Targeting Allosteric Pockets in Protein Kinases Using Molecular Modeling and Simulations

The deregulation of protein kinases is often related with the development of several malignancies such as cancer. Therefore, inhibition of protein kinases is an established and often effective pharmacological strategy. However, point mutations in kinases are frequently the cause of drug resistance. To overcome this issue, many efforts are directed towards the design of allosteric drugs, with the goal to inhibit the mutated forms of kinases. In this regard, the comprehension of the molecular basis of the allosteric control of protein kinases is essential for the design of novel allosteric drugs. In this thesis, we studied the molecular basis that allosterically regulates the function of Abelson (Abl) kinase, a relevant pharmaceutical target for the treatment of several malignancies, as chronic myelogenous leukemia. This study proposes a novel mechanism according to which conserved structural motifs dynamically cooperate to regulate allosterically the function of Abl. The information retrieved from this study can be employed for the rational design of new Abl allosteric inhibitors. In addition, we also developed a new algorithm for the detection of protein pockets in MD simulations. This algorithm has been conceived to identify and analyze all the pockets of a given protein without any user a priori information of their localization. It also enables the detection of pockets’ network, characterizing possible allosteric signaling pathways that connect the functional with the allosteric sites. Overall, this tool allows the study of the dynamic properties of pockets and might be employed in the early stages of the drug discovery process to design both orthosteric and allosteric binders

DEVELOPMENT OF A NOVEL METHOD FOR AMNIOTIC FLUID STEM CELL STORAGE

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.Highlights: Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking; Wastewater analysis protocols are not optimized for trace analysis of viruses; False-positive and -negative errors have consequences for public health responses; Inter-laboratory studies utilizing standardized reference materials and protocols are needed.info:eu-repo/semantics/publishedVersio

Utilization of mechanical power and associations with clinical outcomes in brain injured patients: a secondary analysis of the extubation strategies in neuro-intensive care unit patients and associations with outcome (ENIO) trial

Background: There is insufficient evidence to guide ventilatory targets in acute brain injury (ABI). Recent studies have shown associations between mechanical power (MP) and mortality in critical care populations. We aimed to describe MP in ventilated patients with ABI, and evaluate associations between MP and clinical outcomes. Methods: In this preplanned, secondary analysis of a prospective, multi-center, observational cohort study (ENIO, NCT03400904), we included adult patients with ABI (Glasgow Coma Scale ≤ 12 before intubation) who required mechanical ventilation (MV) ≥ 24 h. Using multivariable log binomial regressions, we separately assessed associations between MP on hospital day (HD)1, HD3, HD7 and clinical outcomes: hospital mortality, need for reintubation, tracheostomy placement, and development of acute respiratory distress syndrome (ARDS). Results: We included 1217 patients (mean age 51.2 years [SD 18.1], 66% male, mean body mass index [BMI] 26.3 [SD 5.18]) hospitalized at 62 intensive care units in 18 countries. Hospital mortality was 11% (n = 139), 44% (n = 536) were extubated by HD7 of which 20% (107/536) required reintubation, 28% (n = 340) underwent tracheostomy placement, and 9% (n = 114) developed ARDS. The median MP on HD1, HD3, and HD7 was 11.9 J/min [IQR 9.2-15.1], 13 J/min [IQR 10-17], and 14 J/min [IQR 11-20], respectively. MP was overall higher in patients with ARDS, especially those with higher ARDS severity. After controlling for same-day pressure of arterial oxygen/fraction of inspired oxygen (P/F ratio), BMI, and neurological severity, MP at HD1, HD3, and HD7 was independently associated with hospital mortality, reintubation and tracheostomy placement. The adjusted relative risk (aRR) was greater at higher MP, and strongest for: mortality on HD1 (compared to the HD1 median MP 11.9 J/min, aRR at 17 J/min was 1.22, 95% CI 1.14-1.30) and HD3 (1.38, 95% CI 1.23-1.53), reintubation on HD1 (1.64; 95% CI 1.57-1.72), and tracheostomy on HD7 (1.53; 95%CI 1.18-1.99). MP was associated with the development of moderate-severe ARDS on HD1 (2.07; 95% CI 1.56-2.78) and HD3 (1.76; 95% CI 1.41-2.22). Conclusions: Exposure to high MP during the first week of MV is associated with poor clinical outcomes in ABI, independent of P/F ratio and neurological severity. Potential benefits of optimizing ventilator settings to limit MP warrant further investigation

Low in‑hospital mortality rate in patients with COVID‑19 receiving thromboprophylaxis: data from the multicentre observational START‑COVID Register

Abstract COVID-19 infection causes respiratory pathology with severe interstitial pneumonia and extra-pulmonary complications; in particular, it may predispose to thromboembolic disease. The current guidelines recommend the use of thromboprophylaxis in patients with COVID-19, however, the optimal heparin dosage treatment is not well-established. We conducted a multicentre, Italian, retrospective, observational study on COVID-19 patients admitted to ordinary wards, to describe clinical characteristic of patients at admission, bleeding and thrombotic events occurring during hospital stay. The strategies used for thromboprophylaxis and its role on patient outcome were, also, described. 1091 patients hospitalized were included in the START-COVID-19 Register. During hospital stay, 769 (70.7%) patients were treated with antithrombotic drugs: low molecular weight heparin (the great majority enoxaparin), fondaparinux, or unfractioned heparin. These patients were more frequently affected by comorbidities, such as hypertension, atrial fibrillation, previous thromboembolism, neurological disease,and cancer with respect to patients who did not receive thromboprophylaxis. During hospital stay, 1.2% patients had a major bleeding event. All patients were treated with antithrombotic drugs; 5.4%, had venous thromboembolism [30.5% deep vein thrombosis (DVT), 66.1% pulmonary embolism (PE), and 3.4% patients had DVT + PE]. In our cohort the mortality rate was 18.3%. Heparin use was independently associated with survival in patients aged ≥ 59 years at multivariable analysis. We confirmed the high mortality rate of COVID-19 in hospitalized patients in ordinary wards. Treatment with antithrombotic drugs is significantly associated with a reduction of mortality rates especially in patients older than 59 years

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Allosteric Communication Networks in Proteins Revealed through Pocket Crosstalk Analysis

The detection and characterization of binding pockets and allosteric communication in proteins is crucial for studying biological regulation and performing drug design. Nowadays, ever-longer molecular dynamics (MD) simulations are routinely used to investigate the spatiotemporal evolution of proteins. Yet, there is no computational tool that can automatically detect all the pockets and potential allosteric communication networks along these extended MD simulations. Here, we use a novel and fully automated algorithm that examines pocket formation, dynamics, and allosteric communication embedded in microsecond-long MD simulations of three pharmaceutically relevant proteins, namely, PNP, A2A, and Abl kinase. This dynamic analysis uses pocket crosstalk, defined as the temporal exchange of atoms between adjacent pockets, along the MD trajectories as a fingerprint of hidden allosteric communication networks. Importantly, this study indicates that dynamic pocket crosstalk analysis provides new mechanistic understandings on allosteric communication networks, enriching the available experimental data. Thus, our results suggest the prospective use of this unprecedented dynamic analysis to characterize transient binding pockets for structure-based drug desig

Novel Bacterial Topoisomerase Inhibitors Exploit Asp83 and the Intrinsic Flexibility of the DNA Gyrase Binding Site

DNA gyrases are enzymes that control the topology of DNA in bacteria cells. This is a vital function for bacteria. For this reason, DNA gyrases are targeted by widely used antibiotics such as quinolones. Recently, structural and biochemical investigations identified a new class of DNA gyrase inhibitors called NBTIs (i.e., novel bacterial topoisomerase inhibitors). NBTIs are particularly promising because they are active against multi-drug resistant bacteria, an alarming clinical issue. Structural data recently demonstrated that these NBTIs bind tightly to a newly identified pocket at the dimer interface of the DNA–protein complex. In the present study, we used molecular dynamics (MD) simulations and docking calculations to shed new light on the binding of NBTIs to this site. Interestingly, our MD simulations demonstrate the intrinsic flexibility of this binding site, which allows the pocket to adapt its conformation and form optimal interactions with the ligand. In particular, we examined two ligands, AM8085 and AM8191, which induced a repositioning of a key aspartate (Asp83B), whose side chain can rotate within the binding site. The conformational rearrangement of Asp83B allows the formation of a newly identified H-bond interaction with an NH on the bound NBTI, which seems important for the binding of NBTIs having such functionality. We validated these findings through docking calculations using an extended set of cognate oxabicyclooctane-linked NBTIs derivatives (~150, in total), screened against multiple target conformations. The newly identified H-bond interaction significantly improves the docking enrichment. These insights could be helpful for future virtual screening campaigns against DNA gyras