Exploring the targeting spectrum of rocaglates among eIF4A homologs.

Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates-two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3-a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA transcripts. We find that rocaglates can also interact with eIF4A3 to induce RNA clamping. Rocaglates also inhibit EJC-dependent NMD in mammalian cells, but this does not appear to be due to induced eIF4A3-RNA clamping, but rather a secondary consequence of translation inhibition incurred by clamping eIF4A1 and eIF4A2 to mRNA.

Synthesis of Neocannabinoids Using Controlled Friedel-Crafts Reactions.

A one-step transformation to produce 8,9-dihydrocannabidiol (H2CBD) and related "neocannabinoids" via controlled Friedel-Crafts reactions is reported. Experimental and computational studies probing the mechanism of neocannabinoid synthesis from cyclic allylic alcohol and substituted resorcinol reaction partners provide understanding of the kinetic and thermodynamic factors driving regioselectivity for the reaction. Herein, we present the reaction scope for neocannabinoid synthesis including the production of both normal and abnormal isomers under both kinetic and thermodynamic control. Discovery and optimization of this one-step protocol between various allylic alcohols and resorcinol derivatives are discussed and supported with density functional theory calculations.

Identification of Small Molecules with Improved Potency against Orthopoxviruses from Vaccinia to Smallpox.

The genus Orthopoxvirus contains several human pathogens, including vaccinia, monkeypox, cowpox, and variola virus, the causative agent of smallpox. Although there are a few effective vaccines, widespread prophylactic vaccination has ceased and is unlikely to resume, making therapeutics increasingly important to treat poxvirus disease. Here, we described efforts to improve the potency of the anti-poxvirus small molecule CMLDBU6128. This class of small molecules, referred to as pyridopyrimidinones (PDPMs), showed a wide range of biological activities. Through the synthesis and testing of several exploratory chemical libraries based on this molecule, we identified several compounds that had increased potency from the micromolar into the nanomolar range. Two compounds, designated (12) and (16), showed inhibitory concentrations of 326 nM and 101 nM, respectively, which was more than a 10-fold increase in potency to CMLDBU6128 with an inhibitory concentration of around 6 µM. We also expanded our investigation of the breadth of action of these molecules and showed that they can inhibit the replication of variola virus, a related orthopoxvirus. Together, these findings highlighted the promise of this new class of antipoxviral agents as broad-spectrum small molecules with significant potential to be developed as antiviral therapy. This would add a small molecule option for therapy of spreading diseases, including monkeypox and cowpox viruses, that would also be expected to have efficacy against smallpox.

Divergent, C-C Bond Forming Macrocyclizations Using Modular Sulfonylhydrazone and Derived Substrates.

A divergent approach to C-C bond forming macrocycle construction is described. Modular sulfonylhydrazone and derived pyridotriazole substrates with three key building blocks have been constructed and cyclized to afford diverse macrocyclic frameworks. Broad substrate scope and functional group tolerance have been demonstrated. In addition, site-selective postfunctionalization allowed for further diversification of macrocyclic cores.

Socioeconomic Disadvantage and Distance to Pediatric Critical Care.

OBJECTIVES: To describe the geography of pediatric critical care services and the relationship between poverty and distance to these services across the United States. DESIGN: Retrospective, cross-sectional study. SETTING: Contiguous United States. PATIENTS: Children less than 18 years as represented in the 2016 American Community Survey. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Pediatric critical care services were geographically concentrated within urban areas, with half of all PICUs located within 9.5 miles of another (interquartile range, 3.4-51.5 miles). Median distances from neighborhoods to the nearest unit increased linearly with Area Deprivation Index (p < 0.001), such that the median distance from the least privileged neighborhoods was nearly three times that of the most privileged neighborhoods (first decile = 7.8 miles [interquartile range, 3.4-15.8 miles] vs tenth decile = 22.6 miles [interquartile range, 4.2-52.5 miles]; p < 0.001). A relationship between neighborhood poverty and distance to a PICU was present across all U.S. regions and within urban/suburban and rural areas. CONCLUSIONS: In the United States, the distance to pediatric critical care services increases with poverty. This carries implications for access to care and health outcome disparities.

Rocaglates as dual-targeting agents for experimental cerebral malaria.

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

Chemical Synthesis Enables Structural Reengineering of Aglaroxin C Leading to Inhibition Bias for Hepatitis C Viral Infection.

As a unique rocaglate (flavagline) natural product, aglaroxin C displays intriguing biological activity by inhibiting hepatitis C viral entry. To further elucidate structure-activity relationships and diversify the pyrimidinone scaffold, we report a concise synthesis of aglaroxin C utilizing a highly regioselective pyrimidinone condensation. We have prepared more than 40 aglaroxin C analogues utilizing various amidine condensation partners. Through biological evaluation of analogues, we have discovered two lead compounds, CMLD012043 and CMLD012044, which show preferential bias for the inhibition of hepatitis C viral entry vs translation inhibition. Overall, the study demonstrates the power of chemical synthesis to produce natural product variants with both target inhibition bias and improved therapeutic indexes.

Intercepted Retro-Nazarov Reaction: Syntheses of Amidino-Rocaglate Derivatives and Their Biological Evaluation as eIF4A Inhibitors.

Rocaglates are a family of natural products isolated from the genus Aglaia which possess a highly substituted cyclopenta[b]benzofuran skeleton and inhibit cap-dependent protein synthesis. Rocaglates are attractive compounds due to their potential for inhibiting tumor cell maintenance in vivo by specifically targeting eukaryotic initiation factor 4A (eIF4A) and interfering with recruitment of ribosomes to mRNA. In this paper, we describe an intercepted retro-Nazarov reaction utilizing intramolecular tosyl migration to generate a reactive oxyallyl cation on the rocaglate skeleton. Trapping of the oxyallyl cation with a diverse range of nucleophiles has been used to generate over 50 novel amidino-rocaglate (ADR) and amino-rocaglate derivatives. Subsequently, these derivatives were evaluated for their ability to inhibit cap-dependent protein synthesis where they were found to outperform previous lead compounds including the rocaglate hydroxamate CR-1-31-B.

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. METHODS: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. RESULTS: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. CONCLUSIONS: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.

Diastereodivergent Synthesis of Chiral Tetrahydropyrrolodiazepinediones via a One-Pot Intramolecular aza-Michael/Lactamization Sequence.

A modular and diastereodivergent synthesis of tetrahydro-1 H-pyrrolo[1,2 d]diazepine-(2,5)-diones is presented. The tetrahydropyrrolodiazepinedione scaffold is obtained via a base-mediated three-step isomerization/tandem cyclization of amino acid-coupled homoallylic amino esters. Diastereoselectivity of the process is mediated by the interplay of a kinetic cyclization event and a propensity for thermodynamic epimerization at two labile chiral centers, giving rise to two distinct major diastereomers dependent on starting material stereochemistry and reaction conditions selected. Herein, we present a synthetic and computational study for this tandem process on a variety of amino ester substrates.

Total Syntheses of the Isomeric Aglain Natural Products Foveoglin†A and Perviridisin†B: Selective Excited-State Intramolecular Proton-Transfer Photocycloaddition.

Selective excited-state intramolecular proton-transfer (ESIPT) photocycloaddition of 3-hydroxyflavones with trans, trans-1,4-diphenyl-1,3-butadiene is described. Using this methodology, total syntheses of the natural products (±)-foveoglin A and (±)-perviridisin B were accomplished. Enantioselective ESIPT photocycloaddition using TADDOLs as chiral hydrogen-bonding additives provided access to (+)-foveoglin A. Mechanistic studies have revealed the possibility for a photoinduced electron-transfer (PET) pathway.

Asymmetric Dearomatization/Cyclization Enables Access to Polycyclic Chemotypes.

Enantioenriched, polycyclic compounds were obtained from a simple acylphloroglucinol scaffold. Highly enantioselective dearomatization was accomplished using a Trost ligand-palladium(0) complex. A computational DFT model was developed to rationalize observed enantioselectivities and revealed a key reactant-ligand hydrogen bonding interaction. Dearomatized products were used in visible light-mediated photocycloadditions and oxidative free radical cyclizations to obtain novel polycyclic chemotypes including tricyclo[4.3.1.01,4]decan-10-ones, bicyclo[3.2.1]octan-8-ones and highly-substituted cycloheptanones.

The effect of sediment mixing on mercury dynamics in two intertidal mudflats at Great Bay Estuary, New Hampshire, USA.

Estuarine sediments store particulate contaminants including mercury (Hg). We studied Hg sediment dynamics in two intertidal mudflats at Great Bay estuary, NH, over multiple years. Sediments at both mudflats were physically mixed down to ~10 cm, as determined by 7Be measurements, albeit via different mechanisms. Portsmouth mudflat (PT) sediments were subject to bioturbation by infaunal organisms and Squamscott mudflat (SQ) sediments were subject to erosion and redeposition. The presence of higher concentrations of fresh Fe(III) hydroxide at PT suggested bioirrigation by the polychaetes (Nereis virens). At depths where infaunal bioirrigation was observed, pore-water inorganic Hg (Hgi) and methylmercury (MeHg) were lower potentially due to their interaction with Fe(III) hydroxide. Methylmercury concentrations increased immediately below this zone in some samples, suggesting that the observed increase in material flux in bioirrigated sediments may initiate from lower depths. Pore water in sediment at PT also had higher fractions of more protein-like and labile DOC than those at SQ that can lead to increased MeHg production in PT, especially at depths where Hgi is not removed from solution by Fe(III) hydroxide. Where sediment erosion and redeposition were observed at SQ, Hg species distribution was extended deeper into the sediment column. Moreover, methyl coenzyme M reductase (MCR) and mercury reductase (mer-A) genes were higher at SQ than PT suggesting differences in conditions for Hg cycling. Results showed that the near-surface region of high MeHg concentrations commonly observed in unmixed sediments does not exist in physically mixed sediments that are common in many estuarine environments.

Discovery of new antimalarial chemotypes through chemical methodology and library development.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Single Cell Profiling Distinguishes Leukemia-Selective Chemotypes.

A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.

A chemical screen identifies structurally diverse metal chelators with activity against the fungal pathogen Candida albicans.

Candida albicans, one of the most prevalent human fungal pathogens, causes diverse diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance toward the drugs and the development of resistance mechanisms such as alterations of the drug's target, modulation of stress responses, and overexpression of efflux pumps. Thus, the need to identify novel antifungal strategies is dire. To address this challenge, we screened 3,049 structurally-diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library against a C. albicans clinical isolate and identified 17 molecules that inhibited C. albicans growth by >80% relative to controls. Among the most potent compounds were CMLD013360, CMLD012661, and CMLD012693, molecules representing two distinct chemical scaffolds, including 3-hydroxyquinolinones and a xanthone natural product. Based on structural insights, CMLD013360, CMLD012661, and CMLD012693 were hypothesized to exert antifungal activity through metal chelation. Follow-up investigations revealed all three compounds exerted antifungal activity against non-albicans Candida, including Candida auris and Candida glabrata, with the xanthone natural product CMLD013360 also displaying activity against the pathogenic mould Aspergillus fumigatus. Media supplementation with metallonutrients, namely ferric or ferrous iron, rescued C. albicans growth, confirming these compounds act as metal chelators. Thus, this work identifies and characterizes two chemical scaffolds that chelate iron to inhibit the growth of the clinically relevant fungal pathogen C. albicansIMPORTANCEThe worldwide incidence of invasive fungal infections is increasing at an alarming rate. Systemic candidiasis caused by the opportunistic pathogen Candida albicans is the most common cause of life-threatening fungal infection. However, due to the limited number of antifungal drug classes available and the rise of antifungal resistance, an urgent need exists for the identification of novel treatments. By screening a compound collection from the Boston University Center for Molecular Discovery (BU-CMD), we identified three compounds representing two distinct chemical scaffolds that displayed activity against C. albicans. Follow-up analyses confirmed these molecules were also active against other pathogenic fungal species including Candida auris and Aspergillus fumigatus. Finally, we determined that these compounds inhibit the growth of C. albicans in culture through iron chelation. Overall, this observation describes two novel chemical scaffolds with antifungal activity against diverse fungal pathogens.

The MYCN 5' UTR as a therapeutic target in neuroblastoma.

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

Amidino-rocaglates (ADRs), a class of synthetic rocaglates, are potent inhibitors of SARS-CoV-2 replication through inhibition of viral protein synthesis.

Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR).

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (eFT226), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.