Binding mode of the activity-modulating C-terminal segment of NS2B to NS3 in the dengue virus NS2B–NS3 protease

The two-component dengue virus NS2B–NS3 protease (NS2B–NS3pro) is an established drug target but inhibitor design is hampered by uncertainties about its 3D structure in solution. Crystal structures reported very different conformations for the functionally important C-terminal segment of the NS2B cofactor (NS2Bc), indicating open and closed conformations in the absence and presence of inhibitors, respectively. An earlier NMR study in solution indicated that a closed state is the preferred conformation in the absence of an artificial linker engineered between NS2B and NS3pro. To obtain direct structural information on the fold of unlinked NS2B–NS3pro in solution, we tagged NS3pro with paramagnetic tags and measured pseudocontact shifts by NMR to position NS2Bc relative to NS3pro. NS2Bc was found to bind to NS3pro in the same way as reported in a previously published model and crystal structure of the closed state. The structure is destabilized, however, by high ionic strength and basic pH, showing the importance of electrostatic forces to tie NS2Bc to NS3pro. Narrow NMR signals previously thought to represent the open state are associated with protein degradation. In conclusion, the closed conformation of the NS2B–NS3 protease is the best model for structure-guided drug design

In silico Strategies to Support Fragment-to-Lead Optimization in Drug Discovery.

Fragment-based drug (or lead) discovery (FBDD or FBLD) has developed in the last two decades to become a successful key technology in the pharmaceutical industry for early stage drug discovery and development. The FBDD strategy consists of screening low molecular weight compounds against macromolecular targets (usually proteins) of clinical relevance. These small molecular fragments can bind at one or more sites on the target and act as starting points for the development of lead compounds. In developing the fragments attractive features that can translate into compounds with favorable physical, pharmacokinetics and toxicity (ADMET-absorption, distribution, metabolism, excretion, and toxicity) properties can be integrated. Structure-enabled fragment screening campaigns use a combination of screening by a range of biophysical techniques, such as differential scanning fluorimetry, surface plasmon resonance, and thermophoresis, followed by structural characterization of fragment binding using NMR or X-ray crystallography. Structural characterization is also used in subsequent analysis for growing fragments of selected screening hits. The latest iteration of the FBDD workflow employs a high-throughput methodology of massively parallel screening by X-ray crystallography of individually soaked fragments. In this review we will outline the FBDD strategies and explore a variety of in silico approaches to support the follow-up fragment-to-lead optimization of either: growing, linking, and merging. These fragment expansion strategies include hot spot analysis, druggability prediction, SAR (structure-activity relationships) by catalog methods, application of machine learning/deep learning models for virtual screening and several de novo design methods for proposing synthesizable new compounds. Finally, we will highlight recent case studies in fragment-based drug discovery where in silico methods have successfully contributed to the development of lead compounds

New Binding Site Conformations of the Dengue Virus NS3 Protease Accessed by Molecular Dynamics Simulation

International audienceDengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4), and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO), which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD) simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation), a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 proteas

Ligand Based Drug Discovery Of Novel Dengue-2 NS2B-NS3 Protease Inhibitors

Kes demam denggi dilaporkan meningkat setiap tahun, namun sehingga kini belum ada ubat anti-denggi boleh didapati di pasaran. Oleh itu, pencarian ejen anti-denggi adalah kritikal. Pemotongan poliprotein pelopor oleh enzim protease NS2B-NS3 merupakan proses yang sangat penting untuk replikasi flavivirus. Oleh itu enzim protease NS2B-NS3 sesuai dijadikan sasaran untuk membangunkan ubat anti-denggi. Di Malaysia, Denggi-2 adalah jenis sera yang paling lazim. Dalam kajian ini, pendekatan berasaskan ligan telah dilaksanakan dalam mencari perencat enzim protease NS2B-NS3 Denggi-2 baharu yang berpotensi. Model farmakofor telah dihasilkan daripada pelbagai struktur perencat enzim protease NS2B-NS3 Denggi-2 yang pernah dilaporkan, yang terdiri daripada molekul peptida dan bukan peptida. Model farmakofor terpilih telah digunakan untuk menyaring senarai sebatian dalam pengkalan data National Cancer Institute, AS (NCI) untuk mencari perencat protease NS2B-NS3 Denggi-2 yang baharu secara maya. The reported dengue cases are increasing yearly, yet no anti-dengue agent is available in the market. Therefore, the search for anti-dengue is critical. In Malaysia, Dengue-2 (DEN-2) is the most prevalent serotype. NS2B-NS3 protease is the enzyme for the cleavage of polyprotein precursor, which is crucial for the flavivirus replications. This makes it a potential target for the development of therapeutics against the dengue virus. In this study, ligand-based approach was implemented in searching for the new potential DEN-2 NS2B-NS3 protease inhibitors. Pharmacophore models were developed from diverse reported structures of DEN-2 NS2B-NS3 protease inhibitors, comprising peptide and non-peptide molecules. The selected pharmacophore models were employed to screen the US National Cancer Institute (NCI) list of compounds to search for new DEN-2 NS2B-NS3 protease inhibitors

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

Mechanistic Elucidation of Protease–Substrate and Protein–Protein Interactions for Targeting Viral Infections

Viral infections represent an old threat to global health, with multiple epidemics and pandemics in the history of mankind. Despite several advances in the development of antiviral substances and vaccines, many viral species are still not targeted. Additionally, new viral species emerge, posing a menace without precedent to humans and animals and causing fatalities, disabilities, environmental harm, and economic losses. In this thesis, we present rational modeling approaches for targeting specific protease-substrate and protein-protein interactions pivotal for the viral replication cycle. Over the course of this work, antiviral research is supported beginning with the development of small molecular antiviral substances, going through the modeling of a potential immunogenic epitope for vaccine development, towards the establishment of descriptors for susceptibility of animals to a viral infection. Notably, all the research was done under scarce data availability, highlighting the predictive power of computational methods and complementarity between in-silico and in-vitro or in-vivo methods

Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis- electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme–inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases

Applications of paramagnetic NMR spectroscopy in drug discovery

NMR spectroscopy of proteins with paramagnetic metal ions, first performed with metalloproteins, is a unique technique to obtain long-range distance information for three-dimensional structure determinations. This thesis focuses on developing applications of paramagnetic NMR spectroscopy, particularly pseudocontact shifts, in drug discovery. The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) from serotype 2 is a well-established drug target, but drug development has been hampered for many years by lack of structural information. In earlier work, pseudocontact shifts (PCSs) induced by lanthanide binding tags at multiple sites had successfully been used to determine the closed conformation of NS2B in the presence of a small inhibitor molecule. Subsequently, PCSs were used to prove that an unlinked construct of NS2B-NS3pro exists predominately in the closed conformation in solution, showing that the open conformation observed previously is an artefact generated by a covalent link between NS2B and NS3 (Paper 1). Next, PCSs generated for NS2B, NS3pro and bovine pancreatic trypsin inhibitor (BPTI) were used to show that the C-terminal segment of NS2B remains in the closed conformation in the presence of BPTI, correcting a crystallographic artefact (Paper 2). The work described in Papers 1 and 2 confirmed that the closed conformation of dengue virus NS2B-NS3pro is the best model for structure-guided drug design. As the sensitivity of NMR spectra of dynamic proteins, such as the dengue virus protease, is compromised by excessive line broadening, alternative NMR tags were sought. O-tert-butyltyrosine incorporated in proteins proved to be an outstanding NMR probe for conformational studies of high-molecular-weight systems and measurement of submicromolar ligand binding affinities in one-dimensional 1H-NMR spectra without any isotope labelling (Paper 3). A tert-butyl probe was also introduced into a tightly binding lead compound against the dengue virus protease. Measurement of ligand PCSs from intense intramolecular NOESY cross-peaks with the tert-butyl group allowed positioning of the ligand on the protein with respect to the paramagnetic centre, while strong intermolecular NOEs validated the structural model of the complex established with the use of PCSs (Paper 4). In summary, the paramagnetic NMR approach, demonstrated on the dengue virus NS2B-NS3 protease, presents a broadly applicable and elegant way for structure-guided drug design at atomic resolution

RNA DEPENDENT RNA POLYMERASE: A VALUABLE TARGET TO BLOCK VIRAL REPLICATION IN SINGLE-STRANDED (+)SENSE RNA VIRUSES.

The (+)strand RNA viruses include a very large group of viruses that cause epidemic diseases in humans, including dengue fever and gastroenteritis. The human (+)RNA viruses include Flaviviruses (FV) and Norovirus (NV). Both encode for proteins essential for viral replication, such as the RNA dependent RNA polymerase (RdRp). Since human cells lack RdRp, it appears as one of the most promising targets for antivirals development. I worked on the identification of new non-nucleotide inhibitors against FV and NV, using RdRp as the main target. In this context, suramin and NF023 have been identified in my lab as NV RdRp inhibitors that, however both are hampered in their application by pharmacokinetics problems. To overcome such problems, I analyzed the potential inhibitory role of Naf2, a fragment derived from these two molecules. Although Naf2 showed a low inhibitory activity, the crystal structures of NV RdRp/Naf2 complex revealed a new binding site. To further map this new site, I tested a Naf2 related molecule, PPNDS. The crystal structures of the RdRp/PPNDS complex revealed interesting features about the new binding site. I also focused on structurally related molecules synthesized following structure-driven information. NV RdRp crystal structures in complex with one of these compounds (Cpd6) were analyzed, providing new knowledge on the interactions between a small fragment and NV RdRps, establishing a platform for structure-guided drug optimization. In parallel to the NV work, I screened in silico a library of compounds against FV RdRp. One of the best compounds identified (HeE1-2Tyr) was able to inhibit the RdRp activity and several FVs in cell-based assays. Although the crystallographic analyses don't reveal clear enough electron density for the inhibitor, indirect evidence suggests that HeE1-2Tyr interferes with the RdRp priming loop that appears disordered

Caractérisation détaillée de l’interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive

Les Flavivirus transmis par les moustiques comme le virus du Nil occidental, le virus de la dengue, le virus de la fièvre jaune, le virus de l’encéphalite japonaise et le virus Zika constituent des préoccupations croissantes de santé publique. Ils se sont répandus dans le monde au cours des dernières décennies, et les épidémies sont devenues plus fréquentes et plus sévères. Chaque année, des millions de personnes sont infectées et environ 50 000 patients décèdent d’infections à Flavivirus. Malgré les nombreux efforts de recherche, il n’y a actuellement aucun médicament antiviral spécifique disponible, et des nouvelles stratégies antivirales sont indispensables. Comprendre comment les Flavivirus fonctionnent au niveau moléculaire aidera à découvrir des nouvelles cibles pour l'intervention thérapeutique. Les Flavivirus ont un génome d'ARN simple brin de polarité positive qui code pour trois protéines structurales et huit protéines non structurales. Seules deux des huit protéines non structurales ont des activités enzymatiques. NS3 possède un domaine protéase et un domaine hélicase, et NS5 a un domaine méthyl- et guanylyltransférase et un domaine ARN polymérase ARN-dépendante. Ensemble, ils répliquent le génome viral. Ici, nous caractérisons l'interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive. Un modèle d'interaction comprenant NS3, NS5 et l’ARN viral a été développé basé sur des structures cristallines connues ainsi que des activités enzymatiques des deux protéines individuelles, et ce modèle a été soumis à des simulations de dynamique moléculaire. Les interactions potentielles entre les protéines NS3 et NS5 ont été identifiées. Les résidus impliqués dans ces interactions ont été mutés dans un réplicon du virus du Nil occidental et les effets de ces mutations sur la réplication virale ont été évalués. Une région particulière à la surface de la protéine NS3 a été identifiée comme étant cruciale pour la réplication virale, très probablement parce qu'elle interagit avec NS5. Cette région pourrait être une cible attrayante pour la recherche de composés qui pourraient interférer avec l'interaction entre NS3 et NS5 et donc posséder un potentiel antiviral intéressant.Abstract : Mosquito-borne Flaviviruses like West Nile virus, Dengue virus, Yellow Fever virus, Japanese encephalitis virus, and Zika virus are increasing public health concerns. They have spread globally during the past decades, and outbreaks have recently become more frequent and more severe. Every year, millions of people are infected, and approximately 50,000 patients die from Flavivirus infections. Despite extensive research efforts, there are currently no specific antiviral drugs available, and new antiviral strategies are greatly needed. Understanding how Flaviviruses work on a molecular level will help in uncovering new points for therapeutic intervention. Flaviviruses have a single-stranded RNA genome of positive polarity that encodes three structural and eight non-structural proteins. Only two of the eight non-structural proteins have enzymatic activities. NS3 has an N-terminal protease domain and a C-terminal helicase domain, and NS5 has an N-terminal capping enzyme domain and a C-terminal RNA-dependent RNA polymerase domain. Together, they replicate the viral genome. Here we characterize the NS3:NS5 interaction within the West Nile virus RNA replicase complex during positive strand synthesis. An interaction model including NS3, NS5 and viral RNA was developed based on the known crystal structures as well as enzymatic activities of the two individual proteins, and this model was subjected to molecular dynamics simulations. Potential interactions between the NS3 and NS5 proteins were identified. Residues involved in these interactions were mutated in a West Nile virus replicon, and the effects of these mutations on viral replication were evaluated. One particular region on the surface of the NS3 protein was identified to be crucial for viral replication, most likely because it mediates the interaction with NS5. This region might be an attractive target for the search of compounds that could interfere with the NS3:NS5 interaction and therefore possess an interesting antiviral potential