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When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67-Mtr2 in the yeast Saccharomyces cerevisiae (TAP-p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.
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Transporte Activo de Núcleo Celular , Núcleo Celular , Regulación Fúngica de la Expresión Génica , Transporte de ARN , ARN sin Sentido , ARN Bicatenario , ARN Mensajero , Saccharomyces cerevisiae , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , ARN sin Sentido/metabolismo , ARN sin Sentido/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Humanos , Transporte Activo de Núcleo Celular , Células Endoteliales/metabolismo , Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Carioferinas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Ribonucleoproteínas/metabolismo , ARN Mensajero/metabolismo , Replicación ViralRESUMEN
Antibacterial resistance is one of the greatest threats to human health. The development of new therapeutics against bacterial pathogens has slowed drastically since the approvals of the first antibiotics in the early and mid-20th century. Most of the currently investigated drug leads are modifications of approved antibacterials, many of which are derived from natural products. In this review, we highlight the challenges, advancements and current standing of the clinical and preclinical antibacterial research pipeline. Additionally, we present novel strategies for rejuvenating the discovery process and advocate for renewed and enthusiastic investment in the antibacterial discovery pipeline.
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Productos Biológicos , Descubrimiento de Drogas , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias/genética , Farmacorresistencia MicrobianaRESUMEN
The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.
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Coenzimas , Klebsiella pneumoniae , Pseudomonas aeruginosa , Transferasas , Humanos , Antibacterianos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Transferasas/química , Transferasas/metabolismo , Conformación Proteica , Coenzimas/metabolismo , Vitamina B 6/biosíntesis , Tiamina/biosíntesis , Apoenzimas/química , Apoenzimas/metabolismo , Tiamina Pirofosfato/metabolismo , Dominio Catalítico , Farmacorresistencia BacterianaRESUMEN
Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act as nuclear retention factors for unprocessed pre-mRNAs. Here we show that the guard protein Npl3 monitors 5'-capping. In its absence, uncapped transcripts resist degradation, because the Rat1-Rai1 5'-end degradation factors are not efficiently recruited to these faulty transcripts. Importantly, in npl3Δ, these improperly capped transcripts escape this quality control checkpoint and leak into the cytoplasm. Our data suggest a model in which Npl3 associates with the Rai1 bound pre-mRNAs. In case the transcript was properly capped and is thus CBC (cap binding complex) bound, Rai1 dissociates from Npl3 allowing the export factor Mex67 to interact with this guard protein and support nuclear export. In case Npl3 does not detect proper capping through CBC attachment, Rai1 binding persists and Rat1 can join this 5'-complex to degrade the faulty transcript.
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Proteínas Nucleares , Caperuzas de ARN , Precursores del ARN , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Caperuzas de ARN/metabolismoRESUMEN
Energy-coupling factor transporters (ECFTs) are membrane-bound ATP-binding cassette (ABC) transporters in prokaryotes that are found in pathogens against which novel antibiotics are urgently needed. To date, just 54 inhibitors of three molecular-structural classes with mostly weak inhibitory activity are known. Target repurposing is a strategy that transfers knowledge gained from a well-studied protein family to under-studied targets of phylogenetic relation. Forty-eight human ABC transporters are known that may harbor structural motifs similar to ECFTs to which particularly multitarget compounds may bind. We assessed 31 multitarget compounds which together target the entire druggable human ABC transporter proteome against ECFTs, of which nine showed inhibitory activity (hit rate 29.0%) and four demonstrated moderate to strong inhibition of an ECFT (IC50 values between 4.28 and 50.2 µM) as well as antibacterial activity against ECFT-expressing Streptococcus pneumoniae. Here, ivermectin was the most potent candidate (MIC95: 22.8 µM), and analysis of five ivermectin derivatives revealed moxidectin as one of the most potent ECFT-targeting antibacterial agents (IC50: 2.23 µM; MIC95: 2.91 µM). Distinct molecular-structural features of avermectins and derivatives as well as the differential biological response of the hit compounds in general provided first indications with respect to the structure-activity relationships and mode of action, respectively.
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Designing novel candidates as potential antibacterial scaffolds has become crucial due to the lack of new antibiotics entering the market and the persistent rise in multidrug resistance. Here, we describe a new class of potent antibacterial agents based on a 5-aryl-N2,N4-dibutylpyrimidine-2,4-diamine scaffold. Structural optimization focused on the 5-aryl moiety and the bioisosteric replacement of the side chain linker atom. Screening of the synthesized compounds focused on a panel of bacterial strains, including gram-positive Staphylococcus aureus strains (Newman MSSA, methicillin- and vancomycin-resistant), and the gram-negative Escherichia coli (ΔAcrB strain). Several compounds showed broad-spectrum antibacterial activity with compound 12, bearing a 4-chlorophenyl substituent, being the most potent among this series of compounds. This frontrunner compound revealed a minimum inhibitory concentration (MIC) value of 1 µg/mL against the S. aureus strain (Mu50 methicillin-resistant S. aureus/vancomycin-intermediate S. aureus) and an MIC of 2 µg/mL against other tested strains. The most potent derivatives were further tested against a wider panel of bacteria and evaluated for their cytotoxicity, revealing further potent activities toward Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis. To explore the mode of action, compound 12 was tested in a macromolecule inhibition assay. The obtained data were supported by the safety profile of compound 12, which possessed an IC50 of 12.3 µg/mL against HepG2 cells. The current results hold good potential for a new class of extended-spectrum antibacterial agents.
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Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Antibacterianos/farmacología , Antibacterianos/química , Staphylococcus aureus , Relación Estructura-Actividad , Bacterias , Pirimidinas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
The authors would like to make the following corrections to the original publication [...].
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The shape and function of ethical imperatives may vary if the context is an interaction between strangers, or those who are well acquainted. This idea, taken up from Stephen Toulmin's distinction between an "ethics of strangers" and an "ethics of intimacy", can be applied to encounters in healthcare. There are situations where healthcare personnel (HCP) know their patients (corresponding to an "ethics of intimacy") and situations where HCP do not know their patients (corresponding to "an ethics of strangers"). Does it make a difference for normative imperatives that follow from central concepts and principles in medical ethics whether HCP know their patients or not? In our view, this question has not yet been answered satisfactorily. Once we have clarified what is meant by "knowing the patient", we will show that the distinction is particularly relevant with regard to some thorny questions of autonomy in healthcare (e.g., regarding advance directives or paternalism in the name of autonomy), whereas the differences with regard to imperatives following from the principles of justice and beneficence seem to be smaller. We provide a detailed argument for why knowing the patient is ethically valuable in encounters in healthcare. Consequently, healthcare systems should provide fertile ground for HCP to get to know their patients, and structures that foster therapeutic continuity. For this to succeed, a number of questions still need to be clarified, which is an important task for medical ethics.
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Autonomía Personal , Humanos , Filosofía Médica , Ética Clínica , Relaciones Médico-Paciente/ética , Beneficencia , Ética Médica , Justicia SocialRESUMEN
Deriving active pharmaceutical agents from renewable resources is crucial to increasing the economic feasibility of modern biorefineries and promises to alleviate critical supply-chain dependencies in pharma manufacturing. Our multidisciplinary approach combines research in lignin-first biorefining, sustainable catalysis, and alternative solvents with bioactivity screening, an in vivo efficacy study, and a structural-similarity search. The resulting sustainable path to novel anti-infective, anti-inflammatory, and anticancer molecules enabled the rapid identification of frontrunners for key therapeutic indications, including an anti-infective against the priority pathogen Streptococcus pneumoniae with efficacy in vivo and promising plasma and metabolic stability. Our catalytic methods provided straightforward access, inspired by the innate structural features of lignin, to synthetically challenging biologically active molecules with the core structure of dopamine, namely, tetrahydroisoquinolines, quinazolinones, 3-arylindoles and the natural product tetrahydropapaveroline. Our diverse array of atom-economic transformations produces only harmless side products and uses benign reaction media, such as tunable deep eutectic solvents for modulating reactivity in challenging cyclization steps.
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Descubrimiento de Drogas , Lignina , Lignina/química , Solventes/química , Catálisis , BiomasaRESUMEN
The natural product chlorotonil displays high potency against multidrug-resistant Gram-positive bacteria and Plasmodium falciparum. Yet, its scaffold is characterized by low solubility and oral bioavailability, but progress was recently made to enhance these properties. Applying late-stage functionalization, we aimed to further optimize the molecule. Previously unknown reactions including a sulfur-mediated dehalogenation were revealed. Dehalogenil, the product of this reaction, was identified as the most promising compound so far, as this new derivative displayed improved solubility and in vivo efficacy while retaining excellent antimicrobial activity. We confirmed superb activity against multidrug-resistant clinical isolates of Staphylococcus aureus and Enterococcus spp. and mature transmission stages of Plasmodium falciparum. We also demonstrated favorable in vivo toxicity, pharmacokinetics and efficacy in infection models with S. aureus. Taken together, these results identify dehalogenil as an advanced lead molecule.
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Antibacterianos , Staphylococcus aureus , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Plasmodium falciparum/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Animales , Enterococcus/efectos de los fármacos , Estructura Molecular , Humanos , RatonesRESUMEN
The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor.
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Metaloendopeptidasas , Factores de Virulencia , Humanos , Factores de Virulencia/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Pseudomonas aeruginosa/metabolismo , Escherichia coli/metabolismo , VirulenciaRESUMEN
Gene knockdown by siRNA offers an unrestricted choice of targets and specificity based on the principle of complementary Watson-Crick base pairing with mRNA. However, the negative charge, large molecular size, and susceptibility to enzymatic degradation of siRNA impede its successful transfection, hence limiting its potential for therapeutic use. The development of efficient and safe siRNA transfection agents is, therefore, critical for siRNA-based therapy. Herein, we developed a protein-based biodynamic polymer (biodynamer) that showed potential as a siRNA transfection vector, owing to its excellent biocompatibility, easy tunability, and dynamic polymerization under acidic environments. The positively charged biodynamers formed stable dynamic nanocomplexes (XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNA via electrostatic interactions and chemical cross-linking. As a proof of concept, the optimized XL-DPs were stable in physiological conditions with serum proteins and demonstrated significant pH-dependent size change and degradability, as well as siRNA release capability. The minimal cytotoxicity and excellent cellular uptake of XL-DPs effectively supported the intracellular delivery of siRNA. Our study demonstrated that the XL-DPs in survivin siRNA delivery enabled potent knockdown of survivin mRNA and induced notable apoptosis of carcinoma cells (2.2 times higher than a lipid-based transfection agent, Lipofectamine 2000). These findings suggested that our XL-DPs hold immense potential as a promising platform for siRNA delivery and can be considered strong candidates in the advancement of next-generation transfection agents.
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Apoptosis , Survivin/genética , ARN Interferente Pequeño , Transfección , Concentración de Iones de Hidrógeno , ARN Mensajero , Línea Celular TumoralRESUMEN
Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.
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Tiamina Pirofosfato , Tiamina , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismo , Tiamina/farmacología , Tiamina/química , Especificidad por Sustrato , Coenzimas/química , BiocatálisisRESUMEN
In order to develop novel inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the UDP binding site of the enzyme, a series of aldotetronic acid-based hydroxamic acids was accessed in chiral pool syntheses starting from 4,6-O-benzylidene-d-glucose and l-arabinitol. The synthesized hydroxamic acids were tested for LpxC inhibitory activity in vitro, revealing benzyl ether 17a ((2S,3S)-4-(benzyloxy)-N,3-dihydroxy-2-[(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)oxy]butanamide) as the most potent LpxC inhibitor. This compound was additionally tested for antibacterial activity against a panel of clinically relevant Gram-negative bacteria, bacterial uptake, and susceptibility to efflux pumps. Molecular docking studies were performed to rationalize the observed structure-activity relationships.
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Amidohidrolasas , Antibacterianos , Inhibidores Enzimáticos , Escherichia coli , Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/química , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
The Sonata System is a minimally invasive, ultrasound-guided transcervical fibroid ablation procedure for the management of symptomatic uterine fibroids. Since its approval by the US Food and Drug Administration in 2018, this procedure has demonstrated an excellent safety profile and postprocedure satisfaction rate. We present the case of a patient treated with Sonata, who subsequently developed bacterial sepsis and Asherman's syndrome-serious complications with long-term sequelae and implications for fertility. A nulligravid woman in her 40s presented in the outpatient setting with dysmenorrhea and bulk symptoms, with imaging showing an enlarged myomatous uterus compressing the urinary bladder. She desired minimally invasive, fertility-preserving management and underwent the Sonata procedure at an outside hospital. On postoperative day 3, she was admitted to our institution with abdominal pain, fever, tachycardia, and Enterococcus faecalis bacteremia. Despite 6 days of culture-directed antibiotic therapy, the patient remained septic with worsening symptoms and imaging findings and with persistent bacteremia. On hospital day 7, the patient underwent laparoscopic myomectomy and excision of hemorrhagic, infected myometrium. She recovered appropriately after surgery and was discharged home on hospital day 11 to continue 2 weeks of intravenous antibiotics. Nine months after myomectomy, the patient was diagnosed as having Asherman's syndrome. She subsequently had an early pregnancy loss with retained products of conception, requiring hysteroscopic lysis of adhesions and dilation and curettage. Ultimately, careful patient selection is critical for the optimal application of the Sonata procedure. Limiting the extent of fibroid necrosis after treatment is a reasonable goal to minimize the risk of secondary bacterial infection and adhesiogenesis as procedural sequelae.
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Bacteriemia , Ginatresia , Leiomioma , Sepsis , Embarazo , Femenino , Humanos , Ginatresia/cirugía , Leiomioma/complicaciones , Leiomioma/cirugía , Sepsis/complicaciones , Bacteriemia/complicacionesRESUMEN
Staphylococcus aureus causes a wide range of infections, and it is one of the leading pathogens responsible for deaths associated with antimicrobial resistance, the rapid spread of which among S. aureus urges the discovery of new antibiotics. The evaluation of in vivo efficacy of novel drug candidates is usually performed using animal models. Recently, zebrafish (Danio rerio) embryos have become increasingly attractive in early drug discovery. Herein, we established a zebrafish embryo model of S. aureus infection for evaluation of in vivo efficacy of novel potential antimicrobials. A local infection was induced by microinjecting mCherry-expressing S. aureus Newman followed by treatment with reference antibiotics via microinjection into different injection sites as well as via waterborne exposure to study the impact of the administration route on efficacy. We successfully used the developed model to evaluate the in vivo activity of the natural product sorangicin A, for which common mouse models were not successful due to fast degradation in plasma. In conclusion, we present a novel screening platform for assessing in vivo activity at the antibiotic discovery stage. Furthermore, this work provides consideration for the choice of an appropriate administration route based on the physicochemical properties of tested drugs.
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Productos Biológicos , Infecciones Estafilocócicas , Animales , Ratones , Staphylococcus aureus , Pez Cebra , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Modelos Animales de Enfermedad , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion.
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We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H2O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O-H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H2O and stigmasterol·H2O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air-water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H2O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H2O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H2O form.
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Colesterol , Agua , Colesterol/química , Cristalización , Agua/químicaRESUMEN
Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available. We aimed to discover new and chemically diverse candidates to enrich the hRSV drug development pipeline. We used a two-step screen that interrogates compound efficacy after primary infection and a consecutive virus passaging. We resynthesized selected hit molecules and profiled their activities with hRSV lentiviral pseudotype cell entry, replicon, and time-of-addition assays. The breadth of antiviral activity was tested against recent RSV clinical strains and human coronavirus (hCoV-229E), and in pseudotype-based entry assays with non-RSV viruses. Screening 6,048 molecules, we identified 23 primary candidates, of which 13 preferentially scored in the first and 10 in the second rounds of infection, respectively. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses, including hCoV-229E. One compound, identified in the second round of infection, did not measurably inhibit hRSV cell entry or replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated BCi-NS1.1 lung cells. In conclusion, we identified four new hRSV inhibitor candidates with different modes of action. Our findings build an interesting platform for medicinal chemistry-guided derivatization approaches followed by deeper phenotypical characterization in vitro and in vivo with the aim of developing highly potent hRSV drugs.