Molecular View on the iRGD Peptide Binding Mechanism: Implications for Integrin Activity and Selectivity Profiles

Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets alpha v beta 3 and alpha v beta 5 but also the alpha v beta 6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties

Identification of a Novel p53 Modulator Endowed with Antitumoural and Antibacterial Activity through a Scaffold Repurposing Approach

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Innovative Computational Approaches in Drug Discovery: Design and Development of Brand New Chemotherapeutic Agents

This PhD thesis is mainly focused on the applications of classical and advanced computational techniques to the medicinal chemistry field. Particularly, the wide armoury of known computational methods was here exploited to facilitate the identification and the development of new potential drug candidates. The manuscript is divided into four chapters. The first one describes the classical drug discovery pipeline and the advantages offered by computer-aided approaches. In the second one, a theoretical overview of the methodologies applied in this work is provided. The final chapters (3 and 4) focus on four research projects, divided into two distinct sections, based on the kind of employed methodologies. In detail, in Chapter 3, two studies centred on the ligand binding problem are presented: i) a successful virtual screening campaign targeting the DNA G-Quadruplex structure of the KRAS proto-oncogene promoter, and ii) a mechanistic insight into the binding mechanism of small molecules to FPR2, a GPCR involved in the resolution of the inflammatory process. In Chapter 4, instead, the importance of an accurate conformational sampling for rationalizing the activity/selectivity profile of peptide ligands is highlighted. In the first case study, due to the availability of NMR-derived data, a mixed computational-experimental approach was adopted to investigate the folding and binding properties of a small cyclopeptide, endowed with remarkable antiviral activity against Herpes Simplex Virus 1 (HSV1) infections. On the other hand, in the last project of this thesis, a purely computational approach was employed for studying the binding mechanism of the well-characterized antitumoral nonapeptide iRGD to integrin receptors

A periodic hybrid DFT approach (including dispersion) to MgCl2-supported Ziegler-Natta catalysts-1: TiCl4 adsorption on MgCl2 crystal surfaces

The adsorption of TiCl4 on the surfaces of MgCl2 crystals has been investigated by means of state-of-the-art periodic hybrid DFT methods, as the first step of a comprehensive study aiming to elucidate the structure of the active species in industrial MgCl2-supported Ziegler-Natta catalysts for ethene and propene polymerization. A first distinctive feature of the approach was the thorough evaluation of dispersion forces, crucial because the binding of TiCl4 on MgCl2 surfaces turned out to be essentially dispersion-driven. Also important was a careful investigation of the effects of different choices on basis set and density functional (DF) on the quantitative aspects of the results; this allowed us to trace the unusually large disagreement in the previous literature and identify unambiguous trends. In particular, three full sets of calculations were run adopting the B3LYP(-D), PBE0(-D) and M06 approximations: to the best of our knowledge, the last represents the first case of M06 functional implementation in a periodic code (CRYSTAL) of widespread use. The results consistently indicated that the adsorption of TiCl4 on well-formed MgCl2 crystals under conditions relevant for catalysis can only occur on MgCl2(1 1 0) or equivalent lateral faces, whereas the interaction with MgCl2(1 0 4) - for decades claimed as the most important surface in stereoselective catalysts - is too weak for the formation of stable adducts. The implications of these findings for catalysis are discussed. (C) 2011 Elsevier Inc. All rights reserved

Design, synthesis and biological evaluation of novel TRβ selective agonists sustained by ADME-toxicity analysis

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the β isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRβ and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as "potentially safe" to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRβ-selective thyromimetics

Disulfide bond replacement with 1,4‐ and 1,5‐disubstituted [1,2,3]‐triazole on C‐X‐C chemokine receptor type 4 (CXCR4) peptide ligands: small changes that make big differences

Here we investigate the structural and biological effects ensuing form the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors