Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Arginine-deprivation-induced oxidative damage sterilizes Mycobacterium tuberculosis.

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

Cytosorb® hemoadsorption of apixaban during emergent cardio-pulmonary bypass: a case report.

BACKGROUND: Peri-operative coagulation management of patients receiving apixaban, a new oral anticoagulant, is difficult. The CytoSorb® hemoadsorption device might represent a therapeutic option to reduce apixaban's pharmacological and inflammatory effects during high-risk surgery. CASE PRESENTATION: An 83-year-old woman treated with Apixaban underwent emergent redo mitral valve replacement for prosthetic valve endocarditis. A CytoSorb® cartridge was added to the cardio-pulmonary bypass (CPB) circuit. Apixaban-specific anti-factor Xa activity (AFXaA) were measured peri-operatively. After 100 minutes of CPB, a 50% AFXaA rate decrease was observed as compared to pre-CPB values. Furthermore, we noticed 39% and 44% reductions of AFXaA levels in comparison to the expected levels in patients with normal or altered renal function, respectively. CONCLUSION: Insertion of a CytoSorb® cartridge in the CPB was safe and associated with rapid correction of Apixaban-associated anticoagulation.

Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility-mass spectrometry.

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility-mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping.

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.

Two Years of Experience in the Implantation of Heartmate III.

INTRODUCTION: Left ventricular assist devices as long-term mechanical circulatory support are increasingly used as an option for medically refractory advanced heart failure. Heartmate III is one of the alternative devices for circulatory support in those patients. OBJECTIVES: Analyze a two years Heartmate III implantation Program. METHODS: From November 2015 to August 2017, Heartmate III was implanted in 16 patients with chronic end-stage heart failure, in 81% (n = 13) as a bridge to transplant and 19% (n = 3) as destination therapy. We did a review off demographic, clinical and surgical data, and we analyzed the overall survival using the Kaplan-Meier method, excluding patients who were transplanted. RESULTS: Heartmate III was implanted in 16 male patients (100%) with age 55.8 ± 11.1 years (limits 38-74 years) and body surface area 2.0 ± 0.19 m2. The baseline hemodynamic data revealed a cardiac index 2.1 ± 0.4 l / min / m2 and a left ventricular ejection fraction of 20.7 ± 7.3%. Ischemic cardiomyopathy was the most common etiology in this chronic heart failure population (n = 9; 56%). Seven patients (44%) were classified INTERMACS 4; five (31%) in profile 2; three (19%) in profile 3 and one (6%) in profile 1. The implantation of the devices was performed under Cardiopulmonary Bypass (78.6 ± 25.7 min), and 25% of the patients (n = 4) had right ventricular dysfunction, requiring postoperative temporary right ventricle support. As complications, 6 patients (38%) manifested bleeding requiring surgery and 2 (12%) reported gastrointestinal bleeding, 4 (25%) developed driveline infection, 3 of them were treated (18%) with conservative therapy and in 1 patient (6%) with driveline transposition. During the total follow-up time (19 months), three patients (18%) were transplanted; two deaths occured due to pulmonary embolism and ischemic stroke respectively; verified by the Kaplan Meier method, an overall survival rate of 92.9 ± 6.9%, stable from 6 months after implantation. CONCLUSION: The 6 months survival rate of 92.9% proves the efficacy of this therapy for our patients and all of them were INTERMACS profils lower than 4. Despite the small number of patients enrolled and the follow-up duration limiting our study, we demonstrated the first experience of our center in the treatment of high-risk population. In conclusion, we show that the Heartmate III was consistent in low INTERMACS profile patients.

Fragment Screening against the EthR-DNA Interaction by Native Mass Spectrometry.

Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry for screening fragments against a protein-DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second-line antitubercular drug used to combat multidrug-resistant Mtb strains. A small-scale fragment screening campaign was conducted against the EthR-DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X-ray crystallography. The screening campaign identified two new fragments that disrupt the EthR-DNA interaction in vitro (IC50 =460-610 µm) and bind to the hydrophobic channel of the EthR dimer.

The molecular biology of mycobacterial trehalose in the quest for advanced tuberculosis therapies.

Trehalose is a natural glucose disaccharide identified in the 19th century in fungi and insect cocoons, and later across the three domains of life. In members of the genus Mycobacterium, which includes the tuberculosis (TB) pathogen and over 160 species of nontuberculous mycobacteria (NTM), many of which are opportunistic pathogens, trehalose has been an important focus of research over the last 60 years. It is a crucial player in the assembly and architecture of the remarkable mycobacterial cell envelope as an element of unique highly antigenic glycolipids, namely trehalose dimycolate ('cord factor'). Free trehalose has been detected in the mycobacterial cytoplasm and occasionally in oligosaccharides with unknown function. TB and NTM infection statistics and death toll, the decline in immune responses in the aging population, human immunodeficiency virus/AIDS or other debilitating conditions, and the proliferation of strains with different levels of resistance to the dated drugs in use, all merge into a serious public-health threat urging more effective vaccines, efficient diagnostic tools and new drugs. This review deals with the latest findings on mycobacterial trehalose biosynthesis, catabolism, processing and recycling, as well with the ongoing quest for novel trehalose-related mechanisms to be targeted by novel TB therapeutics. In this context, the drug-discovery pipeline has recently included new lead compounds directed toward trehalose-related targets highlighting the potential of these pathways to stem the tide of rising drug resistance.

Effective Apixaban removal using hemoadsorption during emergent open-heart surgery: a case report and narrative literature review.

BACKGROUND: The management of hemostasis in patients medicated with apixaban (Eliquis) undergoing emergency cardiac surgery is exceedingly difficult. The body's natural elimination pathways for apixaban prove ineffective in emergency situations, and the impact of hemodialysis is limited. The application of Cytosorb® may attenuate the concentration of apixaban, thereby facilitating the stabilization of these patients. CASE PRESENTATION: An 84-year-old man treated with apixaban, underwent emergency ascending aorta replacement surgery due to an acute type A aortic dissection. To address the challenges induced by apixaban, we integrated Cytosorb® cartridge into the Cardiopulmonary bypass circuit. There was a 63.7% decrease in perioperative apixaban-specific anti-factor Xa activity. The patient's postoperative course was favourable. CONCLUSION: Hemoadsorption with Cytosorb® may offers a safe and feasible approach for reducing apixaban concentration in emergency cardiac surgery, thereby mitigating the risk of hemorrhagic complications.

Hemoperfusion in Cardiac Surgery and ECMO.

Extracorporeal circulation (ECC) such as cardiopulmonary bypass or extracorporeal membrane oxygenation (ECMO) may induce a complex activation of the immune system. To date, strategies to mitigate this activation have failed to translate into meaningful improvement of clinical outcomes. Hemoperfusion is a blood purification technique, which relies on mass separation by a solid agent (hemoadsorption). It can be performed by adding a cartridge filled with adsorptive sorbent in the extracorporeal circuit. These devices have the theoretical advantage to enable the removal of excess pro- and anti-inflammatory molecules. Several studies have demonstrated the feasibility and safety of hemoperfusion during cardiac surgery. They have suggested that the procedure could decrease cytokine levels in situations where they were elevated. However, further studies are required to determine the clinical indications, timing, and duration of hemoperfusion during cardiac surgery. Although a similar rationale can apply to hemoperfusion in ECMO, available data in this situation are even more limited and results are conflicting. In this chapter, we discuss the rationale for hemoperfusion with ECC, how to practically do it, and the current level of evidence supporting this therapy.

Genome sequence of Mycobacterium hassiacum DSM 44199, a rare source of heat-stable mycobacterial proteins.

Mycobacterium hassiacum is a rapidly growing mycobacterium isolated from human urine and so far the most thermophilic among mycobacterial species. Its thermotolerance and phylogenetic relationship to M. tuberculosis render its proteins attractive tools for crystallization and structure-guided drug design. We report the draft genome sequence of M. hassiacum DSM 44199.

Biosynthesis of mycobacterial methylglucose lipopolysaccharides.

Mycobacterial pathogenesis is closely associated with a unique cell envelope rich in complex carbohydrates and unique lipids, among which are the mycolic acids. Mycobacteria also synthesize unique intracellular polymethylated polysaccharides (PMPSs), namely methylglucose lipopolysaccharides (MGLPs), which are acylated with short-chain fatty acids, and methylmannose polysaccharides (MMPs). Since PMPSs modulate the synthesis of long-chain fatty acids in vitro, the possibility of a similar role in vivo and the regulation of mycolic acids assembly have been anticipated. Unlike MGLPs, MMPs have been identified in M. smegmatis and other fast-growing mycobacteria but not in M. tuberculosis, implying an essential role for MGLPs in this pathogen and turning the biosynthetic enzymes into attractive drug targets. The genome of M. tuberculosis was decoded 14 years ago but only recently has the identity of the genes involved in MGLPs biosynthesis been investigated. Two gene clusters (Rv1208-Rv1213 and Rv3030-Rv3037c) containing a few genes considered to be essential for M. tuberculosis growth, have initially been proposed to coordinate MGLPs biosynthesis. Among these genes, only the product of Rv1208 for the first step in the MGLPs pathway has, so far, been crystallized and its three-dimensional structure been determined. However, recent results indicate that at least three additional clusters may be involved in this pathway. The functional assignment of authentic roles to some of these M. tuberculosis H37Rv genes sheds new light on the intricacy of MGLPs biogenesis and renewed interest on their biological role.

Assessment of risk factors for in-hospital mortality after intensive care unit discharge.

CONTEXT: Post-intensive care unit (ICU) mortality predictors are unknown. OBJECTIVE: To assess post-ICU in-hospital mortality predictors. MATERIALS AND METHODS: Analysis of 296 patients discharged alive from a medical-surgical ICU during an 18-month period. RESULTS: Post-ICU in-hospital mortality was 22.6%. Nonsurvivors had significantly higher Charlson comorbidity score and more often had a tracheostomy. C-reactive protein (CRP) "alert measurement", ≥ 6 mg/dL, independently discriminated survivors from nonsurvivors. DISCUSSION: A CRP "alert measurement" or the need for tracheostomy may be used to identify patients with high risk of dying after ICU discharge. CONCLUSIONS: Charlson comorbidity score, CRP and tracheostomy predicted post-ICU in-hospital mortality.

Temporary Right Ventricular Assist Device After Redo Aortic Valve Replacement And Heartmate 3tm Implantation - A Case Report.

The implantation of the left ventricular assist device Abbott HeartMate 3TM is being increasingly performed for management of end-stage heart failure. LVAD implantation might be associated with early or late right ventricular dysfunction. When severe, a temporary right ventricular support device may need to be implanted. However, these situations are associated with higher mortality. We report a successful case of temporary right ventricular support following HeartMate 3 implantation.

Structural Characterization of Mycobacterium abscessus Phosphopantetheine Adenylyl Transferase Ligand Interactions: Implications for Fragment-Based Drug Design.

Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (Kd 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.

Predictive value of D-dimer in the clinical outcome of severe COVID19 patients: Are we giving it too much credit?

BACKGROUND: COVID-19 is a new form of acute respiratory failure leading to multiorgan failure and ICU admission. Gathered evidence suggests that a 3-fold rise in D-dimer concentrations may be linked to poor prognosis and higher mortality. PURPOSE: To describe D-dimer admission profile in severe ICU COVID19 patients and its predictive role in outcomes and mortality. METHODS: Single-center retrospective cohort study. All adult patients admitted to ICU with COVID19 were divided into 3 groups: (1) Lower-values group (D-dimer levels < 3-fold normal range value [NRV] [500ng/mL]), Intermediate-values group (D-dimer ≥3-fold and <10-fold NRV) and Higher-value group (≥10-fold NRV). RESULTS: 118 patients (mean age 63 years, 73% males) were included (N = 73 Lower-values group, N = 31 Intermediate-values group; N = 11 Higher-values group). Mortality was not different between groups (p = 0.51). Kaplan-Meier survival curves revealed no differences (p = 0.52) between groups, nor it was verified even when gender, age, ICU length of stay, and SOFA score were considered as covariables. CONCLUSIONS: In severe COVID19 patients, the D-dimer profile does not retain a predictive value regarding patients' survivability and should not be used as a surrogate of disease severity.

Factors associated with immunization against hepatitis B among primary health care professionals.

Introduction: The hepatitis B virus is the cause of one of the major public health problems worldwide. The infection may affect the entire population equally; however, health care professionals are part of a group that is more vulnerable to the disease, since they are exposed to both occupational and daily hazards. Objectives: To investigate the prevalence and factors associated with the immunization of health care professionals against the hepatitis virus type B, in the city of Montes Claros, state of Minas Gerais, Brazil. Methods: This was a cross-sectional and quantitative study, conducted with primary health care professionals. Using a random cluster sampling, 209 medical professionals, nurses, and nursing technicians who were interested in participating in the research were selected. A structured questionnaire was applied, and blood sampling was performed for the analysis of hepatitis B surface antibody titers. Finally, a descriptive and bivariate statistical analysis was conducted. Results: Data have shown that 91.8% of the professionals had complete immunization against the hepatitis B virus, that is, they had taken the three recommended doses of the vaccine. However, 13.9% of the sample, even after vaccination, was non-reactive (titers < 10 IU/mL hepatitis B surface antibody). Most of the professionals (94.3%) had direct contact with needlesticks/sharps at work and none of the participants reported a previous infection by the virus. Conclusions: Although most participants had complete immunization, the total result of individuals who did not obtain seroconversion was eminent, so the importance of the hepatitis B surface antibody test must be disseminated in the context of public health.

Introdução: O vírus da hepatite B é o causador de um dos grandes problemas de saúde pública mundial. A infecção pode acometer toda a população de igual maneira; entretanto, os profissionais de saúde são parte de um grupo mais vulnerável à doença, visto que são expostos tanto ao risco ocupacional quanto ao risco cotidiano. Objetivos: Investigar a prevalência e os fatores associados à imunização de profissionais de saúde contra o vírus da hepatite viral tipo B na cidade de Montes Claros, Minas Gerais, Brasil. Métodos: Tratou-se de um estudo transversal e quantitativo, realizado com profissionais de saúde da atenção primária. Por amostragem aleatória por conglomerado, foram selecionados 209 profissionais médicos, enfermeiros e técnicos de enfermagem que tiveram interesse em participar da pesquisa. Foi aplicado um questionário estruturado, e foi realizada coleta de sangue para análise de títulos de anticorpo contra o antígeno de superfície da hepatite B. Por fim, a análise estatística descritiva e bivariada foi efetuada. Resultados: Os dados evidenciaram que 91,8% dos profissionais tinham imunização completa contra o vírus da hepatite B - ou seja, haviam tomado as três doses preconizadas da vacina. Entretanto, 13,9% da amostra, mesmo após a vacinação, se mostrou não reagente (títulos < 10 UI/mL de anticorpo contra o antígeno de superfície da hepatite B). A maioria (94,3%) dos profissionais tinha contato direto no trabalho com materiais perfurocortantes, e nenhum participante relatou infecção prévia pelo vírus. Conclusões: Apesar do fato de que a maioria dos participantes tinha imunização completa, o resultado total de indivíduos que não obtiveram soroconversão foi eminente, de modo que há de se divulgar a valia do exame de anticorpo contra o antígeno de superfície da hepatite B no contexto de saúde pública.

Septic shock 3.0 criteria application in severe COVID-19 patients: An unattended sepsis population with high mortality risk.

BACKGROUND: Coronavirus disease 2019 (COVID-19) can be associated with life-threatening organ dysfunction due to septic shock, frequently requiring intensive care unit (ICU) admission, respiratory and vasopressor support. Therefore, clear clinical criteria are pivotal for early recognition of patients more likely to need prompt organ support. Although most patients with severe COVID-19 meet the Sepsis-3.0 criteria for septic shock, it has been increasingly recognized that hyperlactatemia is frequently absent, possibly leading to an underestimation of illness severity and mortality risk. AIM: To identify the proportion of severe COVID-19 patients with vasopressor support requirements, with and without hyperlactatemia, and describe their clinical outcomes and mortality. METHODS: We performed a single-center prospective cohort study. All adult patients admitted to the ICU with COVID-19 were included in the analysis and were further divided into three groups: Sepsis group, without both criteria; Vasoplegic Shock group, with persistent hypotension and vasopressor support without hyperlactatemia; and Septic Shock 3.0 group, with both criteria. COVID-19 was diagnosed using clinical and radiologic criteria with a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive RT-PCR test. RESULTS: 118 patients (mean age 63 years, 87% males) were included in the analysis (n = 51 Sepsis group, n = 26 Vasoplegic Shock group, and n = 41 Septic Shock 3.0 group). SOFA score at ICU admission and ICU length of stay were different between the groups (P < 0.001). Mortality was significantly higher in the Vasoplegic Shock and Septic Shock 3.0 groups when compared with the Sepsis group (P < 0.001) without a significant difference between the former two groups (P = 0.713). The log rank tests of Kaplan-Meier survival curves were also different (P = 0.007). Ventilator-free days and vasopressor-free days were different between the Sepsis vs Vasoplegic Shock and Septic Shock 3.0 groups (both P < 0.001), and similar in the last two groups (P = 0.128 and P = 0.133, respectively). Logistic regression identified the maximum dose of vasopressor therapy used (AOR 1.046; 95%CI: 1.012-1.082, P = 0.008) and serum lactate level (AOR 1.542; 95%CI: 1.055-2.255, P = 0.02) as the major explanatory variables of mortality rates (R 2 0.79). CONCLUSION: In severe COVID-19 patients, the Sepsis 3.0 criteria of septic shock may exclude approximately one third of patients with a similarly high risk of a poor outcome and mortality rate, which should be equally addressed.

Development of Inhibitors of SAICAR Synthetase (PurC) from Mycobacterium abscessus Using a Fragment-Based Approach.

Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.