Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase : impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer

Knowledge of Parents Working in Healthcare Settings Toward Attention-Deficit Hyperactivity Disorder Among Children

Sawsan Abuhammad,1 Manar ALAzzam,2 Yasmine Alabbasi3 1Department of Maternal and Child Health, Faculty of Nursing, Jordan University of Science and Technology, Irbid, Jordan; 2Department of Community and Mental Health Nursing, Princess Salma Faculty of Nursing, Al-Albayt University, Mafraq, 25113 Jordan; 3Department of Maternity and Pediatric Health Nursing, College of Nursing, Princess Nourah Bint Abdulrahman University, Riyadh, 11671 Saudi ArabiaCorrespondence: Yasmine Alabbasi, Email [email protected]: To examine the knowledge of parents who are working as healthcare workers about Attention-Deficit Hyperactivity Disorder (ADHD) and to determine the predictors of ADHD knowledge in children among parents.Methods: A cross-section study was conducted among parents who are working as HCWs. An online survey by google forms facilitated the creation of a 373 participant’s sample.Results: The parents who are working as HCWs toward ADHD Knowledge 14.9 (SD = 7.9). The most questions were answered correctly. ADHD children are frequently distracted by extraneous stimuli 285 (76.8%). ADHD children often fidget or squirm in their seats 272 (73.7%) and HCWs training in managing an ADHD child are generally effective when combined with medication treatment 260 (70.5%).Conclusion: This study found that many parents who working as HCWs had poor knowledge regarding causes and general aspects of this disorder. The results had many implications that include increased efforts to improve the capabilities of parents who working as HCWs and prevent further complications for the children and increase awareness among parents and teachers.Keywords: ADHD, parents who working as HCWs, knowledge, Jorda

“To cross-seed or not to cross-seed”: A pilot study using metallo-β-lactamases

Knowledge of protein structures is of central importance in modern drug discovery and molecular biology, but to be useful the structures, including those obtained in the crystalline state, must be biologically relevant. Small variations in crystallization conditions can lead to alternative crystal forms, conformations, and oligomerization states, causing changes which can lead to altered fold and active site architectures. In the determination of protein structures by X-ray crystallography, crystallization is an essential prerequisite and remains a major bottleneck in drug discovery. Although many methods have been tried in an attempt to improve the production of protein crystals, it is still largely a "trial and error" process. To our knowledge, crystallization by cross-seeding using homologous proteins has previously only been successful for proteins with greater than 61-74% sequence identity. In the study presented here, we explore the effect of low sequence similarity on cross-seeding using metallo-β-lactamases with sequence identities and sequence similarities as low as 24% and 36%, respectively, but with homologous core folds. Despite the low sequence identities, the results show that micro-cross-seeding matrix screening can increase the number of hits obtained and can shorten crystallization time. It can also help in the identification of new crystallization conditions and different crystal forms

CDK4/6 Inhibition Reprograms Mitochondrial Metabolism in BRAF(V600) Melanoma via a p53 Dependent Pathway

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are being tested in numerous clinical trials and are currently employed successfully in the clinic for the treatment of breast cancers. Understanding their mechanism of action and interaction with other therapies is vital in their clinical development. CDK4/6 regulate the cell cycle via phosphorylation and inhibition of the tumour suppressor RB, and in addition can phosphorylate many cellular proteins and modulate numerous cellular functions including cell metabolism. Metabolic reprogramming is observed in melanoma following standard-of-care BRAF/MEK inhibition and is involved in both therapeutic response and resistance. In preclinical models, CDK4/6 inhibitors overcome BRAF/MEK inhibitor resistance, leading to sustained tumour regression; however, the metabolic response to this combination has not been explored. Here, we investigate how CDK4/6 inhibition reprograms metabolism and if this alters metabolic reprogramming observed upon BRAF/MEK inhibition. Although CDK4/6 inhibition has no substantial effect on the metabolic phenotype following BRAF/MEK targeted therapy in melanoma, CDK4/6 inhibition alone significantly enhances mitochondrial metabolism. The increase in mitochondrial metabolism in melanoma cells following CDK4/6 inhibition is fuelled in part by both glutamine metabolism and fatty acid oxidation pathways and is partially dependent on p53. Collectively, our findings identify new p53-dependent metabolic vulnerabilities that may be targeted to improve response to CDK4/6 inhibitors

Assessment on CPR Knowledge and AED Availability in Saudi Malls by Security Personnel: Public Safety Perspective

Security personnel are the first ones who attend the scene in the case of out-of-hospital cardiac arrest (OHCA) at malls. Cardiopulmonary resuscitation (CPR) is not enough for those patients; they need an automated external defibrillator (AED) to bring the heart to function normally. This study aimed to assess the current status of CPR and AED knowledge and availability in Saudi malls by security personnel. Using a descriptive design, a study was conducted at seven malls located in the Eastern Province of Saudi Arabia. Two hundred and fifty participants were surveyed using the American Heart Association (AHA) 2015 guidelines to assess CPR and AED knowledge and availability in Saudi malls. The sample mean age was 32.60 years (SD = 10.02), and 87% of participants were working as security personnel. The majority of the participants had not received training about CPR and AED (75.8% and 95.2%, respectively). Common misconceptions are fallen into all categories of CPR and AED knowledge. Correctly answered statements ranged from 7.2% in the compression rate to 24.2% in hand placement. The study results indicated a poor training knowledge of CPR and AED in public settings. Integrating high-quality CPR and AED knowledge within the school and college curricula is a vital need. However, in order to maximize the survival rate, it is important to set laws and legislation adopted by stakeholders and decision makers to advocate the people who try to help, mandate AED installation in crowded places, and mandate teaching hands-only CPR and AED together as a package