The Coenzyme A Level Modulator Hopantenate (HoPan) Inhibits Phosphopantotenoylcysteine Synthetase Activity

The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease models-especially for pantothenate kinase associated neurodegeneration (PKAN), a genetic disease rooted in impaired CoA metabolism. This use of HoPan was based on reports that it inhibits pantothenate kinase (PanK), the first enzyme of CoA biosynthesis. Using a combination of in vitro enzyme kinetic studies, crystal structure analysis, and experiments in a typical PKAN cell biology model, we demonstrate that instead of inhibiting PanK, HoPan relies on it for metabolic activation. Once phosphorylated, HoPan inhibits the next enzyme in the CoA pathway-phosphopantothenoylcysteine synthetase (PPCS)-through formation of a nonproductive substrate complex. Moreover, the obtained structure of the human PPCS in complex with the inhibitor and activating nucleotide analogue provides new insights into the catalytic mechanism of PPCS enzymes-including the elusive binding mode for cysteine-and reveals the functional implications of mutations in the human PPCS that have been linked to severe dilated cardiomyopathy. Taken together, this study demonstrates that the molecular mechanism of action of HoPan is more complex than previously thought, suggesting that the results of studies in which it is used as a tool compound must be interpreted with care. Moreover, our findings provide a clear framework for evaluating the various factors that contribute to the potency of CoA-directed inhibitors, one that will prove useful in the future rational development of potential therapies of both human genetic and infectious diseases

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies

The clinical relevance of oliguria in the critically ill patient : Analysis of a large observational database

Funding Information: Marc Leone reports receiving consulting fees from Amomed and Aguettant; lecture fees from MSD, Pfizer, Octapharma, 3 M, Aspen, Orion; travel support from LFB; and grant support from PHRC IR and his institution. JLV is the Editor-in-Chief of Critical Care. The other authors declare that they have no relevant financial interests. Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Urine output is widely used as one of the criteria for the diagnosis and staging of acute renal failure, but few studies have specifically assessed the role of oliguria as a marker of acute renal failure or outcomes in general intensive care unit (ICU) patients. Using a large multinational database, we therefore evaluated the occurrence of oliguria (defined as a urine output 16 years) patients in the ICON audit who had a urine output measurement on the day of admission were included. To investigate the association between oliguria and mortality, we used a multilevel analysis. Results: Of the 8292 patients included, 2050 (24.7%) were oliguric during the first 24 h of admission. Patients with oliguria on admission who had at least one additional 24-h urine output recorded during their ICU stay (n = 1349) were divided into three groups: transient - oliguria resolved within 48 h after the admission day (n = 390 [28.9%]), prolonged - oliguria resolved > 48 h after the admission day (n = 141 [10.5%]), and permanent - oliguria persisting for the whole ICU stay or again present at the end of the ICU stay (n = 818 [60.6%]). ICU and hospital mortality rates were higher in patients with oliguria than in those without, except for patients with transient oliguria who had significantly lower mortality rates than non-oliguric patients. In multilevel analysis, the need for RRT was associated with a significantly higher risk of death (OR = 1.51 [95% CI 1.19-1.91], p = 0.001), but the presence of oliguria on admission was not (OR = 1.14 [95% CI 0.97-1.34], p = 0.103). Conclusions: Oliguria is common in ICU patients and may have a relatively benign nature if only transient. The duration of oliguria and need for RRT are associated with worse outcome.publishersversionPeer reviewe

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Developing Pantetheinase-Resistant Pantothenamide Antibacterials: Structural Modification Impacts on PanK Interaction and Mode of Action

Pantothenamides (PanAms) are analogues of pantothenate, the biosynthetic precursor of coenzyme A (CoA), and show potent antimicrobial activity against several bacteria and the malaria parasite in vitro. However, pantetheinase enzymes that normally degrade pantetheine in human serum also act on the PanAms, thereby reducing their potency. In this study, we designed analogues of the known antibacterial PanAm <i>N</i>-heptylpantothenamide (N7-Pan) to be resistant to pantetheinase by using three complementary structural modification strategies. We show that, while two of these are effective in imparting resistance, the introduced modifications have an impact on the analogues’ interaction with pantothenate kinase (PanK, the first CoA biosynthetic enzyme), which acts as a metabolic activator and/or target of the PanAms. This, in turn, directly affects their mode of action. Importantly, we discover that the phosphorylated version of N7-Pan shows pantetheinase resistance and antistaphylococcal activity, providing a lead for future studies in the ongoing search of PanAm analogues that show in vivo efficacy

Overcoming synthetic challenges in targeting coenzyme A biosynthesis with the antimicrobial natural product CJ-15,801

The biosynthesis of the essential metabolic cofactor coenzyme A (CoA) has been receiving increasing attention as a new target that shows potential to counter the rising resistance to established antimicrobials. In particular, phosphopantothenoylcysteine synthetase (PPCS)—the second CoA biosynthesis enzyme that is found as part of the bifunctional CoaBC protein in bacteria, but is monofunctional in eukaryotes—has been validated as a target through extensive genetic knockdown studies in Mycobacterium tuberculosis. Moreover, it has been identified as the molecular target of the fungal natural product CJ-15,801 that shows selective activity against Staphylococcus aureus and the malaria parasite Plasmodium falciparum. As such, CJ-15,801 and 4′-phospho-CJ-15,801 (its metabolically active form) are excellent tool compounds for use in the development of new antimicrobial PPCS inhibitors. Unfortunately, further study and analysis of CJ15,801 is currently being hampered by several unique challenges posed by its synthesis. In this study we describe how these challenges were overcome by using a robust palladium-catalyzed coupling to form the key N-acyl vinylogous carbamate moiety with retention of stereochemistry, and by extensive investigation of protecting groups suited to the labile functional group combinations contained in this molecule. We also demonstrate that using TBAF for deprotection causes undesired off-target effects related to the presence of residual tertiary ammonium salts. Finally, we provide a new method for the chemoenzymatic preparation of 4′-phospho-CJ-15,801 on multi-milligram scale, after showing that chemical synthesis of the molecule is not practical. Taken together, the results of this study advances our pursuit to discover new antimicrobials that specifically target CoA biosynthesis and/or utilization.We are also grateful to the Canberra branch of the Australian Red Cross Blood Service for providing red blood cells. This work was supported by a CPRR grant (#78988) from the National Research Foundation (NRF) of South Africa and a National Institutes of Health (NIH) award (R01AI136836) to ES. RD received grant-holder and free-standing postdoctoral fellowships from the NRF and postdoctoral study support from the Oppenheimer Memorial Trust, RvdW and LB received NRF Scare Skills doctoral bursaries and KJM an NRF Innovation doctoral bursary. ETT was supported by a Research Training Program scholarship from the Australian Government

Vnn1 pantetheinase limits the Warburg effect and sarcoma growth by rescuing mitochondrial activity

Like other tumors, aggressive soft tissue sarcomas (STS) use glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) for growth. Given the importance of the cofactor coenzyme A (CoA) in energy metabolism, we investigated the impact of Vnn1 pantetheinase-an enzyme that degrades pantetheine into pantothenate (vitamin B5, the CoA biosynthetic precursor) and cysyteamine-on tumor growth. Using two models, we show that Vnn1(+) STS remain differentiated and grow slowly, and that in patients a detectable level of VNN1 expression in STS is associated with an improved prognosis. Increasing pantetheinase activity in aggressive tumors limits their growth. Using combined approaches, we demonstrate that Vnn1 permits restoration of CoA pools, thereby maintaining OXPHOS. The simultaneous production of cysteamine limits glycolysis and release of lactate, resulting in a partial inhibition of STS growth in vitro and in vivo. We propose that the Warburg effect observed in aggressive STS is reversed by induction of Vnn1 pantetheinase and the rewiring of cellular energy metabolism by its products

Subcortical brain volume differences in participants with attention deficit hyperactivity disorder in children and adults: a cross-sectional mega-analysis

BACKGROUND: Neuroimaging studies have shown structural alterations in several brain regions in children and adults with attention deficit hyperactivity disorder (ADHD). Through the formation of the international ENIGMA ADHD Working Group, we aimed to address weaknesses of previous imaging studies and meta-analyses, namely inadequate sample size and methodological heterogeneity. We aimed to investigate whether there are structural differences in children and adults with ADHD compared with those without this diagnosis. METHODS: In this cross-sectional mega-analysis, we used the data from the international ENIGMA Working Group collaboration, which in the present analysis was frozen at Feb 8, 2015. Individual sites analysed structural T1-weighted MRI brain scans with harmonised protocols of individuals with ADHD compared with those who do not have this diagnosis. Our primary outcome was to assess case-control differences in subcortical structures and intracranial volume through pooling of all individual data from all cohorts in this collaboration. For this analysis, p values were significant at the false discovery rate corrected threshold of p=0.0156. FINDINGS: Our sample comprised 1713 participants with ADHD and 1529 controls from 23 sites with a median age of 14 years (range 4-63 years). The volumes of the accumbens (Cohen's d=-0.15), amygdala (d=-0.19), caudate (d=-0.11), hippocampus (d=-0.11), putamen (d=-0.14), and intracranial volume (d=-0.10) were smaller in individuals with ADHD compared with controls in the mega-analysis. There was no difference in volume size in the pallidum (p=0.95) and thalamus (p=0.39) between people with ADHD and controls. Exploratory lifespan modelling suggested a delay of maturation and a delay of degeneration, as effect sizes were highest in most subgroups of children (21 years): in the accumbens (Cohen's d=-0.19 vs -0.10), amygdala (d=-0.18 vs -0.14), caudate (d=-0.13 vs -0.07), hippocampus (d=-0.12 vs -0.06), putamen (d=-0.18 vs -0.08), and intracranial volume (d=-0.14 vs 0.01). There was no difference between children and adults for the pallidum (p=0.79) or thalamus (p=0.89). Case-control differences in adults were non-significant (all p>0.03). Psychostimulant medication use (all p>0.15) or symptom scores (all p>0.02) did not influence results, nor did the presence of comorbid psychiatric disorders (all p>0.5). INTERPRETATION: With the largest dataset to date, we add new knowledge about bilateral amygdala, accumbens, and hippocampus reductions in ADHD. We extend the brain maturation delay theory for ADHD to include subcortical structures and refute medication effects on brain volume suggested by earlier meta-analyses. Lifespan analyses suggest that, in the absence of well powered longitudinal studies, the ENIGMA cross-sectional sample across six decades of ages provides a means to generate hypotheses about lifespan trajectories in brain phenotypes. FUNDING: National Institutes of Health

Subcortical brain volume differences in participants with attention deficit hyperactivity disorder in children and adults : a cross-sectional mega-analysis

BACKGROUND: Neuroimaging studies have shown structural alterations in several brain regions in children and adults with attention deficit hyperactivity disorder (ADHD). Through the formation of the international ENIGMA ADHD Working Group, we aimed to address weaknesses of previous imaging studies and meta-analyses, namely inadequate sample size and methodological heterogeneity. We aimed to investigate whether there are structural differences in children and adults with ADHD compared with those without this diagnosis. METHODS: In this cross-sectional mega-analysis, we used the data from the international ENIGMA Working Group collaboration, which in the present analysis was frozen at Feb 8, 2015. Individual sites analysed structural T1-weighted MRI brain scans with harmonised protocols of individuals with ADHD compared with those who do not have this diagnosis. Our primary outcome was to assess case-control differences in subcortical structures and intracranial volume through pooling of all individual data from all cohorts in this collaboration. For this analysis, p values were significant at the false discovery rate corrected threshold of p=0·0156. FINDINGS: Our sample comprised 1713 participants with ADHD and 1529 controls from 23 sites with a median age of 14 years (range 4-63 years). The volumes of the accumbens (Cohen's d=-0·15), amygdala (d=-0·19), caudate (d=-0·11), hippocampus (d=-0·11), putamen (d=-0·14), and intracranial volume (d=-0·10) were smaller in individuals with ADHD compared with controls in the mega-analysis. There was no difference in volume size in the pallidum (p=0·95) and thalamus (p=0·39) between people with ADHD and controls. Exploratory lifespan modelling suggested a delay of maturation and a delay of degeneration, as effect sizes were highest in most subgroups of children (21 years): in the accumbens (Cohen's d=-0·19 vs -0·10), amygdala (d=-0·18 vs -0·14), caudate (d=-0·13 vs -0·07), hippocampus (d=-0·12 vs -0·06), putamen (d=-0·18 vs -0·08), and intracranial volume (d=-0·14 vs 0·01). There was no difference between children and adults for the pallidum (p=0·79) or thalamus (p=0·89). Case-control differences in adults were non-significant (all p>0·03). Psychostimulant medication use (all p>0·15) or symptom scores (all p>0·02) did not influence results, nor did the presence of comorbid psychiatric disorders (all p>0·5). INTERPRETATION: With the largest dataset to date, we add new knowledge about bilateral amygdala, accumbens, and hippocampus reductions in ADHD. We extend the brain maturation delay theory for ADHD to include subcortical structures and refute medication effects on brain volume suggested by earlier meta-analyses. Lifespan analyses suggest that, in the absence of well powered longitudinal studies, the ENIGMA cross-sectional sample across six decades of ages provides a means to generate hypotheses about lifespan trajectories in brain phenotypes. FUNDING: National Institutes of Health