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 Methodologies for Selection and Prioritization of Compounds in Drug Discovery

Ph.DDOCTOR OF PHILOSOPH

RdRp (RNA-dependent RNA polymerase): A key target providing anti-virals for the management of various viral diseases

With the arrival of the Covid-19 pandemic, anti-viral agents have regained center stage in the arena of medicine. Out of the various drug targets involved in managing RNA-viral infections, the one that dominates almost all RNA viruses is RdRp (RNA-dependent RNA polymerase). RdRp are proteins that are involved in the replication of RNA-based viruses. Inhibition of RdRps has been an integral approach for managing various viral infections such as dengue, influenza, HCV (Hepatitis), BVDV, etc. Inhibition of the coronavirus RdRp is currently rigorously explored for the treatment of Covid-19 related complications. So, keeping in view the importance and current relevance of this drug target, we have discussed the importance of RdRp in developing anti-viral agents against various viral diseases. Different reported inhibitors have also been discussed, and emphasis has been laid on highlighting the inhibitor's pharmacophoric features and SAR profile. (c) 2021 Elsevier B.V. All rights reserved

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

A perspective on multi-target drug discovery and design for complex diseases

Diseases of infection, of neurodegeneration (such as Alzheimer's and Parkinson's diseases), and of malignancy (cancers) have complex and varied causative factors. Modern drug discovery has the power to identify potential modulators for multiple targets from millions of compounds. Computational approaches allow the determination of the association of each compound with its target before chemical synthesis and biological testing is done. These approaches depend on the prior identification of clinically and biologically validated targets. This Perspective will focus on the molecular and computational approaches that underpin drug design by medicinal chemists to promote understanding and collaboration with clinical scientists

Computer-aided drug design and synthesis of novel antivirals

The Flaviviridae is a family of 66 viruses of which almost half have been associated with human disease. The most well-known members are: Hepatitis C virus (HCV), Dengue virus (DV), and West Nile virus (WNV). Diseases caused by these viruses are a global health problem that put an estimated 2.5 billion people at risk. At present, there are neither vaccines nor other treatments available to prevent or cure these diseases. Potential targets for the development of therapeutics against the virus are the viral protease and polymerase. The aims of this project are to design and synthesize compounds that can be used as inhibitors for these two key enzymes for Dengue. Structure-based drug design methods utilize knowledge of a three dimensional structure of an enzyme/receptor to develop small molecules able to bind to the desired target, generating a specific biological response. These computer-based methodologies are now becoming an integral part of the drug discovery process and, although the principles of molecular recognition are far from being completely understood, some marketed compounds (i.e. Zanamivir, Lopinavir) have been developed with the help the of successful application of structure-based design techniques. Different structure-based drug design approaches have been used to identify putative new inhibitors for the Dengue protease and polymerase. A pharmacophore query has been built based on the active site of the Dengue protease enzyme and then used for screening different databases for identification of potential inhibitors. For the polymerase, a fragment-based approach has been used to find the fragments that would interact more efficiently with a specific binding pocket on the enzyme. The virtual library obtained by linking the best scored fragment was then docked to identify the most promising structures to be synthesized. The identification of potent small molecules that bind to receptors and enzymes is one of the major goals of chemical and biological research

Computer aided drug design

Hepatitis C virus (HCV) chronic infection represents one of the major and still unresolved health problems. HCV infecting 3% of the world population, leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to the extrahepatic manifestations. No efficient therapy exists; the standard dual treatment with peg IFN-alpha and ribavirin is effective only in 55% of the selected cases with substantial side effects in addition to the high cost. To date, there is no vaccine against HCV due to the high variability of the RNA genome. NS3 helicase is one of the non-structural proteins whose activity is indispensable for viral RNA replication and its inhibition is estimated to arrest viral proliferation and indirectly stimulate a cellular antiviral response against ds RNA. In our project we proposed to use structure based knowledge of the x-ray crystal structure of helicase enzyme to design and synthesise different scaffolds of novel potential HCV NS3 helicase inhibitors. Using different computer software packages, we manage to design a number of small focused libraries of compounds, which were used for docking simulations. The results obtained in silico guided the selection of two series of promising compounds for synthesis. In the first series; several quinazoline derivatives were prepared and evaluated for antiviral activity in subgenomic replicon assay showing EC50 in the low muM range with relatively high selectivity index. In the second series of pyrrole or phenyl based compounds, irreversible inhibition of helicase is assumed through addition to the electrophilic warheads of the alpha,beta-unsaturated ketones, thiols or 1,2,4 thiadiazoles based inhibitors. Among the synthesised compounds a number showed a sub muM activity in the helicase enzyme assay. These promising findings are considered to be a starting point for further optimisation of structure, activity and toxicity relationships