Application of Medicinal Chemistry Methods on Different Classes of Drugs

The present doctoral thesis is the result of the work carried out during the three years of my PhD scholarship at the Rome Center for Molecular Design laboratory (RCMD, Department of Chemistry and Drug Technologies, Sapienza University of Rome), under the supervision of Prof. Rino Ragno. The research activity was focused mainly on the design, optimization and application of computational strategies to derive quantitative structure-activity relationships (QSAR, 3-D QSAR, and COMBINE) on different molecular classes of current interest, such as: opioid receptor antagonists (OPAs), Hepatitis C Virus NS5B-Polymerase Inhibitors (NS5B-NNIs), Hystone Deacetylase Inhibitors (HDACIs), Anti- tubercular agents, vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors, HSP90 inhibitors, HIV-1 reverse transcriptase inhibitors (NNRTIs), Bovine Serum Amine Oxidase (BSAO) substrates, etc... Moreover two research periods abroad were performed: the first framed in a LLP Erasmus program collaboration, was conducted for six months at the Laboratoire d'Ingénierie et Moléculaire Pharmacologique Biochimie (LIMBP) of the Université de Lorraine Metz (France), directed by Prof. Gilbert Kirsch, and characterized by the application of organic synthesis to obtain new thienopyrimidinone derivatives as potential inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2); the second took place, for three months, at the Department of Biochemistry and Molecular Biophysics in Washington University School of Medicine in Saint Louis (MO, USA), under the supervision of Prof. Garland R. Marshall, investigating the activity profile of new Histone Deacetylases (HDACs) inhibitors by the application of the Mobility Shift Assay Technology. Main purpose of this doctoral thesis is to highlight the activities carried out in the different research projects, the applied methodologies and the obtained results. The text starts describing those studies whose results were published in scientific journals (chapters I-VI): the author decided to omit some procedural details, completely reported in the published papers, that would make the text too long, tedious and redundant; therefore readers who want to delve these aspects can also refer to Chapter XII in which is possible to read the original papers; on the contrary for studies that have not yet been published, as those characterizing the Chapters VII and VIII, discussion is adequately detailed. Chapters IX and X report the scientific activities carried out in France and in USA respectively; Chapter XI summarizes all the scientific activities accomplished during the entire PhD course, whereas Chapter XII, as mentioned, contains the published articles

Application of Medicinal Chemistry Methods on Different Classes of Drugs

The present doctoral thesis is the result of the work carried out during the three years of my PhD scholarship at the Rome Center for Molecular Design laboratory (RCMD, Department of Chemistry and Drug Technologies, Sapienza University of Rome), under the supervision of Prof. Rino Ragno. The research activity was focused mainly on the design, optimization and application of computational strategies to derive quantitative structure-activity relationships (QSAR, 3-D QSAR, and COMBINE) on different molecular classes of current interest, such as: opioid receptor antagonists (OPAs), Hepatitis C Virus NS5B-Polymerase Inhibitors (NS5B-NNIs), Hystone Deacetylase Inhibitors (HDACIs), Anti- tubercular agents, vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors, HSP90 inhibitors, HIV-1 reverse transcriptase inhibitors (NNRTIs), Bovine Serum Amine Oxidase (BSAO) substrates, etc... Moreover two research periods abroad were performed: the first framed in a LLP Erasmus program collaboration, was conducted for six months at the Laboratoire d'Ingénierie et Moléculaire Pharmacologique Biochimie (LIMBP) of the Université de Lorraine Metz (France), directed by Prof. Gilbert Kirsch, and characterized by the application of organic synthesis to obtain new thienopyrimidinone derivatives as potential inhibitors of vascular endothelial growth factor receptor-2 (VEGFR-2); the second took place, for three months, at the Department of Biochemistry and Molecular Biophysics in Washington University School of Medicine in Saint Louis (MO, USA), under the supervision of Prof. Garland R. Marshall, investigating the activity profile of new Histone Deacetylases (HDACs) inhibitors by the application of the Mobility Shift Assay Technology. Main purpose of this doctoral thesis is to highlight the activities carried out in the different research projects, the applied methodologies and the obtained results. The text starts describing those studies whose results were published in scientific journals (chapters I-VI): the author decided to omit some procedural details, completely reported in the published papers, that would make the text too long, tedious and redundant; therefore readers who want to delve these aspects can also refer to Chapter XII in which is possible to read the original papers; on the contrary for studies that have not yet been published, as those characterizing the Chapters VII and VIII, discussion is adequately detailed. Chapters IX and X report the scientific activities carried out in France and in USA respectively; Chapter XI summarizes all the scientific activities accomplished during the entire PhD course, whereas Chapter XII, as mentioned, contains the published articles

Εικονικός Βιολογικός Έλεγχος Βάσεων Δεδομένων Χημικών Μορίων με Τριδιάστατα Μοντέλα Ποσοτικών Σχέσεων Δομής-Δράσης (3D-QSAR): Εξερεύνηση Καινοτόμων Φαρμακευτικών Μορίων για τη Θεραπεία της Ηπατίτιδας C και της Μεσογειακής Αναιμίας

Σκοπός της διδακτορικής διατριβής ήταν ο in silico εντοπισμός νέων ενώσεων κατά των νόσων της ηπατίτιδας C και της μεσογειακής αναιμίας. Η ηπατίτιδα C, η οποία τις τελευταίες δεκαετίες διαδόθηκε παγκόσμια, οφείλεται στον ιό της ηπατίτιδας C (Hepatitis C Virus, HCV) που προσβάλει κυρίως το ήπαρ. Μέχρι σήμερα, δεν έχει ανακαλυφθεί το εμβόλιο κατά της ηπατίτιδας C, ενώ η σύγχρονη φαρμακευτική αγωγή είναι αποτελεσματική μόνο σε περιορισμένες περιπτώσεις. Η RNA-εξαρτώμενη RNA πολυμεράση NS5B του HCV είναι το κλειδί λειτουργίας της αντιγραφής του ιικού RNA, αποτελώντας θεραπευτικό στόχο της νόσου. Η αναζήτηση αναστολέων της RNA πολυμεράσης NS5B του HCV έχει οδηγήσει στη διερεύνηση των νουκλεοζιτικών (NIs) και μη νουκλεοζιτικών αναστολέων (NNIs), που δρουν στο καταλυτικό κέντρο και στις αλλοστερικές θέσεις («παλάμη», «αντίχειρας», «δάχτυλα») του ενζύμου, αντίστοιχα, με τους τελευταίους να έχουν προσελκύσει το ιδιαίτερο ενδιαφέρον των ερευνητών. Στην παρούσα εργασία, συνδυάστηκε η μοριακή πρόσδεση, το 3D-QSAR CoMSIA (Three Dimensional Quantitative Structure-Activity Relationship Comparative Molecular Similarity Indices Analysis) και η αναζήτηση ομοιότητας, με σκοπό να εντοπιστούν ισχυρά παράγωγα του ινδολίου στη βάση δεδομένων της ChEMBL, ως αναστολείς της αντιγραφής του HCV, μέσω της εικονικής διαλογής. Σε πρώτο στάδιο πραγματοποιήθηκαν υπολογισμοί μοριακής πρόσδεσης 41 παραγώγων στο ενεργό κέντρο του ενζύμου, «παλάμη» ΙΙ. Σε δεύτερο στάδιο χρησιμοποιήθηκε η πόζα πρόσδεσης της κάθε ένωσης για την ευθυγράμμιση με βάση τον υποδοχέα και την παραγωγή των πεδίων CoMSIA. Στη συνέχεια, κατασκευάστηκε ένα επικυρωμένο μοντέλο 3D-QSAR CoMSIA, ώστε να υπολογιστούν με ακρίβεια οι τιμές δραστικότητας. Η ροή εργασίας έδωσε πληροφορίες για τα δομικά χαρακτηριστικά που επηρεάζουν την πρόσδεση και την ανασταλτική δραστικότητα αυτών των παραγώγων στην πολυμεράση του HCV. Το ληφθέν in silico μοντέλο χρησιμοποιήθηκε για την πρόβλεψη της δραστικότητας νέων ενώσεων πριν από τη σύνθεση και βιολογική δοκιμή τους, μέσω της διαδικασίας της εικονικής διαλογής. Η βάση δεδομένων της ChEMBL που χρησιμοποιήθηκε έδωσε 18 νέες ενώσεις που περιέχουν το ινδολικό σκελετό και προβλέπεται να έχουν υψηλή δραστικότητα και, ως εκ τούτου, να τους δοθεί προτεραιότητα για βιολογική εξέταση. Ακολουθήθηκε μία παρόμοια ροή εργασίας με συνδυασμό των υπολογιστικών μεθόδων: (i) μοριακής πρόσδεσης, (ii) 3D-QSAR CoMFA (Comparative Molecular Fields Analysis), (iii) αναζήτησης ομοιότητας και (iv) εικονικής διαλογής της βάσης δεδομένων της PubChem για τον εντοπισμό νέων δυνάμει αναστολέων του HCV με δομή βασισμένη στο ανθρανιλικό οξύ. Αυτή τη φορά, 53 ενώσεις προσδέθηκαν στην αλλοστερική θέση της RNA πολυμεράσης, στον «αντίχειρα» II. Τα πεδία CoMFA δημιουργήθηκαν μέσω της ευθυγράμμισης των προσδεμένων δομών στο ένζυμο, ώστε να κατασκευαστεί ένα επικυρωμένο και σταθερό μοντέλο 3D-QSAR CoMFA. Το προτεινόμενο μοντέλο έδωσε μία πρώτη εικόνα για τα μοριακά χαρακτηριστικά που προάγουν τη βιοδραστικότητα, και στη συνέχεια μέσω της εικονικής διαλογής, εκτιμήθηκε η δραστικότητα νέων δυνάμει βιοδραστικών ενώσεων. Η μεσογειακή αναιμία (ή β-θαλασσαιμία) είναι μια κοινή διαταραχή του αίματος, που μειώνει την παραγωγή της αιμοσφαιρίνης (Hb) και εμφανίζεται σε όλες τις περιοχές της υφηλίου. Η φαρμακολογική επανενεργοποίηση του γονιδίου της γ- σφαιρίνης για την παραγωγή της εμβρυϊκής αιμοσφαιρίνης (HbF) αποτελεί μια πολλά υποσχόμενη θεραπευτική οδό για τη νόσο. Η ερυθρολευχαιμική ανθρώπινη κυτταρική σειρά Κ562 έχει τη δυνατότητα να εκφράσει τη γ- αλλά όχι τη β-σφαιρίνη. Η διαφοροποίηση των αιμοποιητικών κυττάρων Κ562 σχετίζεται με την αύξηση στην έκφραση των γονιδίων της εμβρυϊκής σφαιρίνης, όπως των γονιδίων της ζ, ε, και γ- σφαιρίνης. Αυτό το χαρακτηριστικό καθιστά τα κύτταρα Κ562 ένα ευρέως χρήσιμο μοντέλο κυτταρικής σειράς για τη μελέτη ενώσεων που είναι δυνητικοί επαγωγείς της γ-σφαιρίνης στη θεραπεία της β-θαλασσαιμίας. Σε μια προσπάθεια σχεδιασμού νέων χημειοτύπων με ενισχυμένη κυτταροτοξικότητα εναντίον των κυττάρων της σειράς Κ562, παράχθηκαν 3D φαρμακοφόρα μοντέλα, ενώ διεξήχθησαν μελέτες 3D-QSAR CoMFA και CoMSIA σε 33 (Ε)-α-βενζυλο-θειο χαλκόνες, ως νέοι αναστολείς της BCR-ABL. Η BCR-ABL είναι μια μονίμως ενεργή κινάση τυροσίνης, η οποία είναι υπεύθυνη για τον κακοήθη μετασχηματισμό και τη χρόνια μυελογενή λευχαιμία (CML). Αναπτύχθηκε ένα φαρμακοφόρο πέντε τοποθεσιών (AHHRR), με ένα δέκτη δεσμού υδρογόνου, δύο υδρόφοβες ομάδες, και δύο αρωματικούς δακτυλίους ως φαρμακοφόρα χαρακτηριστικά, και προέκυψε ένα σημαντικό στατιστικά μοντέλο 3D-QSAR με εξαιρετική δυνατότητα πρόβλεψης. Το φαρμακοφόρο μοντέλο χρησιμοποιήθηκε επίσης για την ευθυγράμμιση των 33 ενώσεων σε μία ανάλυση CoMFA/CoMSIA. Οι ισοϋψείς χάρτες για τα πεδία CoMFA και CoMSIA παρείχαν μία δομική εικόνα για το πώς αυτά τα μόρια προωθούν την τοξικότητα τους. Συζητήθηκε η δυνατότητα χρήσης αυτού του μοντέλου για τον σχεδιασμό φαρμάκων στη θεραπεία της β-θαλασσαιμίας, δεδομένου ότι αρκετοί αναστολείς της BCR-ABL είναι σε θέση να επάγουν τη διαφοροποίηση των αιμοποιητικών κυττάρων Κ562 (κυτταρική σειρά της CML BCR-ABL) και, ως εκ τούτου την ενεργοποίηση της γ- σφαιρίνης.The aim of this dissertation was the in silico identification of new compounds against hepatitis C and beta-thalassaemia diseases. Hepatitis C, caused by hepatitis C virus (HCV), mainly affects the liver and has spread worldwide in recent decades. To date, no vaccine has been discovered against hepatitis C virus, while the current therapy is effective only in limited cases. The HCV NS5B RNA-dependent RNA polymerase is the key function of the replication of viral RNA, constituting a therapeutic target of disease. Search of inhibitors of HCV NS5B RNA polymerase has led to the investigation of the nucleoside (NIs) and non-nucleoside inhibitors (NNIs), targeting on the catalytic site and allosteric sites (palm, thumb, fingers) of the enzyme, respectively. NNIs have attracted the particular interest of researchers. Molecular docking, 3D-QSAR CoMSIA and similarity search were combined in a multi-step framework with the ultimate goal to identify potent indole analogs, in the ChEMBL database, as inhibitors of HCV replication, in a virtual screening procedure. Initially, 41 known inhibitors were docked into the enzyme ‘‘Palm II’’ active site. In a second step, the docking pose of each compound was used in a receptor-based alignment for the generation of the CoMSIA fields. A validated 3D-QSAR CoMSIA model was subsequently built to accurately estimate the activity values. The proposed framework gave insight into the structural characteristics that affect the binding and the inhibitory activity of these derivatives on HCV polymerase. The obtained in silico model was used to predict the activity of novel compounds prior to their synthesis and biological testing, within a virtual screening procedure. The ChEMBL database was mined to afford 18 compounds containing the indole scaffold that are predicted to possess high activity and thus can be prioritized for biological screening. A similar combination of the computational methods: (i) molecular docking, (ii) 3D-QSAR CoMFA, (iii) similarity search and (iv) virtual screening using PubChem database was applied to identify new anthranilic acid-based inhibitors of HCV replication. 53 known inhibitors were initially docked into the ‘‘Thumb Pocket 2’’ allosteric site of the crystal structure of the RNA polymerase. Then, the CoMFA fields were generated through a receptor-based alignment of docking poses to build a validated and stable 3D-QSAR CoMFA model. The proposed model was utilized to get insight into the molecular features that promote bioactivity, and then a virtual screening procedure was used to estimate the activity of novel potential bioactive compounds. Beta-thalassaemia is a common blood disorder spread worldwide, that reduces the production of hemoglobin (Hb). Pharmacological reactivation of the γ-globin gene for the production of fetal haemoglobin (HbF) is a very promising therapeutic avenue for the disease. K562 human erythroleukemic cell line has the potential to highly express the γ- but not the β-globin gene. Erythroid differentiation of K562 cells is associated with an increase in the expression of embryo-fetal globin genes such as ζ, ε, and γ-globin genes. This characteristic makes K562 cells a widely useful model cell line for the study of compounds that are potential γ-globin inducers for use β–thalassaemia In an attempt to aid the design of new chemotypes with enhanced cytotoxicity against K562 cells, 3D pharmacophore models were generated and 3D-QSAR CoMFA and CoMSIA studies were carried out on the 33 novel ABL kinase inhibitors (E)-α- benzylthio chalcones. BCR-ABL is a constitutively active tyrosine kinase that is responsible for the malignant transformation and chronic myelogenous leukemia (CML). A five-point pharmacophore (AHHRR) with a hydrogen bond acceptor, two hydrophobic groups, and two aromatic rings as pharmacophore features, and a statistically significant 3D-QSAR model with excellent predictive power were developed. The pharmacophore model was also used for alignment of 33 compounds in a CoMFA/CoMSIA analysis. The contour maps of the fields of CoMFA and CoMSIA models were utilized to provide structural insight into how these molecules promote their toxicity. The possibility of using this model for the design of drugs for the treatment of β–thalassaemia since several BCR-ABL inhibitors are able to promote erythroid differentiation and γ–globin expression in CML cell lines and primary erythroid cells was discussed

In silico investigation of hepatitis c virus: a novel perspective into targeted viral inhibition of NS3 helicase, NS 3/4a protease and NS5b RNA dependent RNA polymerase.

Doctoral Degrees (Pharmaceutical Sciences). University of KwaZulu-Natal. Westville, 2019.Hepatitis C Virus (HCV) is an escalating global healthcare and economic burden that requires extensive intervention to alleviate its control. Over the years, drug design efforts have produced many anti-HCV drugs; however, due to drug resistance brought on by numerous genetic variations of the virus and lack of specificity and stability, current drugs are rendered ineffective. The situation has been further intensified by the absence of a viable vaccine. For these reasons, continuous HCV research is imperative for the design and development of promising inhibitors that address the challenges faced by present antiviral therapies. Moreover, exposure of previously neglected viral protein targets can offer another potentially valuable therapeutic route in drug design research. Structure-based drug design approaches accentuate the development of small inhibitor molecules that interact with therapeutic targets through non-covalent interactions. The unexpected discovery of covalent inhibitors and their distinctive nature of instigating complete and irreversible inhibition of targets have shifted attention away from the use of non-covalent drugs in antiviral treatment. This has led to significant progress in understanding covalent inhibition regarding their underlying mechanism of action and in the design of novel covalent inhibitors that work against biological targets. However, due to difficulties arising in its application and resultant safety, the pharmaceutical industry were reluctant to pursue this strategy. With the use of rational drug design, a novel strategy was then proposed known as selective covalent inhibition. Due to the lack of competent protocols and information, little is known regarding selective covalent inhibition This study investigates three biological HCV targets, NS3 protease, RNA helicase and NS5B RNAdependent RNA polymerase. With constantly evolving viruses like HCV, computational methods including molecular modelling and docking, virtual screening and molecular dynamic simulations have allowed chemists to screen millions of compounds to filter out potential lead drugs. These in silico approaches have allowed Computer-Aided Drug Design as a cost-effective strategy to accelerate the process of drug discovery. The above techniques, with numerous other computational tools were employed in this study to fill the gap in HCV drug research by providing insights into the structural and dynamic changes that describe the mechanism of selective covalent inhibition and pharmacophoric features that lead to unearthing of potential small inhibitor molecules against Hepatitis C. v The first study (Chapter 4) provides a comprehensive review on HCV NS3/4A protein, current therapies and covalent inhibition as well as introduces a technical guideline that provides a systematic approach for the design and development of potent, selective HCV inhibitors. The second study (Chapter 5) provides a comprehensive understanding concerning the implications of selective covalent inhibition on the activity of HCV NS5B RNA-dependent RNA polymerase, with respect to key components required for viral replication, when bound to a target-specific small inhibitor molecule. The third study (Chapter 6) is preliminary investigation that uses Pharmacophore-based virtual screening as an efficient tool for the discovery of improved potential HCV NS3 helicase inhibitors. The pharmacophoric features were created based on the highly contributing amino acid residues that bind with highest affinity to the weak inhibitor, quercetin. These residues were identified based on free energy footprints obtained from molecular dynamic and thermodynamic calculations. Post molecular dynamic analysis and appropriate drug-likeness properties of the three top-hit compounds revealed that ZINC02495613 could be a more effective potential HCV helicase inhibitor; however, further validation steps are still required. This study offers a comprehensive in silico perspective to fill the gap in rational drug design research against HCV, thus providing an insight into the mechanism of selective covalent inhibition, uncovering a previously neglected viral target and identifying possible antiviral drugs. To this end, the work presented in this report is considered a fundamental platform to advance research toward the design and development of novel and selective anti-HCV drugs

In Silico Methodologies for Selection and Prioritization of Compounds in Drug Discovery

Ph.DDOCTOR OF PHILOSOPH

Identification and characterisation of small molecule inhibitors targeted to the hepatitis C virus NS2 autoprotease.

Hepatitis C virus (HCV) is a positive-strand RNA virus present in 2-3% of the global population and commonly establishing a chronic infection, leading to long term diseases such as liver cirrhosis and hepatocellular carcinoma. Recent advances have led to the development of a range of direct-acting anti-viral drugs (DAAs), some of which are already improving outcomes in the clinic. It is clear however, that effective therapy for the treatment of HCV will most likely require a combination of DAAs to overcome the rapid onset of viral resistance. In this regard additional inhibitors of the virus lifecycle, which act through a novel molecular target, are required. The autoprotease activity encoded within the C-terminus of the non-structural 2 (NS2) protein is essential for processing of a precursor to the mature viral proteins, and as a consequence is also required for the onset of viral genome replication and the establishment of HCV infection. Despite representing an attractive target for anti-virals, no inhibitors of the NS2 autoprotease have been reported. In order to identify small molecule inhibitors of the NS2 autoprotease, two independent assays were optimised as a measure of NS2-mediated proteolysis. These assays were employed to demonstrate that inhibitors of the NS2 autoprotease were able to block HCV genome replication. The assays were subsequently used to identify a lead-like small molecule inhibitor by screening an in silico enriched library. This compound was further characterised in the context of NS2 activity in vitro and cell culture models of the virus lifecycle. The resultant series represent the first documented inhibitors capable of exerting an anti-viral effect by targeting the NS2 autoprotease