The increasing use of information technology in the discovery of new molecular entities encourages the use of modern molecular-modeling tools to help teach important concepts of drug design to chemistry and pharmacy undergraduate students. In particular, statistical models such as quantitative structure–activity relationships (QSAR)—often as its 3D QSAR variant—are commonly used in the development and optimization of a leading compound. We describe how these drug discovery methods can be taught and learned by means of free and open-source web applications, specifically the online platform . This new suite of web applications has been integrated into a drug design teaching course, one that provides both theoretical and practical perspectives. We include the teaching protocol by which pharmaceutical biotechnology master students at Pharmacy Faculty of Sapienza Rome University are introduced to drug design. Starting with a choice among recent articles describing the potencies of a series of molecules tested against a biological target, each student is expected to build a 3D QSAR ligand-based model from their chosen publication, proceeding as follows: creating the initial data set (Py-MolEdit); generating the global minimum conformations (Py-ConfSearch); proposing a promising mutual alignment (Py-Align); and finally, building, and optimizing a robust 3D QSAR models (Py-CoMFA). These student activities also help validate these new molecular modeling tools, especially for their usability by inexperienced hands. To more fully demonstrate the effectiveness of this protocol and its tools, we include the work performed by four of these students (four of the coauthors), detailing the satisfactory 3D QSAR models they obtained. Such scientifically complete experiences by undergraduates, made possible by the efficiency of the 3D QSAR methodology, provide exposure to computational tools in the same spirit as traditional laboratory exercises. With the obsolescence of the classic Comparative Molecular Field Analysis Sybyl host, the 3dqsar web portal offers one of the few available means of performing this well-established 3D QSAR method.
Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
Background Diffuse gliomas display heterogeneous biology, natural history, response to treatments, and outcome. According to the 2021 WHO Classification, an integration of histological and molecular factors is needed for the diagnosis of diffuse gliomas. The Italian Association of Neuro-Oncology (AINO), with the participation of the Italian Society of Neurosurgery (SINch), promoted a survey to explore how the 2021 WHO molecular diagnostic criteria are integrated into clinical practice in a national framework. Material and Methods A web-based survey containing 38-item multiple-choice questions was sent to members of the AINO and SINch in February 2022 via the respective email listings of these organizations. Results We collected 152 answers. Most attendants were < 45-year-old (117, 77.0%). Participants from North, Centre and South of Italy were 85 (55.9%), 38 (25.0%), and 29 (19.1%). Academic and non-academic hospitals were 35 (46.1%) and 82 (53.9%). The presence of an institutional Brain Tumour Board was reported in 108 cases (71.7%). One hundred forty attendees (92.1%) reported that IDH mutation was assessed in all glioma patients regardless of age. The 1p19q-codeletion was assessed routinely in all IDH-mutant gliomas in 88 (57.9%) or when TP53 mutation and/or ATRX expression was found (45, 29.6%). The MGMTp methylation was assessed, regardless of grading, at diagnosis in 110 (72.4%), and at second surgery in 82 (53.9%). Eighty (52.6%) performed a quantitative analysis of MGMTp status. The CDKN2A/B homozygous deletion in IDH-mutant lower-grade astrocytomas was routinely investigated in 53 (34.9%). Assessment of EGFR amplification, pTERT status or +7/-10 chromosome alterations to stratify IDH-wildtype lower-grade astrocytomas was reported in 76 (50.0%), 43 (28.3%), and 16 (10.5%) cases. Rarer alterations were less commonly investigated (H3K27M: 34, 22.4%; H3G34: 11, 7.2%; BRAF: 18, 11.8%; NTRK: 16, 10.5%), being usually evaluated in selected cases (e.g., younger patients). Academic vs non-academic hospitals treated more patients per year (> 300 in 22/70, 31.4% vs 3/82, 3.7%, p<0.001), had more available molecular technologies (53/70, 75.5% vs 37/82, 45.1, p<0.001), had a higher availability of molecular markers, such as CDKN2A/B deletion (34/70, 48.6% vs 19/82, 23.2%, p=0.001), MGMTp at second surgery (48/69, 69.6% vs 34/72, 47.2%, p=0.008), EGFR/pTERT/+7-10 (46/70, 65.7% vs 32/77, 41.6%, p=0.003), BRAF (14/70, 20.0% vs 4/82, 4.9%, p=0.002), NTRK (14/70, 20.0% vs 2/81, 2.5%, p<0.001). Conclusion The availability of new molecular markers is increasing among Italian Neuro-Oncology Centres. However, there is still a gap with the proposed criteria of the 2021 WHO Classification and the real-life application. A critical issue remains how to select patients who might benefit from the identification of some extremely rare mutations in light of targeted therapies.
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