Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.

Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs

Comparison of Patient Satisfaction with Telehealth and In-Clinic Post-Operative Visits

Introduction: Telehealth has grown rapidly in the healthcare industry as a time efficient, affordable, and accessible means to provide care. Jefferson’s Department of Urology currently employs telehealth for post-operative visits but its efficacy in post-nephrectomy patients has not been well studied. We aim to demonstrate that patient satisfaction is the same for telehealth versus in-clinic post-operative visits in nephrectomy patients. Secondarily we will examine distance saved for telehealth patients. Methods: We will administer an IRB approved questionnaire to all nephrectomy patients via the EPIC® EMR platform after their post-operative visit. Each questionnaire will be specific to telehealth or in-clinic and asks the patients to rate their experiences on a 5-point Likert scale. The study time frame is from May 2019-November 2022. Time and distance data for secondary outcomes was extrapolated from the EPIC® EMR. Results: Thus far there have been 35 in-clinic and 9 telehealth post-operative appointments. Patient satisfaction data will be collected in the future. Each telehealth patient saved a mean of 19 miles (6.9-32.5 miles) while each in-clinic patient traveled a mean of 30 miles (0.8-60.9 miles). Discussion: Delays of the project have left us unable to determine patient satisfaction at present. Future establishment of equal satisfaction may lead to increased provider utilization of telehealth. Our secondary outcomes are positive, reporting that telehealth patients saved both time and travel distance on their appointments. If the questionnaire ultimately reveals satisfaction with telehealth, the combination with travel distance saved leads telehealth in a forward-looking direction

Comparison of Patient Satisfaction between Telehealth and In-Clinic Post-Operative Visits

Introduction: Telehealth has the potential to be an efficient, affordable, and accessible means to give care. In the field of Urology, its use has large potential but has not been well studied. We hypothesize that the use of telehealth for post-op nephrectomy patients will maintain the same level of patient satisfaction. We also hypothesize that telehealth visits will take less time and save patients money by eliminating the need to travel. Methods: From May through July 2019, post-nephrectomy patients who consented to the study were given either a telehealth or in-clinic post-operative visit. Outcome metrics and demographics information were obtained through the Epic® EMR Platform. Results: There were a total of 6 telehealth patients and 15 in-clinic patients in the time frame. Due to issues with the Epic® survey system, the satisfaction data cannot be presented at this time. The telehealth patient saved a mean of 19 miles of travel (range 6.9-32.5 miles). The in-clinic patient traveled a mean of 30.4 miles (range 0.8-60.9). Discussion: Due to insufficient data, it is difficult to make any reliable comparisons. Telehealth visits provide some benefit for patients by eliminating the need to travel. With more data, we expect the telehealth patients to be equally satisfied with their visits compared to in-clinic patients. We also expect telehealth visits to take significantly less time for the patients compared to in-clinic visits. This is an ongoing study, and we hope our data will be more robust in the future

Predicting Binding to P-Glycoprotein by Flexible Receptor Docking

P-glycoprotein (P-gp) is an ATP-dependent transport protein that is selectively expressed at entry points of xenobiotics where, acting as an efflux pump, it prevents their entering sensitive organs. The protein also plays a key role in the absorption and blood-brain barrier penetration of many drugs, while its overexpression in cancer cells has been linked to multidrug resistance in tumors. The recent publication of the mouse P-gp crystal structure revealed a large and hydrophobic binding cavity with no clearly defined sub-sites that supports an “induced-fit” ligand binding model. We employed flexible receptor docking to develop a new prediction algorithm for P-gp binding specificity. We tested the ability of this method to differentiate between binders and nonbinders of P-gp using consistently measured experimental data from P-gp efflux and calcein-inhibition assays. We also subjected the model to a blind test on a series of peptidic cysteine protease inhibitors, confirming the ability to predict compounds more likely to be P-gp substrates. Finally, we used the method to predict cellular metabolites that may be P-gp substrates. Overall, our results suggest that many P-gp substrates bind deeper in the cavity than the cyclic peptide in the crystal structure and that specificity in P-gp is better understood in terms of physicochemical properties of the ligands (and the binding site), rather than being defined by specific sub-sites

Sexual Harassment or Just Coaching? Sport Students Making Sense of Possibly Sexualising Coach Behaviours

Research has shown that athletes are divided in their assessment of possibly sexualising behaviours from coaches towards athletes. How they arrive at their conclusions has received less attention—yet it is crucial to understand as a basis for safeguarding measures. Using video-elicitation focus group interviews with sport students, we zoomed in on different types of ‘grey area’ situations involving coaches and athletes. We drew on social script theory to highlight the cultural tools sport students use to distinguish between acceptable and unacceptable coaching behaviours. Our analyses showed that the students drew on two types of scripts in their interpretative work: (1) sport scripts, denoting templates for ‘normal’ coach–athlete interactions (typically with a performance and/or caring rationale), and (2) sexual harassment scripts, encompassing beliefs and expectations of how sexual transgressions play out and among whom. We discuss how the students evaluated concrete grey area situations by comparing and contrasting them with both scripts. In these assessments, the students relied on cues and clues from the portrayed interactions, including the gender of the coach and athlete and knowledge about the specific sport setting. Our analyses demonstrate how views about sexual harassment in sport relate to the specificities of the sport setting and the gendered social dynamics in the situation

Protease inhibitors targeting coronavirus and filovirus entry.

In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics

Organic synthesis in supercritical carbon dioxide

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1998.Includes bibliographical references.by Adam Robert Renslo.Ph.D

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 μM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development. </p

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography

Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three sub-Angstrom resolution X-ray crystal structures of CTX-M \u3b2-lactamase, representing three stages of the enzymatic pathway, apo protein (0.79 \uc5), pre-covalent complex (0.89 \uc5), and acylation transition state analog (0.84 \uc5). The binding of a non-covalent ligand induces a proton transfer from the catalytic Ser70 to the general base Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73. QM/MM reaction path calculations determined the proton transfer barrier between Ser70 and Lys73 to be 1.53 kcal/mol, further confirming the presence of a LBHB. This LBHB is absent in the other two structures. Our data represents the first evidence of a direct and transient LBHB stabilizing a nucleophilic serine, as hypothesized by Cleland and Kreevoy. These results have important implications for the study of enzyme mechanisms as well as protein-inhibitor interactions