Ribosomal selection of mRNAs with degenerate initiation triplets

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present

Deciphering the Molecular Recognition Mechanism of Multidrug Resistance Staphylococcus aureus NorA Efflux Pump Using a Supervised Molecular Dynamics Approach

The use and misuse of antibiotics has resulted in critical conditions for drug-resistant bacteria emergency, accelerating the development of antimicrobial resistance (AMR). In this context, the co-administration of an antibiotic with a compound able to restore sufficient antibacterial activity may be a successful strategy. In particular, the identification of efflux pump inhibitors (EPIs) holds promise for new antibiotic resistance breakers (ARBs). Indeed, bacterial efflux pumps have a key role in AMR development; for instance, NorA efflux pump contributes to Staphylococcus aureus (S. aureus) resistance against fluoroquinolone antibiotics (e.g., ciprofloxacin) by promoting their active extrusion from the cells. Even though NorA efflux pump is known to be a potential target for EPIs development, the absence of structural information about this protein and the little knowledge available on its mechanism of action have strongly hampered rational drug discovery efforts in this area. In the present work, we investigated at the molecular level the substrate recognition pathway of NorA through a Supervised Molecular Dynamics (SuMD) approach, using a NorA homology model. Specific amino acids were identified as playing a key role in the efflux pump-mediated extrusion of its substrate, paving the way for a deeper understanding of both the mechanisms of action and the inhibition of such efflux pumps

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors

: Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones

p38α MAPK and Type I Inhibitors: Binding Site Analysis and Use of Target Ensembles in Virtual Screening

Mitogen-activated protein kinase p38α plays an essential role in the regulation of pro-inflammatory signaling, and selective blockade of this kinase could be efficacious in many pathological processes. Despite considerable research efforts focused on the discovery and development of p38α MAPK inhibitors, no drug targeting this protein has been approved for clinical use so far. We herein analyze the available crystal structures of p38α MAPK in complex with ATP competitive type I inhibitors, getting insights into ATP binding site conformation and its influence on automated molecular docking results. The use of target ensembles, rather than single conformations, resulted in a performance improvement in both the ability to reproduce experimental bound conformations and the capability of mining active molecules from compound libraries. The information gathered from this study can be exploited in structure-based drug discovery programs having as the ultimate aim the identification of novel p38α MAPK type I inhibitors

Structure-based insights into p38α MAPK: from crystal structures analysis to hit optimization

p38α mitogen-activated protein kinase (MAPK) is a serine/threonine kinase involved in the regulation of pro-inflammatory signaling networks and in the biosynthesis of cytokines, representing a well-recognized therapeutic target for the treatment of autoimmune and inflammatory diseases.1 Over the past two decades, tremendous efforts have been focused on the discovery and development of small-molecule acting as p38α MAPK inhibitors, although no drugs targeting this protein are currently available.2 Thus, the discovery and development of novel small molecule p38α inhibitors represent an exciting area of research. In this context, we have recently identified the pyrazolobenzothiazine core as a new p38αMAPK inhibitor chemoptype.3 Some derivatives showed interesting activity in both p38α MAPK and TNF-α release assays, coupled with a promising kinase selectivity profile. The binding mode prediction of the best hits (Figure 1) has provided clues for further chemical optimization. Towards this aim, first an in depth analysis of the available crystal structures of p38α MAPK in complex with ATP competitive type I inhibitors (TI-Is)4 has been carried out, getting insights into ATP binding site conformations and key inhibitor features. Building on this, we have analysed the impact of protein target conformations on automated molecular docking and docking-based virtual screening. The information gathered from such modelling studies is currently used to aid both our hit-to-lead optimization strategy and the rational identification of new p38a MAPK inhibitor chemotypes through virtual screening campaigns

Intossicazione acuta da metadone a seguito di ingestione accidentale in un bambino di due anni

Il Centro Antiveleni del Policlinico Umberto I di Roma viene contattato per un bambino di 30 mesi che aveva accidentalmente ingerito una quantità imprecisata di metadone, avendo bevuto, un’ora e mezza prima, dallo stesso bicchiere in cui era presente un residuo imprecisato di metadone cloridrato sciroppo, alla concentrazione 0,5%, assunto dal padre. Il bambino viene ammesso al DEA in arresto cardiorespiratorio, cianotico e con pupille isocoriche e isocicliche. Inoltre il paziente risulta essere affetto da Varicella e presenta alterazione febbrile (T °: 38.5). Viene consigliata dal CAV la somministrazione di Naloxone cloridrato 0.2 mg in bolo da ripetere fino a risveglio e respirazione spontanea. A seguito della somministrazione della prima dose di naloxone, il bambino riprende coscienza. Successivamente viene instaurata una terapia infusionale con naloxone 1 gr in 250 ml di soluzione fisiologica (0.9%NaCl in acqua) alla velocita di 60 ml/ora ed un’ulteriore fiala di naloxone 0.4 mg viene somministrata per via intramuscolare. Stabilizzate le condizioni cliniche, il paziente viene trasferito nel reparto di osservazione pediatrica dove vengono riscontrati i seguenti parametri vitali: PA 90/60 mm/Hg, FC 116 battiti/min, FR 28 atti respiratori/min, e saturazione del 100% con O2 L/min. L’EGA venoso mostra acidosi (ph 7,28), mentre gli esami ematochimici sono nella norma. Successivamente il bambino viene valutato dai medici del CAV; all’E.O. risulta risvegliabile, parametri vitali nella norma , la FR è di 22 atti respiratori/minuto e le pupille sono miotiche ma reagenti alla luce. Viene somministrata infusione continua di glucosata e fisiologica e ripetuto naloxone 0.2 mg in 24 ml di glucosata al 5% alla velocità di 1 ml/ora. Il giorno successivo viene richiesto un esame tossicologico delle urine ed una determinazione della metadonemia. Si riscontra metadone nel campione urinario esaminato a mezzo di EMIT (1380 ng/ml (cut-off 300 ng/ml); la metadonemia indagata con metodica HPTLC è di 1.99 microg/ml, quella con LC-MS/MS è di 1.09 ng/ml. Nonostante le elevate concentrazioni di metadone nel sangue, le condizioni del bambino risultano stabili e viene così sospesa la ventilazione assistita. Il bambino rimane sotto osservazione in fluidoterapia. Viene giornalmente monitorata l’escrezione di metadone nelle urine e la concentrazione ematica, che risultano entrambe in progressiva diminuzione (nel campione ematico: 485 ng/ml). Quattro giorni dopo il ricovero il bambino viene dimesso in quanto l’esame tossicologico delle urine mostra risultato negativo e i livelli ematici di metadone si sono normalizzati. Di particolare interesse è la discordanza tra i risultati del quadro laboratoristico e il quadro clinico del paziente. Infatti, dopo l’iniziale sintomatologia tipica delle intossicazioni acute da metadone, le condizioni cliniche del bambino sono sempre state stabili e nonostante gli elevati livelli urinari ed ematici di metadone, il bambino è sempre stato cosciente, reattivo, senza segni e sintomi d’impregnazione da oppiacei e bisogno di ventilazione assistita. Particolare attenzione, inoltre, va posta riguardo agli affidi ai pazienti in terapia sostitutiva con metadone nella formulazione con concentrazione 0,5%, attualmente maggiormente utilizzata rispetto a quella allo 0,1%, soprattutto se custodiscono il metadone in luoghi frequentati da bambini

A 1,8-naphthyridone derivative targets the HIV-1 Tat-mediated transcription and potently inhibits the HIV-1 replication

The emergence of multidrug resistant HIV-1 strains and the inability of the HAART to eradicate HIV-1 virus from infected patients demand new drugs able to interfere with an alternative step of the replicative cycle. The naphthyridone 3 (HM13N), described in the present study, is a promising anti-HIV agent due to its ability to inhibit the HIV-1 Tat-mediated transcription and the potent antiviral activity observed in acutely, chronically, and latently infected cells. The absence of any tendency to select for resistance mutations in vitro adds to the potential clinical value of this type of compounds, especially as these compounds are drug-like and obey the Lipinski rules.status: publishe

Echocardiographic Evaluation in Paediatric Sickle Cell Disease Patients: A Pilot Study

Cardiovascular involvement has a great impact on morbidity and mortality in sickle cell disease (SCD). Currently, few studies are available regarding the paediatric setting and, moreover, current guidelines for the echocardiogram screening program in the asymptomatic paediatric population are controversial. We performed a retrospective observational monocentric study on 64 SCD patients (37 male and 27 female, median age 10) at the Bambino Gesù Childrens’ Hospital, who had undergone a routine transthoracic echocardiogram. In total, 46 (72%) patients had at least one cardiac abnormality. Left atrial dilatation (LAD) was present in 41 (65%) patients and left ventricular hypertrophy (LVH) was found in 29 (45%) patients. Patients with LAD showed lower median haemoglobin levels (p = 0.009), and a higher absolute reticulocyte count (p = 0.04). LVH was negatively correlated with the median haemoglobin value (p = 0.006) and positively with the reticulocyte count (p = 0.03). Moreover, we found that patients with cardiac anomalies had higher transfusion needs and a lower frequency of pain crises. In our setting, cardiac involvement has a high prevalence in the paediatric cohort and seems to be associated with specific laboratory findings, and with a specific clinical phenotype characterized by complications related to high haemodynamic load

Pivaloylcodeine, a new codeine derivative, for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC50 were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC50 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors. © 2013 Elsevier Ltd. All rights reserved