plasma concentration versus time profiles of the above three components were then inputted into the Phoenix WinNonlin IVIVC Toolkit to obtain their in vivo absorption profiles using numerical deconvolution. The best-fit dissolution profiles of each marker component in the seven studied media were further used to correlate with its obtained in vivo absorption profile by the linear correlation models to establish the corresponding IVIVCs in each studied medium. Finally, the best in vivo correlated medium for each investigated marker component was selected based on their adjusted correlation coefficients, Akaike Information Criterion (AIC) and Schwarz's Bayesian Criterion (SBC) values. As a result, the dissolution profiles of bilobalide, ginkgolide A, ginkgolide B from EGb 761 tablet in 0.1 M HCl, FaSSGF, FaSSIF-V2 demonstrated the best correlation with their in vivo absorption profiles, respectively. Our current studies for the first time applied the concept of IVIVC to EGb 761 tablet and successfully identified the in vivo relevant dissolution media for its three active marker components to improve its quality control.

Epigenetic CRISPR Screens Identify Npm1 as a Therapeutic Vulnerability in Non-Small Cell Lung Cancer.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. SIGNIFICANCE: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

In Vivo Epigenetic CRISPR Screen Identifies Asf1a as an Immunotherapeutic Target in Kras-Mutant Lung Adenocarcinoma.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D/Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161.

Post-translational site-selective protein backbone α-deuteration.

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-1H → C-2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine.

Selective Radical Trifluoromethylation of Native Residues in Proteins.

The incorporation of fluorine can not only significantly facilitate the study of proteins but also potentially modulate their function. Though some biosynthetic methods allow global residue-replacement, post-translational fluorine incorporation would constitute a fast and efficient alternative. Here, we reveal a mild method for direct protein radical trifluoromethylation at native residues as a strategy for symmetric-multifluorine incorporation on mg scales with high recoveries. High selectivity toward tryptophan residues enhanced the utility of this direct trifluoromethylation technique allowing ready study of fluorinated protein constructs using 19F-NMR.

Concise and Stereodivergent Synthesis of Carbasugars Reveals Unexpected Structure-Activity Relationship (SAR) of SGLT2 Inhibition.

Carbasugar sodium-glucose cotransporter 2 (SGLT2) inhibitors are highly promising drug candidates for the treatment of Type 2 diabetes mellitus (T2DM). However, the clinical usage of carbasugar SGLT2 inhibitors has been underexplored, due to the lengthy synthetic routes and the lack of structure-activity relationship (SAR) studies of these compounds. Herein, we report a concise and stereodivergent synthetic route towards some novel carbasugar SGLT2 inhibitors, featuring an underexploited, regioselective, and stereospecific palladium-catalyzed allyl-aryl coupling reaction. This synthetic strategy, together with computational modeling, revealed the unexpected SAR of these carbasugar SGLT2 inhibitors, and enabled the discovery of a highly selective and potent SGLT2 inhibitor.

Chemical Probes for Visualizing Intact Animal and Human Brain Tissue.

Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain.

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity.

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)-C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cß-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

Palladium-Catalyzed Arylation of Carbasugars Enables the Discovery of Potent and Selective SGLT2 Inhibitors.

Selective inhibition of the transporter protein sodium-glucose cotransporter 2 (SGLT2) has emerged as a promising way to control blood glucose level in diabetes patients. Reported herein is a short and convergent synthetic route towards some small-molecule SGLT2 inhibitors by a chemo- and diastereospecific palladium-catalyzed arylation reaction. This synthetic strategy enabled the discovery of two highly selective and potent SGLT2 inhibitors, thereby paving the way towards the development of carbasugar SGLT2 inhibitors as potential antidiabetic/antitumor agents.

Rationalisation and Validation of an Acrylamide-Free Procedure in Three-Dimensional Histological Imaging.

Three-dimensional visualization of intact tissues is now being achieved by turning tissues transparent. CLARITY is a unique tissue clearing technique, which features the use of detergents to remove lipids from fixed tissues to achieve optical transparency. To preserve tissue integrity, an acrylamide-based hydrogel has been proposed to embed the tissue. In this study, we examined the rationale behind the use of acrylamide in CLARITY, and presented evidence to suggest that the omission of acrylamide-hydrogel embedding in CLARITY does not alter the preservation of tissue morphology and molecular information in fixed tissues. We therefore propose a novel and simplified workflow for formaldehyde-fixed tissue clearing, which will facilitate the laboratory implementation of this technique. Furthermore, we have investigated the basic tissue clearing process in detail and have highlighted some areas for targeted improvement of technologies essential for the emerging subject of three-dimensional histology.