Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

An estimated 2.5 billion people are at risk of diseases caused by dengue and West Nile virus. As of today, there are neither vaccines to prevent nor drugs to cure the severe infections caused by these viruses. The NS3 protease is one of the most promising targets for drug development against West Nile virus because it is an essential enzyme for viral replication and because success has been demonstrated with the closely related hepatitis C virus protease. We have discovered a small molecule that inhibits the NS3 protease of West Nile virus by computer-aided high-throughput docking, and validated it using three experimental techniques. The inhibitor has potential to be developed to a drug candidate to combat West Nile virus infections

Hepatitis C Virus Non-Structural Protein 3/4A: A Tale of Two Domains: A Dissertation

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure

New Direct Acting Anti-Virals Inhibiting Hepatitis C Virus Helicase and Insights into How ATP Fuels Helicase Action

According to the World Health Organization, Hepatitis C Virus (HCV) has infected 130-150 million people worldwide. Approximately 700,000 of those die each year from chronic HCV related causes such as cirrhosis or cancer. Currently, there are numerous HCV drugs on the market; they target the protease, polymerase and NS5A proteins encoded by of HCV. These drugs are expensive and HCV can become resistant, thus there is constant need for new DAAs. The first part of this thesis examines the search for additional drugs that function by inhibiting the NS3 helicase, which have been challenging to develop. Part of the reason for a lack of helicase inhibitors can be due to the difficulty of understanding its mechanism. The helicase is a motor protein that couples ATP hydrolysis to DNA or RNA unwinding. The second part of this thesis examine the role of a cysteine residue in the helicase ATP binding site. When the cysteine was replaced with other amino acids, the protein possessed unusual features not seen in the wildtype helicase. Helicase proteins lacking the cysteine, were able to hydrolyze ATP in the absence of nucleic acid 15times faster than wildtype. This finding may provide future information into the coupling mechanism of chemical energy to physical motions of the enzyme

In silico design of selective high affinity ligands against HCV using novel computational diology tools

Background: Hepatitis C virus (HCV) infects 170 million patients worldwide. The absence of an effective mean of treatment or prophylaxis makes HCV infection a serious public health problem. Generating selective high affinity ligands (SHALs) against HCV could provide a solution to this public health burden. HCV E2 glycoprotein is required for HCV entry into host cells. It binds to CD81, a host receptor protein that belongs to the tetraspanin family and plays a critical role in viral invasion. HCV protease is essential for the cleavage of non structural proteins, in addition to preventing the phosphorylation of human interferon regulatory factor 3 and thus prevents the anti-viral response. Objective: To design a cocktail of SHAL-based inhibitors against several target proteins such as CD81, HCV E2, and HCV protease and optimize the currently available E2 homology models. Methods and findings: Different homology modeling techniques such as AS2TS, Phyre, ROSETTA and TASSER, were used to obtain reliable models of HCV NS3 serine protease and HCV E2 glycoprotein. LGA was used to structurally analyze the models in addition of clustering the obtained models and finding the closest structural templates using StralCP. Auto Dock Tools 1.5.6 was used to prepare the crystal structures of the CD81-LEL protein (1G8Q and 1IV5), HCV protease, HCV polymerase and the homology model of HCV E2 by deleting water molecules, adding polar hydrogens, and assigning Gasteiger charges and to create a grid bounding box, which provided the desired grid parameter file using 0.375 A spacing. Autoligand, an AutoDock tool, was used to identify several binding sites on the protein targets. Fill points were created using a 1 A grid, and the calculations were performed using 10 to 210 fill points. AutoDock 4.2 was used to screen 30,000 ligands obtained from different libraries (NCI_DSII, Sigma and Asinex) and identify small molecules that might bind to each site. The docking results were analyzed and the top 20 ligands for each binding site on the target proteins were ranked according to selection criteria required for the design of promising SHALs. Distances between pairs of bound ligands were estimated and used to design several SHALs that should bind selectively to the target proteins. Conclusion: New computational tools have been used to design in silico several SHAL-based inhibitors that might have the potential to prevent both HCV entry into hepatocytes and the production of inflammatory cytokines that accelerate liver damage when targeting E2-CD81 interaction. It might alter protein processing and viral replication if HCV protease was targeted. By targeting HCV RNA dependent RNA polymerase, the HCV replication could be blocked. In addition, blocking the complexation of NS3 with the NS4A co-factor will render it non functional and thus block the replication process and disrupt the HCV life cycle. If a reliable E2 homology model was developed based on different studies and validations, it could help generate selective high affinity ligands that block the earliest phase of the HCV life cycle

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

Identification of drug leads against HCV and malaria using different target proteins

Hepatitis C Virus (HCV) infects 170 million individuals worldwide. Although several newly FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating all HCV genotypes and subtypes to be used in an interferon-free regimen. On the other hand, malaria is another public health burden that causes 219 million clinical episodes, and 660,000 deaths per year. In addition, 3.3 billion people live in areas at risk of malaria transmission in 106 countries. It is alarming that 86% of deaths caused by malaria globally were in children. Several challenges are faced when treating malaria, such as resistance against drugs that are used in treatment. This necessitates the development of new classes of drugs to overcome resistance. CD81 is a target protein that plays an essential role in the internalization of HCV into hepatocytes. Thus it was also targeted to identify sets of small molecule ligands predicted to bind to several sites that were identified to be involved in HCV infection. Thirty-six ligands predicted by AutoDock to bind to these sites were tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 23 out of 36 of the ligands bound in vitro to the recombinant CD81-LEL protein. In an effort to create new drugs that block hepatitis C virus entry into hepatocytes, we have designed and synthesized a small molecule that targets the HCV E2 glycoprotein binding site on CD81. A selective high affinity ligand (SHAL) (11) was created by linking together two small molecules that were predicted by docking and were shown by experimental methods to bind to the same site on CD81 where E2 binds. SH7153 was found to bind to recombinant CD81-LEL with a Kd of 21 µM but wasn’t found to inhibit HCV infection when tested using Raji cells (antibody neutralizing assays) and HCV infection inhibition assays. This led to the conclusion that the linkers’ lengths should be optimized so as to have a SHAL that fits properly in the desired binding sites. The HCV glycoprotein E2 has also been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. Recently, 2 research groups were able to resolve the core structure of HCV E2 which will largely help providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. By targeting conserved E2 residues among different genotypes and subtypes in the CD81 binding site on HCV E2, one might also be able to develop drugs that block HCV infection in a genotype-independent manner. Using the E2c structure as a template, we have used homology modeling methods to develop a structural model of the E2 protein core (residues 421-645) that includes the three amino acid segments that are not present in the E2c structure. Blind docking to this model was then performed using a library of ~4000 small molecules and a set of 40 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction were selected for experimental testing. Surface Plasmon resonance was used to screen the ligands for binding to recombinant E2 protein and the best binders were subsequently tested to identify compounds that inhibit the infection of hepatocytes by HCV. One compound, 281816, inhibited infection by HCV genotypes 1a, 1b, 2a, 2b, 4a and 6a with IC50’s ranging from 2.2 uM to 4.6 uM. Such inhibitors may represent a new paradigm for HCV treatment. In an attempt to make 281816 more promising, a SHAL prototype was designed using an analogue of 281816 (SH2216). It would be tempting to test the SHAL inhibitory effect and compare it to the 281816’s inhibitory effect. To date, human CD81 (hCD81) is the only human surface protein known to play a role in the process by which sporozoites of several Plasmodium species infect human hepatocytes. Blocking a human receptor that is exploited for the entry process of pathogens has been proven to be a good strategy for fighting drug-resistant mutants. Hence, we targeted the 21 amino acid stretch on CD81 large extracellular loop that was found to be involved in Plasmosium yoleii invasion via virtual screening runs, preliminary binding assays and sporozoite invasion assays. This led to the identification of 4 drug leads that range between moderate and strong inhibitors of infection by Plasmodium yoleii and Plamodium falciparum. Additionally one ligand was found to potentiate the invasion of Plasmodium yoleii

Hepatiit C viiruse ja Chikungunya viiruse vastased lähenemised

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tänapäeval on võimalik ennast erinevate viiruste vastu vaktsineerida ning ka viirushaiguste ravi on muutunud oluliselt tõhusamaks. Samas leidub endiselt meditsiiniliselt olulisi viiruseid, mille vastu puudub vaktsiin ja/või mille poolt põhjustatud haigustele pole siiani adekvaatset ravi. Viirus-vastaste ühendite ja vaktsiinide väljatöötamist raskendavad nii viiruste suur mitmekesisus kui ka nende keeruline elutsükkel. Üheks selliseks viiruseks on C hepatiidi viirus (HCV), mis on kroonilise maksahaiguse levinuimaks tekkepõhjuseks. Hinnanguliselt on selle viirusega krooniliselt nakatunud ~3% inimkonnast. Kuigi HCV infektsiooni ravis on toimunud suur läbimurre, on viiruse geneetilise mitmekesisuse, ravimresistentsete vormide tekkimise ning patsientide ravile mitteallumise tõttu endiselt väga oluline uute HCV vastaste ravimite väljatöötamine. Antud uurimustöö üheks eesmärgiks oli analüüsida erinevaid tehnoloogilisi lahendusi HCV vastaste ühendite loomiseks. Ühe lähenemisena valiti FQSAR arvutiprogrammi põhiselt välja madal-molekulaarsed ühendid, mis seondudes HCV NS3/4A proteaasiga inhibeerivad HCV replikatsiooni, ja iseloomustati nende mõju viiruse infektsioonile. Kõik analüüsitud seitse ühendit omasid HCV-vastast efekti, kuid ainult üks ühend (23332) oli kasutatavas kontsentratsioonis mitte-toksiline. Teine lähenemisviis seisnes looduslikult esineva modifikatsiooni (8-oxo-dG) mõju analüüsimises oligonukleotiidsete (ON) inhibiitorite efektiivsusele. Kombineerides erinevaid modifikatsioone leiti ON ühend, mis inhibeeris HCV replikatsiooni nanomolaarsetel kontsentratsioonidel. Lisaks HCV uurimisele on võimalik käsitletud lähenemise kasutada ka teiste viiruste vastu suunatud ühendite väljatöötamisel. Chikungunya viirus (CHIKV, perekond Alfaviirus) on troopilistes piirkondades leviv arboviirus, mis on viimasel aastakümnel korduvalt väljunud oma tavalisest levialast ja põhjustanud epideemiaid erinevates maailmajagudes. Antud töö kolmandaks eesmärgiks oli analüüsida uudsete CHIKV-vastaste vaktsiinikandidaatide geneetilist stabiilsust ning uurida nendes sisalduvate viirust nõrgestavate mutatsioonide mõju CHIKV elutsüklile. Leiti, et viirustel CHIKVΔ5nsP3 ja CHIKVΔ6K on nõrgestatud fenotüüp ka pärast mitmekordset passeerimist koekultuuri rakkudes. Mitmetest analüüsitud CHIKV-vastastest vaktsiini kandidaatidest osutus kõige efektiivsemaks CHIKVΔ5nsP3. See nõrgestatud viirus sisaldab suurt deletsiooni nsP3 valgu C-terminaalses regioonis. Katsetest selgus, et nimetatud regioon interakteerub sama valgu keskmise domeeni ning nsP2 valgu C-terminaalse osaga ja need kontaktid on olulised viiruse replikatsioonil. Need avastused võimaldavad edaspidi välja selgitada CHIKVΔ5nsP3 mitte-patogeense fenotüübi põhjused. CHIKV Δ5nsP3 vaktsiini tüvi on kasutusele võetud edasiseks arendamiseks farmatseutilise firma poolt.Viruses have been and will be an important part of every ecosystem. In the past, viral outbreaks have left painful marks on mankind. Using vaccines and antivirals has greatly reduced the number of infections and virus-caused pathology. Despite extensive research, some viruses and viral diseases are still lacking any good vaccine or treatment. Viral features like high mutation rate, complexity of viral lifecycle and genome diversity are only some of the obstacles needed to overcome for antivirals and vaccines to be safe and efficient. Hepatitis C virus (HCV) is associated with different liver pathologies and it is estimated that approximately 3% of the world population is chronically infected with HCV. It is lacking efficient vaccine and the options for combating HCV infection, HCV-induced pathology, spread and persistence are limited to the use of antiviral drugs. One part of this dissertation is focused on the development of anti-HCV inhibitors using two different technological approaches. Firstly, a new FQSAR method based approach allowed rapid prediction of hit compounds targeting the NS3/4A protease of HCV. Seven compounds analysed in this project displayed some anti-HCV properties but only the effect caused by the non-cytotoxic compound 23332 can be considered to be direct. Secondly, a novel technology – incorporation of naturally occurring minimally modified nucleobases into ASOs – was evaluated using ASOs binding to the HCV non-structural region. This approach led to the development of ASO compounds with high anti-HCV activity. The technology based on the use of novel modified ONs is promising as well for the development antivirals for other viruses and diseases. Chikungunya virus (CHIKV) re-emerged in the past decade and is currently spreading around the world, affecting millions of people. The second part of this study is focused on the analysis of a laboratory-developed attenuated CHIKV vaccine strain. CHIKVΔ5nsP3 and CHIKVΔ6K viruses were found to have a stably attenuated phenotype and the introduced molecular changes were maintained during serial passages. From all studied vaccine candidates the CHIKVΔ5nsP3 was the most potent. Further studies revealed that the region removed from CHIKVΔ5nsP3 vaccine candidate, is apparently involved in interactions with another domain of nsP3 as well as with the C-terminal region of nsP2. These findings provide a platform for further analysis of biological reasons for the attenuation of CHIKVΔ5nsP3 vaccine candidate

Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors

In Silico Design and Selection of CD44 Antagonists:implementation of computational methodologies in drug discovery and design

Drug discovery (DD) is a process that aims to identify drug candidates through a thorough evaluation of the biological activity of small molecules or biomolecules. Computational strategies (CS) are now necessary tools for speeding up DD. Chapter 1 describes the use of CS throughout the DD process, from the early stages of drug design to the use of artificial intelligence for the de novo design of therapeutic molecules. Chapter 2 describes an in-silico workflow for identifying potential high-affinity CD44 antagonists, ranging from structural analysis of the target to the analysis of ligand-protein interactions and molecular dynamics (MD). In Chapter 3, we tested the shape-guided algorithm on a dataset of macrocycles, identifying the characteristics that need to be improved for the development of new tools for macrocycle sampling and design. In Chapter 4, we describe a detailed reverse docking protocol for identifying potential 4-hydroxycoumarin (4-HC) targets. The strategy described in this chapter is easily transferable to other compounds and protein datasets for overcoming bottlenecks in molecular docking protocols, particularly reverse docking approaches. Finally, Chapter 5 shows how computational methods and experimental results can be used to repurpose compounds as potential COVID-19 treatments. According to our findings, the HCV drug boceprevir could be clinically tested or used as a lead molecule to develop compounds that target COVID-19 or other coronaviral infections. These chapters, in summary, demonstrate the importance, application, limitations, and future of computational methods in the state-of-the-art drug design process