Computational Studies on Pharmaceutical Targets in Human Diseases

Bacterial multidrug resistance (i.e. the ability of some bacterial species to survive in presence of various drugs) has become a primary challenge at a global level. Due to various factors, such as the overuse of antibiotics in human activities like health care and farming or inadequate diagnostic, many bacteria have indeed evolved acquiring novel and highly efficient resistance mechanisms. Some species, in particular, have become resistant to almost all in-use drugs. Among the several mechanisms of resistance, efflux pumps of the RND superfamily (resistance-nodulation-cell division) play a major role. These complexes span the cell wall and are able to expel a wide range of noxious compounds, including antibiotics of many different classes. In order to reinvigorate the action of these drugs, a viable route is to hinder their transport out of the cell through co-administration of efflux pumps inhibitors (EPIs). At present several EPIs have been identified, but none of them is usable in clinical therapies due to adverse effects. Moreover, several questions are still open regarding the mode of action of known EPIs as well as the functioning mechanism of RND efflux pumps. Further research in this field is thus needed. In order to characterize the mode of action of several EPIs of this pump, we applied computational techniques such as molecular docking and molecular dynamics (MD) simulations. Specifically, we focused on the EPIs: (i) amitriptyline and chlorpromazine, repurposed drugs which were proven to act as inhibitors against AcrB; (ii) PAβN, a known inhibitor of the pump whose mode of action is not fully understood. This thesis focuses on the inhibition of the AcrB efflux pump, the best known representative of the RND superfamily. High-resolution structural data are indeed available for this protein (specifically, for its Escherichia coli orthologue). Moreover, a fluoroquinolone resistant variant of this pump has been detected in clinical environments. With regard to amitriptyline and chlorpromazine, our in silico investigations revealed that both compounds tend to occupy a known binding pocket of AcrB. Their binding mode presents considerable similarities with that of several substrates and other EPIs of the pump, indicating that amitriptyline and chlorpromazine may inhibit the AcrB pump through competitive binding. In the case of PAβN, MD simulations were compared with experimental data from hydrogen-deuterium exchange mass spectrometry. From these analyses, it emerged that PAβN can significantly restrain the conformational dynamics of AcrB and its fluoroquinolone resistant variant. This EPI, therefore, may act by preventing conformational changes that are functional for AcrB. Importantly, our MD simulations revealed that PAβN and the antibiotic ciprofloxacin can simultaneously occupy the same binding pocket, suggesting that the EPI does not act by competitive binding. Further computational analyses were conducted on structural models of Salmonella Typhimurium AcrB. Experimental structural data on this wt protein are indeed missing, while the structure of its fluoroquinolone resistant variant has recently been solved through cryo-electron microscopy (cryo-EM). In order to assess the structural differences between the two proteins, we derived their structural models through homology modelling and MD simulations (modeling of the fluoroquinolone resistant variant was integrated with cryo-EM data). Structural analyses were then performed, with focus on the binding pockets of the protein. Considerable differences were detected regarding the volume as well as the hydration properties of the pockets. Although not strictly related to EPI development, this information may be valuable for the design of novel drugs and/or inhibitors of AcrB from Salmonella

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics

Combination therapy of high-dose rabeprazole plus metronomic capecitabine in advanced gastrointestinal cancer: a randomized phase II trial

Abstract: Background: In recent years, proton pump inhibitors (PPIs) have been investigated at high-dose tomodulate tumormicroenvironment acidification thus restoring chemotherapeutic sensitivity. This is the first trial to study activity and safety of repurposing high dose rabeprazole combined with metronomic capecitabine (mCAP). Methods: A phase II study in which patients with gastrointestinal cancer, refractory to standard treatments, who had a life expectancy >3 months, were blind randomized 1:1 to mCAP, 1500 mg/daily, continuously with or without rabeprazole 1.5 mg/kg bid, three days a week. The primary endpoint was 3-months progression-free survival (PFS). The secondary endpoints were clinical benefit (CB) and overall survival (OS). Safety and plasma concentrations of capecitabine and its metabolites (50-DFUR and 5-FU) were also evaluated. Results: Sixty-seven (median age 69 years; 63% male; 84% colorectal cancer, 76% ECOG-PS 1; 84% pretreated with two or more lines of chemotherapy) out of 90 patients screened for eligibility, were randomized to receive mCAP+rabeprazole (n = 32) vs. mCAP (n = 35). All patients were evaluable for response. No significant dierence between mCAP+rabeprazole vs. mCAP, in terms of 3-months PFS rate (HR = 1.43, 95%CI 0.53–3.85; p = 0.477), median PFS (HR = 1.22, 95%CI 0.75–2.00, p = 0.420), CB (RR = 0.85, 95%CI 0.29–2.44; p = 0.786) and median OS (HR = 0.89, 95%CI 0.54–1.48; p = 0.664) was observed. However, a 3-year OS rate of 10% and 12% was reported in the mCAP-rabeprazole and mCAP groups, respectively. Overall, no grade 3 or 4 toxicity occurred but grade 1 or 2 adverse event of any type were more frequently in the mCAP+rabeprazole group than in the mCAP (OR 2.83, 95%CI 1.03–7.79; p = 0.043). Finally, there was not statistically significant dierence in the plasma concentration of capecitabine and its metabolites between the two groups. Conclusions: Although the adjunct of high dose rabeprazole to mCAP was not shown to aect mCAP activity, as PPI are being investigated worldwide as drugs to be repositioned in cancer treatment and also considering the limited sample size as well as the favorable safety profile of the combination in the present study, further clinical investigations are desirable

Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity

An unusual case of corpse concealment driven by emotional distress

: Corpse concealment involves hiding a body for criminal purposes for many different reasons, such as destroy evidence of a murder or avoid the discovery of the victim. Although defendants could argue that they did not conceal the corpse with any criminal intent, but rather to spare themselves or others from emotional distress or to honor the wishes of the deceased. However, these arguments are often challenging to substantiate, and defendants may encounter significant legal obstacles when attempting to justify their actions. Herein, we report a case involving the concealment of a woman's corpse by her father. Autopsy and histological investigations were significantly limited due to the advanced decomposition of the body. Nevertheless, by integrating these data with radiological findings obtained from total body CT and micro-CT of the larynx-hyoid complex, hanging was deemed the cause of death. Additionally, the psychological evaluation of the father indicated that the act of concealment was motivated by emotions rather than criminal intent

L'olio di oliva ozonizzato come strumento di prevenzione della carie: studio preliminare in vitro

L’ozono (O3) è un gas composto da tre atomi di ossigeno e, rispetto a quest’ultimo, è notevolmente più reattivo. Ha la capacità di ossidare composti organici e inorganici, reagendo immediatamente con essi; è in grado, pertanto, di ossidare anche le membrane plasmatiche dei microrganismi, quali batteri, virus e funghi, provocandone l’eliminazione (1). L’ozono può essere applicato sui tessuti in diverse forme: miscela gassosa di ossigeno e ozono, acqua ozonizzata, oppure veicolato sottoforma di olii (2). L’olio ozonizzato è stato testato nel trattamento di differenti patologie come nelle patologie articolari e cutanee e a livello orale, in ambito parodontale, endodontico e nella cura di alcune infezioni (1). Molti studi sono stati focalizzati sulla sua attività antimicrobica, infatti l’O3 si è dimostrato notevolmente efficace sotto forma di gas contro i Gram positivi e, soprattutto, Gram negativi quali Porphyromonas endodontalis e Porphyromonas gingivalis (2). L'acqua ozonata ha mostrato ottimi risultati nei confronti della Candida albicans, riducendone la crescita in vitro in maniera molto più efficace rispetto ad un antimicotico topico (3). In odontoiatria l'olio d’oliva ozonizzato (OZ) è stato proposto in diversi campi: rigenerazione ossea, remineralizzazione delle white spot, ritrattamenti canalari dovuti a patogeni persistenti nell’endodonto, disinfezione delle tasche parodontali, sbiancamento, gestione e risoluzione dell’ipersensibilità e dolori articolari dell’ATM (4). In uno studio clinico in doppio cieco, randomizzato e controllato è stata confrontata l'azione terapeutica dell'olio di oliva ozonizzato con altre terapie nell'ambito della parodontologia. Sono stati divisi i pazienti in quattro gruppi: il primo gruppo prevedeva solo una terapia causale con levigature radicolari; il secondo gruppo l’associazione tra levigature e olio ozonizzato; il terzo solo con olio; il quarto disinfezione tramite clorexidina. Dai risultati emerge che l'olio di oliva ozonizzato risulta efficace nella riduzione degli indici parodontali (5). Da un punto di vista chirurgico, l'olio di oliva ozonizzato promuove una miglior guarigione degli innesti prelevati dal palato per la chirurgia muco-gengivale. Tale funzione dell'olio ozonizzato è stata dimostrata da due studi in cui veniva messo a confronto con un gruppo controllo trattato con olio placebo, dai quali risultava una prognosi dell'innesto significativamente più favorevole (6,7). L'obiettivo del nostro studio in vitro è quello di valutare l'efficacia dell'olio di oliva ozonizzato nei confronti di uno dei principali agenti eziologici della carie dentale: lo Streptococcus mutan

Micro-Computed Tomography (Micro-CT) Features of Laryngeal Fractures in a Case of Fatal Manual Strangulation

After attending this presentation, attendees will be aware of a new application of micro-Computed Tomography (CT) for dealing with the identification of the signs of pressure of the neck in cases of subtle manual strangulation. This presentation will impact the forensic science community by suggesting a novel approach for the identification of laryngeal fractures by means of micro-CT analysis. It is widely accepted that there are no specific or fully determinate signs of asphyxia. In cases of subtle fatal neck compression, postmortem findings on external examination of a body as well as on autopsy may vary considerably depending on the type of violent trauma, the strength of the resistance exerted by the victim, and on the intensity and duration of neck compression. Based on the detection of a combination of morphological hallmarks such as external injuries to the skin, hyoid or laryngeal fractures, hemorrhages of the overlying soft tissues, and subconjuctiveal petechiae, the diagnosis of homicidal neck compression is generally accepted. A case is presented of a 45-year-old schizophrenic patient found on the floor of the bedroom of a psychiatric ward in cardiopulmonary arrest. The patient was successfully resuscitated; nevertheless, brain death was declared after two days in a vegetative state. The roommate of the deceased, who was also schizophrenic, stated that \u201cthe voice of God told me to kill my roommate\u201d and death occurred after a prolonged scuffle. The body showed only vague signs of violence at external inspection (i.e., a subtle bruise on the forehead and a minute scratch on the left arm). At autopsy, the identification of subepicardial petechiae and moderate pulmonary emphysema were not useful for the diagnosis of asphyxial death because these signs might be related to resuscitative maneuvers with prolonged artificial ventilation. Since focal hemorrhagic infiltration of the tracheal mucosa and the superior horns of the thyroid cartilage were observed, a CT scan of the entire larynx was performed, with the goal of identifying laryngeal fractures. Moreover, both superior horns of the thyroid cartilage were analyzed thorough a micro-CT, a radiological technique for tissue analysis with a spatial resolution of a few microns. The 3D reconstruction allowed for identification of multiple cartilage fragments of the larynx. Consequently, the cause of death was identified as asphyxia due to manual strangulation. Considering that micro-CT analysis is able to generate images with a high level of detail compared to conventional radiological techniques, in cases of subtle fatal neck compression this type of investigation could be useful for the morphological identification of comminuted bilateral fractures. This may be particularly useful in cases involving children and young adults where fractures or cartilage damages may be minimal or absent as a consequence of the elasticity of the ligaments around the larynx and hyoid bone as well as the early stage of ossification

A case of sudden cardiac death following Domperidone self-medication

The phenomenon of sudden cardiac death is usually related to the worsening of existing heart conditions leading to ventricular arrhythmia (VA). One of the well-known triggers of SCD is drug-induced prolongation of the QT interval, such as that caused by Domperidone (D). Despite its risk to prolong the QT interval and associated narrow therapeutic index, D is available as an over-the-counter (OTC) drug in many countries such as Italy, Ireland, Netherlands, China, South Africa, Mexico, New Zealand and Chile to treat gastroesophageal reflux and functional dyspepsia. The present paper reports a case of SCD that occurred some hours after D self-administration in a 47-year-old female subject with mitral valve prolapse, thus, predisposed to both VA and SCD. Despite the risks related to D administration, to the best of our knowledge, this particular issue has not been discussed in the medico-legal literature. For this reason, the forensic implications of D administration are discussed focusing on issues related to the self-administration as an OTC drug (as seen in this case), administration to incapacitated subjects, prescription to patients with contraindications and the off-label drug use of D at high and hazardous concentrations to stimulate lactation

Determinazione di residui di sparo in ferite d\u2019arma da fuoco putrefatte mediante micro-TC

In cadaveri in avanzato stato di putrefazione l\u2019analisi ispettiva delle ferite d\u2019arma da fuoco pu\uf2 risultare confusa e difficoltosa a causa delle alterazioni indotte da fenomeni autolitici-putrefattivi. Nel presente studio gli Autori propongono l\u2019applicazione della Micro-TC all\u2019identificazione e quantificazione del GSR su ferite d\u2019arma da fuoco sottoposte a putrefazione standardizzata. La micro-TC risulta essere un valido strumento per la diagnosi differenziale tra foro di ingresso e foro di uscita, ma non per la determinazione della distanza di sparo