Discovery of potent small-molecule inhibitors of lipoprotein(a) formation.

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).

Vital Pulp Therapy in Permanent Teeth Diagnosed with Symptomatic Irreversible Pulpitis: Reports with Long-Term Controls.

The aim of this study was to describe the treatment of permanent teeth diagnosed with irreversible pulpitis, which can be effectively managed with partial or total pulpotomy. This alternative approach has shown great clinical and radiographic success in the long term compared to traditional pulpectomies. In this series of clinical cases, all the teeth exhibited symptoms of intense pain upon exposure to cold and at night. The clinical examination revealed extensive caries, while radiographic imaging showed radiolucent lesions in contact with the pulp chamber, indicating symptomatic irreversible pulpitis. The chosen treatment approach was either partial or total pulpotomy. The tooth was anesthetized, and the operative field was isolated and disinfected. After removing caries with a sterile round drill, the area was rinsed with sodium hypochlorite. In some cases, a portion of the pulp tissue was removed, while in others, the entire tissue of the pulp chamber was extracted using diamond burs. Hemostasis was achieved by applying sterile cotton pellets for 2 to 6 minutes. Following that, the tissue exhibited no signs of bleeding. Bioceramic cements were used, and the tooth was definitively restored. Periodic follow-up examinations were conducted, consistently showing positive pulp responses and no evidence of periradicular radiolucent lesions on radiographs.

In Silico Searching for Alternative Lead Compounds to Treat Type 2 Diabetes through a QSAR and Molecular Dynamics Study.

Free fatty acid receptor 1 (FFA1) stimulates insulin secretion in pancreatic ß-cells. An advantage of therapies that target FFA1 is their reduced risk of hypoglycemia relative to common type 2 diabetes treatments. In this work, quantitative structure-activity relationship (QSAR) approach was used to construct models to identify possible FFA1 agonists by applying four different machine-learning algorithms. The best model (M2) meets the Tropsha's test requirements and has the statistics parameters R2 = 0.843, Q2CV = 0.785, and Q2ext = 0.855. Also, coverage of 100% of the test set based on the applicability domain analysis was obtained. Furthermore, a deep analysis based on the ADME predictions, molecular docking, and molecular dynamics simulations was performed. The lipophilicity and the residue interactions were used as relevant criteria for selecting a candidate from the screening of the DiaNat and DrugBank databases. Finally, the FDA-approved drugs bilastine, bromfenac, and fenofibric acid are suggested as potential and lead FFA1 agonists.

Prognosis Stratification Tools in Early-Stage Endometrial Cancer: Could We Improve Their Accuracy?

There are three prognostic stratification tools used for endometrial cancer: ESMO-ESGO-ESTRO 2016, ProMisE, and ESGO-ESTRO-ESP 2020. However, these methods are not sufficiently accurate to address prognosis. The aim of this study was to investigate whether the integration of molecular classification and other biomarkers could be used to improve the prognosis stratification in early-stage endometrial cancer. Relapse-free and overall survival of each classifier were analyzed, and the c-index was employed to assess accuracy. Other biomarkers were explored to improve the precision of risk classifiers. We analyzed 293 patients. A comparison between the three classifiers showed an improved accuracy in ESGO-ESTRO-ESP 2020 when RFS was evaluated (c-index = 0.78), although we did not find broad differences between intermediate prognostic groups. Prognosis of these patients was better stratified with the incorporation of CTNNB1 status to the 2020 classifier (c-index 0.81), with statistically significant and clinically relevant differences in 5-year RFS: 93.9% for low risk, 79.1% for intermediate merged group/CTNNB1 wild type, and 42.7% for high risk (including patients with CTNNB1 mutation). The incorporation of molecular classification in risk stratification resulted in better discriminatory capability, which could be improved even further with the addition of CTNNB1 mutational evaluation.

Prognostic implications of tumor-infiltrating T cells in early-stage endometrial cancer.

Patients with endometrial cancer differ in terms of the extent of T-cell infiltration; however, the association between T-cell subpopulations and patient outcomes remains unexplored. We characterized 285 early-stage endometrial carcinoma samples for T-cell infiltrates in a tissue microarray format using multiplex fluorescent immunohistochemistry. The proportion of T cells and their subpopulations were associated with clinicopathological features and relapse-free survival outcomes. CD3+ CD4+ infiltrates were more abundant in the patients with higher grade or non-endometrioid histology. Cytotoxic T cells (CD25+, PD-1+, and PD-L1+) were strongly associated with longer relapse-free survival. Moreover, CD3+ PD-1+ stromal cells were independent of other immune T-cell populations and clinicopathological factors in predicting relapses. Patients with high stromal T-cell fraction of CD3+ PD-1+ cells were associated with a 5-year relapse-free survival rate of 93.7% compared to 79.0% in patients with low CD3+ PD-1+ fraction. Moreover, in patients classically linked to a favorable outcome (such as endometrioid subtype and low-grade tumors), the stromal CD3+ PD-1+ T-cell fraction remained prognostically significant. This study supports that T-cell infiltrates play a significant prognostic role in early-stage endometrial carcinoma. Specifically, CD3+ PD-1+ stromal cells emerge as a promising novel prognostic biomarker.

Tularemia Outbreaks in Spain from 2007 to 2020 in Humans and Domestic and Wild Animals.

In this study, tularemia outbreaks associated with humans and several domestic and wild animals (Iberian hares, wild rabbits, voles, mice, grey shrews, sheep, dogs, foxes, wolves, ticks, and river crayfish) are reported in Spain from 2007 to 2020. Special attention was paid to the outbreaks in humans in 2007-2009 and 2014-2015, when the most important waves occurred. Moreover, positive rates of tularemia in lagomorphs were detected in 2007-2010, followed by negative results in 2011-2013, before again returning to positive rates in 2014 and in 2017 and in 2019-2020. Lagomorphs role in spreading Francisella tularensis in the epidemiological chain could not be discarded. F. tularensis is described for the first time infecting the shrew Crocidura russula worldwide, and it is also reported for the first time infecting wild rabbits (Oryctolagus cuniculus) in Spain. Serological positives higher than 0.4% were seen for sheep only from 2007-2009 and again in 2019, while serological rates greater than 1% were revealed in dogs in 2007-2008 and in wild canids in 2016. F. tularensis were detected in ticks in 2009, 2014-2015, 2017, and 2019. Lastly, negative results were achieved for river crayfish and also in environmental water samples from 2007 to 2020.

Modeling the Antileukemia Activity of Ellipticine-Related Compounds: QSAR and Molecular Docking Study.

The antileukemia cancer activity of organic compounds analogous to ellipticine representes a critical endpoint in the understanding of this dramatic disease. A molecular modeling simulation on a dataset of 23 compounds, all of which comply with Lipinski's rules and have a structure analogous to ellipticine, was performed using the quantitative structure activity relationship (QSAR) technique, followed by a detailed docking study on three different proteins significantly involved in this disease (PDB IDs: SYK, PI3K and BTK). As a result, a model with only four descriptors (HOMO, softness, AC1RABAMBID, and TS1KFABMID) was found to be robust enough for prediction of the antileukemia activity of the compounds studied in this work, with an R2 of 0.899 and Q2 of 0.730. A favorable interaction between the compounds and their target proteins was found in all cases; in particular, compounds 9 and 22 showed high activity and binding free energy values of around -10 kcal/mol. Theses compounds were evaluated in detail based on their molecular structure, and some modifications are suggested herein to enhance their biological activity. In particular, compounds 22_1, 22_2, 9_1, and 9_2 are indicated as possible new, potent ellipticine derivatives to be synthesized and biologically tested.

Role of the sugar moiety on the opioid receptor binding and conformation of a series of enkephalin neoglycopeptides.

Glycosylation by simple sugars is a drug discovery alternative that has been explored with varying success for enhancing the potency and bioavailability of opioid peptides. Long ago we described two O-glycosides having either ß-Glucose and ß-Galactose of (d-Met2, Pro5)-enkephalinamide showing one of the highest antinociceptive activities known. Here, we report the resynthesis of these two analogs and the preparation of three novel neoglycopeptide derivatives (α-Mannose, ß-Lactose and ß-Cellobiose). Binding studies to cloned zebrafish opioid receptors showed very small differences of affinity between the parent compound and the five glycopeptides thus suggesting that the nature of the carbohydrate moiety plays a minor role in determining the binding mode. Indeed, NMR conformational studies, combined with molecular mechanics calculations, indicated that all glycopeptides present the same major conformation either in solution or membrane-like environment. The evidences provided here highlight the relevance for in vivo activity of the conjugating bond between the peptide and sugar moieties in opioid glycopeptides.

Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to 1H CPMG Ligand-Observed NMR.

To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods' hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the 19F chemical shift to 1H in 1D-protein observed 1H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed 1H NMR may also be effective; however, the 19F chemical shift remains significantly more responsive.

[Psychometric properties of an instrument to evaluate the nurse attitude towards good practice guides]. / Propiedades psicométricas de un instrumento PARA EVALUAR LA ACTITUD, ENFERMERA HACIA LAS GUÍAS DE BUENAS PRÁCTICAS.

The Guides of Good Practices (GGP) are necessary tools in the universal healthcare and in the clinical management, providing the user/patient with a major quality in the assistance, in order to optimize and reinforce an individualized attention into action, taking into account the best scientific evidence. The literature provides different references to the development of the GGP, but there is little knowledge about the attitude of professionals towards them, since most of the studies that exist are qualitative. Therefore, the aim of this work is to construct and validate a Likert scale which could assess the attitude of the nurse towards GGP. The methodology used was quantitative, descriptive, cross, opinion, anonymous and also it could validate a scale via the following measurements: content validation by experts, correlation between items, external reliability, internal consistency, stability and exploratory factor analysis. The result was a scale consisting of 20 items that refer to the attitude toward the GGP, with a percentage of agreement among experts over 75 % on all the items, and a significant Pearson correlation between the pre-test and post-test in all variables, but for three. The internal consistency measured by Cronbach's alpha was 0.878. These results are acceptable in terms of the psychometric characteristics of the instrument, with easy and fast administration and simple in their interpretation, allowing quantifying and generating knowledge about the attitudes of nurses towards GGP.

Influence of polar side chains modifications on the dual enkephalinase inhibitory activity and conformation of human opiorphin, a pain perception related peptide.

The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study involving minor modifications on the polar side chains of Arg residues and glycosylation with monosaccharides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin. However, the correlations deduced offer further proof for the key role of these residues upon the binding and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopeptides suggest that they are still very flexible compounds that may attain their respective bioactive conformations.

Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly.

Escherichia coli ß-galactosidase inhibitors through modifications at the aglyconic moiety: experimental evidence of conformational distortion in the molecular recognition process.

Herein, we describe the use of thioglycosides as glycosidase inhibitors by employing novel modifications at the reducing end of these glycomimetics. The inhibitors display a basic galactopyranosyl unit (1→4)-bonded to a 3-deoxy-4-thiopentopyranose moiety. The molecular basis of the observed inhibition has been studied by using a combination of NMR spectroscopy and molecular modeling techniques. It is demonstrated that these molecules are not recognized by Escherichia coli ß-galactosidase in their ground-state conformation, with a conformational selection process taking place. In fact, the observed conformational distortion depends on the chemical nature of the compounds and results from the rotation around the glycosidic linkage (variation of Φ or Ψ) or from the deformation of the six-membered ring of the pentopyranose. The bound conformations of the ligand are adapted in the enzymatic pocket with a variety of hydrogen-bond, van der Waals, and stacking interactions.

Molecular recognition of the Thomsen-Friedenreich antigen-threonine conjugate by adhesion/growth regulatory galectin-3: nuclear magnetic resonance studies and molecular dynamics simulations.

Nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods have been strategically combined to elucidate the molecular recognition features of the binding of threonine O-linked Thomsen-Friedenreich (TF) antigen to chimera-type avian galectin-3 (CG-3). Saturation transfer difference (STD) NMR experiments revealed the highest intensities for the H4 protons of both the ß-D-Galp and α-D-GalpNAc moieties, with 100 and 71% of relative STD, respectively. The methyl protons of the threonine residue exhibited a small STD effect, <15%, indicating that the interaction of the amino acid with the protein is rather transient. Two-dimensional transferred nuclear Overhauser effect spectroscopy NMR experiments and molecular modeling suggested some differences in conformer populations between the free and bound states. A dynamic binding mode for the TF antigen-CG-3 complex consisting of two poses has been deduced. In one pose, intermolecular interactions were formed between the terminal threonine residue and the receptor. In the second pose, intermolecular interactions involved the internal GalpNAc. The difference in the trend of some shifts in the heteronuclear single-quantum coherence titration spectra indicates some disparities in the binding interactions of CG-3 with lactose and TF antigen. The results obtained from this model of the avian orthologue of human galectin-3 will allow detailed interspecies comparison to give sequence deviations in phylogeny a structural and functional meaning. Moreover, the results indicate that the peptide scaffold presenting TF antigen could be relevant for binding and thus provides a possible route for the design of galectin-3 inhibitors with improved affinity and selectivity.

Structure-activity relationship study of opiorphin, a human dual ectopeptidase inhibitor with antinociceptive properties.

Toward developing new potential analgesics, this first structure-activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe(3) is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe(3)-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe(3) derivatives showed that replacing l-Phe(3) for d-Phe(3) increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH-π stacking interactions between the aromatic ring of d-Phe(3) and the Hγ protons of Arg(2). This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.

Characterization of exopolysaccharides produced by Bifidobacterium longum NB667 and its cholate-resistant derivative strain IPLA B667dCo.

Bifidobacteria are natural members of the human intestinal microbiota and some strains are being used as probiotics. Adaptation to bile can allow them to increase survival in gastrointestinal conditions, thus improving their viability. Bifidobacterium longum NB667 and the cholate-resistant strain B. longum IPLA B667dCo produced exopolysaccharides (EPS) that were partially characterized. Analysis by size exclusion chromatography-multiangle laser light scattering indicated that the EPS crude fractions of both strains contained two polymer peaks of different molar mass. On the basis of chromatographic techniques both peaks appeared to be heteropolysaccharides. The smaller peak was mainly composed of glucose, galactose and rhamnose whose molar ratios and linkage types showed slight variations between the EPS fractions of both strains. The bigger peak consisted of glucose and galactose; the monosaccharide composition was identical in the EPS fractions of the two microorganisms, but their infrared spectra presented some differences regarding compounds other than carbohydrates that seem to be associated to the polymer. Differences in the composition of EPS fractions did not affect the capability of crude EPS from B. longum to be fermented by the human intestinal microbiota in fecal batch cultures.

Synthesis, biological evaluation and structural characterization of novel glycopeptide analogues of nociceptin N/OFQ.

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr(5)-O-α-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser(10)-O-α-D-GalNAc]-N/OFQ (glycopeptide 2) and [Ser(10)-O-ß-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.

Application of NMR methods to the study of the interaction of natural products with biomolecular receptors.

In this review, we present applications of NMR spectroscopy as a potent tool for the study of molecular interactions. It is clear that a variety of NMR methods may be employed to deduce key features of ligand­receptor molecular recognition processes, looking at the process from the perspective of the receptor or the ligand. We have not provided an exhaustive review, but we have tried to focus on describing the different aspects within this research topic. We have therefore selected examples accordingly, depending on the particular problem under study or the application/development of protocols to circumvent the technical problems that may be found when working in this field.