Evolutionary conservation of influenza A PB2 sequences reveals potential target sites for small molecule inhibitors.

The influenza A basic polymerase protein 2 (PB2) functions as part of a heterotrimer to replicate the viral RNA genome. To investigate novel PB2 antiviral target sites, this work identified evolutionary conserved regions across the PB2 protein sequence amongst all sub-types and hosts, as well as ligand binding hot spots which overlap with highly conserved areas. Fifteen binding sites were predicted in different PB2 domains; some of which reside in areas of unknown function. Virtual screening of ~50,000 drug-like compounds showed binding affinities of up to 10.3 kcal/mol. The highest affinity molecules were found to interact with conserved residues including Gln138, Gly222, Ile529, Asn540 and Thr530. A library containing 1738 FDA approved drugs were screened additionally and revealed Paliperidone as a top hit with a binding affinity of -10 kcal/mol. Predicted ligands are ideal leads for new antivirals as they were targeted to evolutionary conserved binding sites

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein

Strategies against nonsense: oxadiazoles as translational readthrough-inducing drugs (TRIDs)

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs

Virtual screening of the inhibitors targeting at the viral protein 40 of Ebola virus

Multilingual abstracts in the six official working languages of the United Nations. (PDF 373 kb

Identifying Inhibitors Targeting the Nonstructural Protein 15 and Main Protease of Coronaviruses Using Molecular Docking and Molecular Dynamics Simulation

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 has impacted daily life globally for over a year. While multiple vaccines have been authorized for emergency use and one oral medication has entered clinical trials, we are still seeking antiviral drugs for a long-term treatment for SARS-CoV-2 as well as other coronaviruses. Computational drug screenings of two SARS-CoV-2 protein target candidates are presented in this thesis: the nidoviral RNA uridylate-specific endoribonuclease (Nsp15) and the main protease (Mpro) of SARS-CoV-2. Nonstructural proteins of coronaviruses were selected as targets as they are more conserved across coronavirus strains than structural proteins. High throughput virtual screening of small molecule libraries including DrugBank and ZINC 15 resulted in several promising compounds for each of these targets. Molecular dynamics simulation allowed us to predict the binding energies for these compounds using molecular mechanics with generalized born surface area solvation calculations (MM-GBSA). Four top compounds were discovered for Nsp15 and eight compounds for Mpro

Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space?

The organization and mining of malaria genomic and post-genomic data is highly motivated by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should therefore be as reliable and versatile as possible. In this context, we examined five aspects of the organization and mining of malaria genomic and post-genomic data: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from X-omic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Progresses toward a grid-enabled chemogenomic knowledge space are discussed.Comment: 43 pages, 4 figures, to appear in Malaria Journa

Biophysical and computational investigations into G-quadruplex structural polymorphism and interaction with small molecules.

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to investigate G-quadruplex structures, biophysical properties, small molecule interaction, and the thermodynamics of G-quadruplex formation. Current approaches to study G-quadruplex structures often employ sequence modifications or changes to the experimental condition, as a way of resolving the structural polymorphism associated with many G-quadruplex-forming sequences, to select for a single conformation for high-resolution structural studies. Our strategy for resolving G-quadruplex structural polymorphism is superior in that the experimental approaches do not result in drastic perturbation of the system. In the first approach, we employed size exclusion chromatography to separate a mixture of G-quadruplex structures formed from a G-quadruplex-forming sequence. We demonstrated that it is possible to isolate distinct species of G-quadruplex structures for further biophysical studies. In the second approach, we employed hydrodynamic bead modeling to study the structural polymorphism of a G-quadruplex-forming sequence. We showed that properties calculated from models agreed with experimentally determined values and could be used to predict the folding of G-quadruplex-forming oligonucleotides whose high-resolution structures are ambiguous or not available. In our third approach, we presented a virtual screening platform that was successful in identifying a new Gquadruplex-interacting small molecule. The results of the virtual screen were validated with extensive biophysical testing. Our target for the virtual screen was a G-quadruplex structure generated in silico, which represents one approach to receptor-based drug discovery when high-resolution structures of the binding site are not available. Taken together, our three approaches represent a new paradigm for drug discovery from guaninerich sequence to anti-cancer drugs

Interfering with mRNA methylation by the 2′O-Methyltransferase (NSP16) from SARS-CoV-2 to tackle the COVID-19 disease

The pandemic associated to Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has resulted in a huge number of deaths and infected people. Although several vaccine programmes are currently underway and have reached phase 3, and a few small size drugs repurposed to aid treatment of severe cases of COVID-19 infections, effective therapeutic options for this disease do not currently exist. NSP16 is a S-adenosyl-L-Methionine (SAM) dependent 2′O-Methyltransferase that converts mRNA cap-0 into cap-1 structure to prevent virus detection by cell innate immunity mechanisms. NSP16 methylates the ribose 2′O-position of the first nucleotide of the mRNA only in the presence of an interacting partner, the protein NSP10. This feature suggests that inhibition of the NSP16 may represent a therapeutic window to treat COVID-19. To test this idea, we performed comparative structural analyses of the NSP16 present in human coronaviruses and developed a sinefungin (SFG) similarity-based virtual screening campaign to assess the druggability of the SARS-CoV-2 NSP16 enzyme. Through these studies, we identified the SFG analogue 44601604 as a promising more potent inhibitor of NSP16 to limit viral replication in infected cells, favouring viral clearance

Tailoring Toll-like Receptor 8 Ligands for Balancing Immune Response and Inflammation

Toll-like receptors (TLRs) play a central role in innate immunity by recognising invading pathogens and host-derived danger signals and initiating the inflammatory response. Aberrant TLR response is involved in the pathogenesis of cancers, infections, autoimmune disorders and allergic diseases. Therefore, TLRs represent attractive targets for novel therapeutic agents. The PhD project's main research aim is to discover novel small molecule modulators of Toll-like receptor 8 (TLR8) and understand their mechanisms of action using computational approaches. TLR8 crystal structure is solved, and several modulators are known from previous drug screens. Therefore, TLR8 is a promising target for rational computer-aided development of novel drug candidates. In the initial phase of the project, the main goal was to study relevant structural features in available crystal structures of TLR8. The focus was on the dimerisation interface because of its role in the binding of ligands and subsequent activation of the receptor. Additionally, we studied the conservation of the relevant structural features across the closely related TLRs. The second part shifts the focus to the binding of the small molecules to TLR8. We investigated interactions between the known ligands and TLR8 and used it to develop the most plausible 3D pharmacophore model. Subsequently, we employed the developed 3D pharmacophore model in virtual screening to identify novel modulators of TLR8. We identified a pyrimidine-based compound that inhibits TLR8-mediated signalling in the micromolar concentration range. The potent anti-inflammatory and dose-dependent response has been confirmed in a series of derivatives of this initial virtual hit, which allowed for a detailed elucidation of structure-activity relationships (SAR) and more precise description of the binding mode. Conclusively, we have developed a novel and promising pyrimidine-based TLR8 inhibitors in silico and confirmed their biological activity, selectivity and low cytotoxicity in vitro. Results from the study on TLR8 represent a solid basis for the future design of small molecule TLR modulators as novel therapeutic agents for modulating immune response and inflammation.Toll-like Rezeptoren (TLRs) spielen eine zentrale Rolle in angeborenen Immunsystem, indem sie eindringende Pathogene sowie endogene Gefahrensignale erkennen und Entzündungsreaktionen einleiten. TLRs sind an der Pathogenese von Krebserkrankungen, Infektionen, Autoimmunerkrankungen und allergischen Erkrankungen beteiligt. Aus diesem Grund stellen TLRs attraktive Ziele für neue, niedermolekulare Wirkstoffe dar. Das Hauptziel dieses Promotionsprojekts ist die Entdeckung neuer niedermolekularer Modulatoren des Toll-like-Rezeptors 8 (TLR8) und das Verständnis ihrer Wirkmechanismen mit Hilfe computergestützter Ansätze. Die Kristallstruktur von TLR8 ist verfügbar und mehrere Modulatoren sind aus früheren Wirkstoffscreens bekannt. Daher ist TLR8 ein vielversprechendes Ziel für die rationale computergestützte Entwicklung neuer Wirkstoffkandidaten. Am Beginn des Projekts bestand das Hauptziel darin, relevante strukturelle Merkmale in den verfügbaren Kristallstrukturen von TLR8 zu untersuchen. Der Fokus lag dabei auf dem Dimerisierungsbereich, da dieser eine wichtige Rolle bei der Bindung von Liganden und der anschließenden Aktivierung des Rezeptors spielt. Zusätzlich untersuchten wir die Konservierung der relevanten Strukturmerkmale über die eng verwandten TLRs hinweg. Der zweite Teil verlagert den Fokus auf die Bindung kleiner Moleküle an TLR8. Wir untersuchten die Interaktionen zwischen den bekannten Liganden und TLR8 und entwickelten daraus systemtisch ein 3D-Pharmakophormodell. Anschließend setzten wir das entwickelte 3D-Pharmakophormodell im virtuellen Screening ein, um neuartige Modulatoren des TLR8 zu identifizieren. Wir identifizierten ein Pyrimidin-Analogon, das die TLR8- vermittelte Signalweiterleitung im mikromolaren Konzentrationsbereich hemmt. Die potente entzündungshemmende und dosisabhängige Wirkung wurde in einer kleinen Serie von Analoga bestätigt. Schließlich optimierten wir die identifizierten Pyrimidinverbindungen weiter, was eine detailliertere Struktur-Aktivitäts-Analyse und eine genauere Aufklärung des Bindungsmodus ermöglichte. Zusammenfassend haben wir neuartige und vielversprechende TLR8-Inhibitoren auf Pyrimidinbasis in silico entwickelt und ihre in vitro biologische Aktivität, Selektivität und geringe Zytotoxizität bestätigt. Die Ergebnisse der Studie zu TLR8 helfen uns, die Prozesse zu verstehen, die für ein erfolgreiches Wirkstoffdesign auch bei anderen TLR notwendig sind und stellen eine gute Ausgangsbasis dar, um in Zukunft optimierte, niedermolekulare TLR- Modulatoren zu entwickeln und damit Entzündung und die Immunreaktion effizient zu modulieren