Molecular and Structural Aspects of Clinically Relevant Mutations of SARS-CoV-2 RNA-Dependent RNA Polymerase in Remdesivir-Treated Patients

(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp ( Delta Gbind(WT) = 122.70 kcal/mol; Delta Gbind(P323L+ 671S+M899I) = 84.78 kcal/mol; Delta Gbind(P323L+ G671S+L838I+D738Y+K91E) = 96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding

Selenium-analogs based on natural sources as cancer-associated carbonic anhydrase isoforms IX and XII inhibitors

AbstractIn the relentless search for new cancer treatments, organoselenium compounds, and carbonic anhydrase (CA) inhibitors have emerged as promising drug candidates. CA isoforms IX and XII are overexpressed in many types of cancer, and their inhibition is associated with potent antitumor/antimetastatic effects. Selenium-containing compounds, particularly selenols, have been shown to inhibit tumour-associated CA isoforms in the nanomolar range since the properties of the selenium atom favour binding to the active site of the enzyme. In this work, two series of selenoesters (1a–19a and 1b–19b), which gathered NSAIDs, carbo/heterocycles, and fragments from natural products, were evaluated against hCA I, II, IX, and XII. Indomethacin (17b) and flufenamic acid (19b) analogs exhibited selectivity for tumour-associated isoform IX in the low micromolar range. In summary, selenoesters that combine NSAIDs with fragments derived from natural sources have been developed as promising nonclassical inhibitors of the tumour-associated CA isoforms

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase

How dynamical motions in enzymes might be linked to catalytic function is of significant general interest, although almost all relevant experimental data, to date, has been obtained for enzymes with a single active site. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating dynamical motions for proteins that are not amenable to study using solution-phase NMR methods. Here we use 3D variability analysis (3DVA) of an EM structure for human asparagine synthetase (ASNS) in combination with atomistic molecular dynamics (MD) simulations to detail how dynamic motions of a single side chain mediates interconversion of the open and closed forms of a catalytically relevant intramolecular tunnel, thereby regulating catalytic function. Our 3DVA results are consistent with those obtained independently from MD simulations, which further suggest that formation of a key reaction intermediate acts to stabilize the open form of the tunnel in ASNS to permit ammonia translocation and asparagine formation. This conformational selection mechanism for regulating ammonia transfer in human ASNS contrasts sharply with those employed in other glutamine-dependent amidotransferases that possess a homologous glutaminase domain. Our work illustrates the power of cryo-EM to identify localized conformational changes and hence dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics regulate function in metabolic enzymes with multiple active sites

A New Protocol to Treat Abdominal Subcutaneous Fat Combining Microwaves and Flat magnetic stimulation

Background: A healthy lifestyle is not always able to improve the abdomen’s appearance, especially in those patients who have undergone sudden weight changes. Objective: We aimed at evaluating the efficacy of combined microwaves and flat magnetic stimulation (FMS) to treat abdominal localized adiposity and laxity. Methods: Twenty-five patients were subjected to two treatment sessions per month on the abdominal area with microwaves. FMS was also performed twice per week, with a minimum of two days between each session for two months. The technology uses three types of different protocols: massage, muscle definition (shaping), and muscular strengthening. Measurements, including body mass index (BMI) and waist, and abdominal ultrasound were performed at baseline and three months after the last treatment session. Blood examinations were performed, and a 5-Likert scale questionnaire was used to assess patient satisfaction. Results: At follow-up, three months after the last treatment, the mean waist circumference (WC) was significantly reduced, and skin laxity improved in all patients (p < 0.001). A significant improvement in abdominal muscle tissue thickness was also shown in all abdominal areas, and the thickness of the adipose tissue evaluated by ultrasound was reduced. Conclusions: This study proves that the combination of microwaves and FMS treatment is secure and efficient for treating abdominal subcutaneous fat and skin laxity

Food safety and nutritional quality for the prevention of non communicable diseases: The Nutrient, hazard Analysis and Critical Control Point process (NACCP)

Background: The important role of food and nutrition in public health is being increasingly recognized as crucial for its potential impact on health-related quality of life and the economy, both at the societal and individual levels. The prevalence of non-communicable diseases calls for a reformulation of our view of food. The Hazard Analysis and Critical Control Point (HACCP) system, first implemented in the EU with the Directive 43/93/CEE, later replaced by Regulation CE 178/2002 and Regulation CE 852/2004, is the internationally agreed approach for food safety control. Our aim is to develop a new procedure for the assessment of the Nutrient, hazard Analysis and Critical Control Point (NACCP) process, for total quality management (TMQ), and optimize nutritional levels.Methods: NACCP was based on four general principles: i) guarantee of health maintenance; ii) evaluate and assure the nutritional quality of food and TMQ; iii) give correct information to the consumers; iv) ensure an ethical profit. There are three stages for the application of the NACCP process: 1) application of NACCP for quality principles; 2) application of NACCP for health principals; 3) implementation of the NACCP process. The actions are: 1) identification of nutritional markers, which must remain intact throughout the food supply chain; 2) identification of critical control points which must monitored in order to minimize the likelihood of a reduction in quality; 3) establishment of critical limits to maintain adequate levels of nutrient; 4) establishment, and implementation of effective monitoring procedures of critical control points; 5) establishment of corrective actions; 6) identification of metabolic biomarkers; 7) evaluation of the effects of food intake, through the application of specific clinical trials; 8) establishment of procedures for consumer information; 9) implementation of the Health claim Regulation EU 1924/2006; 10) starting a training program.Results and discussion: We calculate the risk assessment as follows: Risk (R) = probability (P) x damage (D). The NACCP process considers the entire food supply chain "from farm to consumer"; in each point of the chain it is necessary implement a tight monitoring in order to guarantee optimal nutritional quality