Design of Small Molecules with Antitumor Activity through Computational Methodologies

Today, there are multiple targeted therapies against cancer. The most relevant ones are those aimed at the stop of cancer cells from growing, or at the halting of signals that stimulate blood vessels, or at helping the immune system destroy cancer cells, and many others. The last one, has achieved impressive results to date. Indeed, the immuno- oncology field is entering a new, exciting phase having the potential to change the current cancer treatment either as a standalone therapy or in combination. Recently, many innovative strategies exist to overcome tumor-induced immunosuppression. Currently the main ones are checkpoint blockade inhibitors, adoptive T cell transfers, and vaccination strategies. To date, the immuno-oncology therapeutics on the market are mostly biologic products (e.g. monoclonal antibodies (mAbs), proteins, engineered cells, and oncolytic viruses). However, for example, antibodies have specific drawbacks: high production costs, lack of oral bioavailability, poor tumor penetrating capacity, Fc-related toxicities, and immunogenic properties. In this perspective, small molecules could potentially overcome many of these issues and be complementary to, and potentially synergistic with, biologic therapeutics too. In this context, my PhD work was focused on discovery of small molecules targeting three different proteins: MDM2 (Mouse Double Minute 2) the PD-1/PD-L1 axis (Programmed cell Death protein-1/ Programmed Death-ligand 1), and STING protein (STimulator of INterferon Genes). For all targets, a tandem approach of computational studies/NMR spectroscopy was applied

Cytosine epigenetic modifications and conformational changes in G-quadruplex DNA: An ultraviolet resonance Raman spectroscopy study

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G-quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G-quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G-quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G-quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G-quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G-quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G-quadruplex structure elucidation under biologically relevant solution conditions

HOPPI-NMR: Hot-Peptide-Based Screening Assay for Inhibitors of Protein-Protein Interactions by NMR

Protein-protein interactions (PPIs) contribute to the onset and/or progression of several diseases, especially cancer, and this discovery has paved the way for considering disruption of the PPIs as an attractive anti-tumor strategy. In this regard, simple and efficient biophysical methods for detecting the interaction of the inhibitors with the protein counterpart are still in high demand. Herein, we describe a convenient NMR method for the screening of putative PPI inhibitors based on the use of "hot peptides" (HOPPI-NMR). As a case study, HOPPI-NMR was successful applied to the well-known p53/MDM2 system. Our outcomes highlight the main advantages of the method, including the use of a small amount of unlabeled proteins, the minimization of the risk of protein aggregation, and the ability to identify weak binders. The last leaves open the possibility for application of HOPPI-NMR in tandem with fragment-based drug discovery as a valid strategy for the identification of novel chemotypes acting as PPI inhibitors

Theoretical and experimental studies on the interaction of biphenyl ligands with human and murine PD-L1: Up-to-date clues for drug design

: Today it is widely recognized that the PD-1/PD-L1 axis plays a fundamental role in escaping the immune system in cancers, so that anti-PD-1/PD-L1 antibodies have been evaluated for their antitumor properties in more than 1000 clinical trials. As a result, some of them have entered the market revolutionizing the treatment landscape of specific cancer types. Nonetheless, a new era based on the development of small molecules as anti PD-L1 drugs has begun. There are, however, some limitations to advancing these compounds into clinical stages including the possible difficulty in counteracting the PD-1/PD-L1 interaction in vivo, the discrepancy between the in vitro IC50 (HTFR assay) and cellular EC50 (immune checkpoint blockade co-culture assay), and the differences in ligands' affinity between human and murine PD-L1, which can affect their preclinical evaluation. Here, an extensive theoretical study, assisted by MicroScale Thermophoresis binding assays and NMR experiments, was performed to provide an atomistic picture of the binding event of three representative biphenyl-based compounds in both human and murine PD-L1. Structural determinants of the species' specificity were unraveled, providing unprecedented details useful for the design of next generation anti-PD-L1 molecules

Discovery of the First-in-Class GSK-3β/HDAC Dual Inhibitor as Disease-Modifying Agent to Combat Alzheimer's Disease

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3β (GSK-3β), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H 2 O 2 and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (<100 μM) and high water solubility

Interfering with the Tumor-Immune Interface: Making Way for Triazine-Based Small Molecules as Novel PD-L1 Inhibitors

: The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 (1) as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications

Impact of asthma and comorbid allergic rhinitis on quality of life and control in patients of italian general practitioners

Objective. Asthma is a disease with elevated prevalence within the general population. Although general practitioners (GPs) are among the first health-care professionals to whom patients refer for their symptoms, there are few evaluations of this disease based on data provided by the GPs. The aim of this observational study is to assess the impact of asthma and comorbid allergic rhinitis on individual/social burden, quality of life, and disease control in asthmatic patients of Italian GPs. Methods. Throughout Italy, 107 GPs enrolled 995 patients diagnosed with asthma and using anti-asthmatic drug prescriptions, or with asthma-like symptoms during the previous 12 months. Data were collected through questionnaires filled out by GPs and patients. Results. Of the 995 asthmatic patients, 60.6 had concomitant allergic rhinitis (RA), 39.4 had asthma alone. The latter, compared to those with RA, showed significantly lower prevalence of intermittent asthma (37.5 vs. 55.6) and higher prevalence of mild, moderate, and severe persistent asthma (28.4 vs. 23.2, 28.7 vs. 18.8, and 5.4 vs 2.4, respectively). Individual/social burden due to asthma was frequent and increased with disease severity: 87.5 of severe persistent asthma patients reported at least one medical consultation in the last 12 months, 37.5 emergency department visits, 26.7 hospitalization, and 62.5 limitations in daily activities. Control and quality of life were inversely associated with disease severity and were worse in patients with RA than in those with asthma alone. Conclusions. This study showed the negative impact of high severity levels and comorbid allergic rhinitis on quality of life of asthmatic patients and on individual/social burden due to asthma in an Italian GPs setting. \uc2\ua9 2012 Informa Healthcare USA, Inc

The ARGA study with general practitioners: Impact of medical education on asthma/rhinitis management

Aim: To evaluate the impact of a medical education course (MEC) on the behaviour of general practitioners (GPs) to treat asthma and allergic rhinitis (AR). Methods: Data on 1820 patients (mean age 41yrs \uc2\ub1 17yrs) with asthma or AR were collected by 107 Italian GPs: 50% attended a MEC and 50% didn't (group B). The adherence for AR and asthma treatment was evaluated according to ARIA and GINA guidelines (GL). Results: AR and asthma were diagnosed in 78% and 56% of patients; 34% had concomitant AR and asthma. Regardless of the MEC, the adherence to GL was significantly higher for AR than for asthma treatment (52 versus 19%). Group B GPs were more compliant to ARIA guidelines in the treatment of mild AR, whereas group A were more compliant in the treatment of moderate-severe AR; the adherence didn't differ between the groups for AR patients with comorbid asthma. Adherence to GINA GL for asthma treatment did not differ between GPs of groups A and B, independently from concomitant AR. Though insignificantly, group A were more compliant to GINA GL in the treatment of patients with only severe persistent asthma (63 versus 46%) as group B were for patients with severe persistent asthma and concomitant AR. Conclusions: GPs often tend to treat patients independently from GL. The impact of a single MEC did not improve adherence to GL in treating less severe AR and asthma patients, while there was a trend towards the opposite attitude in more severe AR patients without concomitant asthma. \uc2\ua9 2012 Elsevier Ltd. All rights reserved

Prescriptive adherence to GINA guidelines and asthma control: An Italian cross sectional study in general practice

Background: Although general practitioners (GPs) are frequently the first healthcare professionals whom asthma patients refer to for their symptoms, few studies have explored the extent of adherence to guidelines for asthma management based on data provided directly by GPs. Aims of the present study were to assess drug prescriptions for asthma by GPs and to evaluate prescriptive adherence to GINA guidelines (GL) and its relationship with disease control in real life. Methods: 995 asthmatic patients (45% males, mean age 43.3 ± 17.7 yrs) were enrolled by 107 Italian GPs distributed throughout the country. Data on diagnosis, disease severity, prescribed anti-asthmatic drugs and control were collected through questionnaires filled out by GPs taking into consideration the 2009 GINA Guidelines. Data on drug use and chronic sinusitis, nasal polyposis, chronic bronchitis, emphysema were reported by patients through a self-administered questionnaire. Results: The large majority of patients were classified by GPs as having intermittent (48.4%) or mild persistent asthma (25.3%); 61% had co-morbid allergic rhinitis (AR). The prevalent therapeutic regimen used by patients was a combination of inhaled corticosteroids (ICS) plus long-acting β2-agonists (LABA) (54.1%), even in the intermittent/mild persistent group. ICS as mono-therapy or in combination with other drugs but LABA, was the second most frequently adopted treatment (14.4%). In general, the GPs adherence to GL treatment indications was 28.8%, with a significant association with a good asthma control (OR 1.85, 95% CI 1.18–2.92). On the other hand, comorbidity (OR 0.52, 95% CI 0.32–0.84), moderate (0.44, 0.28–0.69) and severe (0.06, 0.02–0.20) persistent asthma showed significant negative effects on asthma control. Conclusions: Our results show that over-treatment of intermittent/mild persistent asthma is frequent in the GPs setting while therapeutic regimens are more appropriately applied for moderate/severe asthma. In general, we found low adherence to GINA GL treatment recommendations even if its relevance in asthma control was confirmed