Evaluation of docking performance in a blinded virtual screening of fragment-like trypsin inhibitors

International audienceIn this study, we have "blindly" assessed the ability of several combinations of docking software and scoring functions to predict the binding of a fragment-like library of bovine trypsine inhibitors. The most suitable protocols (involving Gold software and GoldScore scoring function, with or without rescoring) were selected for this purpose using a training set of compounds with known biological activities. The selected virtual screening protocols provided good results with the SAMPL3-VS dataset, showing enrichment factors of about 10 for Top 20 compounds. This methodology should be useful in difficult cases of docking, with a special emphasis on the fragment-based virtual screening campaigns

Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and in Vitro Assay

A multi-step cascade strategy using integrated ligand-and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K-i) in the low micromolar range (3-60 mu M) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 mu M), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. in order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. the IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6 +/- 0.1 mu M, tenfold lower than that obtained for benznidazole, which was taken as positive control. in addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Sao Carlos, Dept Quim, BR-13560 Sao Carlos, SP, BrazilUniv São Paulo, Inst Quim Sao Carlos, Grp Quim Med IQSC USP, Sao Carlos, SP, BrazilUniv Calif San Francisco, Dept Pathol, Ctr Discovery & Innovat Parasit Dis, San Francisco, CA 94140 USAUniv São Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, BR-14049 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilFAPESP: 2011/01893-3,CNPq: 301614/2010-5CAPES: 5985/11-0Web of Scienc

Fragment based ligand discovery : library design and screening by thermal shift analysis

The central idea in Fragment Based Ligand Discovery (FBLD) is to identify small, low molecular weight compounds (MW < 250) that bind to a particular protein active site. Hits can be used to efficiently design larger compounds with the desired affinity and selectivity. Three approaches to FBLD are described in this thesis. The first topic is the development and assessment of different chemoinformatics procedures to select those fragments that maximally represent the chemical features of a larger compound library. Such a fragment library could be of great value in the so-called “SAR by Catalogue" approach, where the initial stage of fragment growth is by selecting existing compounds that contain sub-structures of the hit fragments. Five schemes implemented in the Pipeline Pilot software are described. The second project was to develop improved approaches to processing Thermal Shift Analysis (TSA) data. The shift in melting temperature can indicate that a ligand binds and thus stabilises a protein. A program, MTSA, has been written which allows more straightforward processing of the experimental data than existing available software. However, detailed analysis of fragment screening data highlighted difficulties in defining the melting temperature and suggest that TSA is not sufficiently reliable for routine screening use. Finally, a number of proteins were assessed experimentally for suitability for FBLD: N-myristoyl transferase (NMT), the bacterial homologue of a GlcNAcase enzyme (BtGH84) and the model system hen egg white lysozyme (HEWL). It was not possible to produce suitable NMT material due to the inherent instability of the protein produced in York. The screening results of HEWL with a new Surface Plasmon Resonance (SPR) assay, a cell based activity assay and TSA were inconsistent and difficult to interpret. However, BtGH84 was suitable for screening by both TSA and SPR. The resulting fragment hits are suitable starting points for further evolution.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Exploration of fragment-derived modulators of glycoside hydrolases

Previous work at York demonstrated that fragment molecules can increase the activity of the glycoside hydrolase, BtGH84. The initial aim of this project was to use fragment-based discovery methods to identify activators of several enzymes used in cellulose degradation where low activity is one of the limiting steps in the industrial process. This was successful for one enzyme, the fungal glycoside hydrolase, TrBgl2. The characterisation of the mechanism of activation for this enzyme is the main focus of this thesis. A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule activator of TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate binding site, stabilizing the enzyme-substrate complex

Applications of nuclear magnetic resonance spectroscopy: from drug discovery to protein structure and dynamics.

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors currently in clinical trials. This work combines screening of an FDA-approved drug panel and the identification of binders by protein-observed NMR. FDA-approved drugs salirasib and candesartan were identified as potent inhibitors in in vitro inhibition and migration assays while a weak inhibitor, olsalazine, was identified by NMR as the first small molecule inhibitor to directly bind PRL3. NMR was also used to validate the binding of additional compounds identified as experimental PRL3 inhibitors. Thienopyridone, a potent experimental inhibitor, did not show direct binding to PRL3 but instead inhibited phosphatase activity via redox mechanism. NMR also revealed that other experimental inhibitors did not engage PRL3. Thus, there remains a need to identify potent PRL3-directed inhibitors. Meanwhile, molecular modeling revealed a putative druggable site that has not been thoroughly explored before. The current study provides some scaffolds such as candesartan and particularly, olsalazine, the only binder identified, that could be the starting point of further drug discovery efforts, as well as a putative site that can be targeted in silico. MDMX, a negative regulator of p53, is another important therapeutic target in cancer, along with the homologous protein, MDM2. Inhibitors that block the MDM2-p53 interaction have been identified and despite similarities in the binding site of these homologous proteins, these inhibitors are ineffective against MDMX. It is hypothesized that the flexibility of MDMX contributes to this significant difference in response to inhibitors, despite comparable affinity to their endogenous target, p53. Examination of available inhibitor-bound structures of MDMX reveal a conserved pharmacophore but the structures adopt distinct conformations away from the binding site. This implies that global motions of the protein might contribute to molecular recognition. The conformational heterogeneity in MDMX was further confirmed by collecting residual dipolar couplings (RDCs). Further investigations on both MDMX and MDM2 are necessary to uncover whether the flexibility of MDMX contributes to the differential binding to inhibitors. Finally, NMR relaxation methods and state-of-the-art high-power Carr-Purcell-Meiboom Gill (CPMG) relaxation dispersion measurements, the first documented application on an enzyme, were used to characterize the solution dynamics of GMPK and the changes in dynamics upon GMP binding. Substrate binding resulted in restricting the amplitudes of motion for backbone amide bonds within the picosecond-nanosecond timescale. Meanwhile, CPMG showed dispersion in both in the absence and presence of GMP, such that substrate binding did not quench dynamics within the microsecond-millisecond timescale. Interestingly, more residues are observed to have dispersion in the bound form, some near the C-terminal of helix 3, which has previously been proposed to be involved in product release. Current studies show that substrate binding affect different timescales of protein motion. Future work shall follow how motions within different timescales are affected as GMPK processes its substrates – such as, for instance, binding of ATP analogs within the ATP binding site or simultaneous occupancy of both substrate binding pockets. This paves the way for a complete picture of the relationship of function and dynamics in the conformational enzymatic cycle of a bi-substrate enzyme using GMPK as a model. The current work illustrates some of the diverse applications of NMR on three unique systems that are also drug targets. Information collected here can be leveraged on future structure and dynamics studies as well as drug discovery efforts targeting any of these proteins

Crystallographic fragment screening - improvement of workflow, tools and procedures, and application for the development of enzyme and protein-protein interaction modulators

One of the great societal challenges of today is the fight against diseases which reduce life expectancy and lead to high economic losses. Both the understanding and the addressing of these diseases need research activities at all levels. One aspect of this is the discovery and development of tool compounds and drugs. Tool compounds support disease research and the development of drugs. For about 20 years, the discovery of new compounds has been attempted by screening small organic molecules by high-throughput methods. More recently, X-ray crystallography has emerged as the most promising method to conduct such screening. Crystallographic fragment-screening (CFS) generates binding information as well as 3D-structural information of the target protein in complex with the bound fragment. This doctoral research project is focused primarily on the optimization of the crystallographic fragment screening workflow. Investigated were the requirements for more successful screening campaigns with respect to the crystal system studied, the fragment libraries, the handling of the crystalline samples, as well as the handling of the data associated with a screening campaign. The improved CFS workflow was presented as a detailed protocol and as an accompanying video to train future CFS users in a streamlined and accessible way. Together, these improvements make CFS campaigns a more high-throughput method, offering the ability to screen larger fragment libraries and allowing higher numbers of campaigns performed per year. The protein targets throughout the project were two enzymes and a spliceosomal protein-protein complex. The enzymes comprised the aspartic protease Endothiapepsin and the SARS-Cov-2 main protease. The protein-protein complex was the RNaseH-like domain of Prp8, a vital structural protein in the spliceosome, together with its nuclear shuttling factor Aar2. By performing the CFS campaigns against disease-relevant targets, the resulting fragment hits could be used directly to develop tool compounds or drugs. The first steps of optimization of fragment hits into higher affinity binders were also investigated for improvements. In summary, a plethora of novel starting points for tool compound and drug development was identified

Targeting Trypanosoma brucei FPPS by Fragment-based drug discovery

Trypanosoma brucei (T. brucei) is the causative agent of the Human African Trypanosomiasis (HAT), which is a neglected disease with an endemic occurrence in 36 sub-Saharan African countries. The current standard of care suffers from low efficacy and severe side effects. Therefore, new drugs with better safety and efficacy profiles are urgently needed. Nitrogen-containing bisphosphonates, a current treatment for bone diseases, have been shown to block the growth of the T. brucei parasites by inhibiting farnesyl pyrophosphate synthase (FPPS); however, due to their particular pharmacokinetic properties they are not well suited for parasitic therapy. Recently, an additional allosteric site was discovered at the surface of human FPPS that, based on sequence analysis, is likely also present in T. brucei FPPS. The high unmet medical need combined with the discovery of a potential new target site prompted a fragment-based drug discovery approach to identify non-bisphosphonate binders on T. brucei FPPS, which is presented in this work. Fragment screening was performed by NMR and X-ray crystallography. To this end, a robust T. brucei FPPS crystallization system was established enabling high-throughput determination of crystal structures up to 1.67 Å resolution. Structural superimpositions revealed that the allosteric site found on human FPPS is in fact present in T. brucei FPPS. This observation enabled subsequent protein-observed NMR and crystal soaking experiments with established human FPPS binders resulting in three protein-ligand complex structures with bound fragments in the previously unknown allosteric site. For most of the tested binders, Kd by SPR was outside of experimental range for T. brucei FPPS and only for one fragment the Kd on T. brucei FPPS was determined three orders of magnitude higher than the IC50 value on human FPPS. Crystal structural analysis revealed a different binding mode on human and T. brucei FPPS with reduced protein-ligand interactions on T. brucei FPPS, which explains the significantly reduced binding affinity. Encouraged by the detection of first allosteric binders on T. brucei FPPS, fragment pools were screened by ligand-observed NMR and identified hits were followed-up by single compound ligand observed NMR and protein-observed NMR resulting in 25 validated fragment hits for T. brucei FPPS. Validated hits were followed-up by crystal soaking and co-crystallization experiments and seven protein-ligand complex structures were solved using PanDDA. Out of the seven fragments, four fragments were bound in the active site, one fragment was detected in the allosteric site that was identified as part of this thesis, and two fragments were bound in surface exposed binding sites. Notably, an active site bound fragment with a four atom long flexible linker adopted an orthogonal binding mode along αD when compared to the other three ligands. Sixteen fragment analogues of the elongated flexible active site fragment were tested by SAR using additional test compounds retrieved from catalogue and archive, and one crystal structure with a fragment analogue was solved and was surprisingly found in the allosteric site. In addition to the NMR fragment screen, an X-ray screen was performed at XChem (Diamond, UK) and at EMBL/ESRF (Grenoble, FR) resulting in seven protein-ligand structures. One fragment was positioned in the active site, three fragments in the allosteric site, two fragments in a cryptic site between helices αI and αH and one fragment at the opposite side of the allosteric site close to αG and αF. Fragment binding was further validated in protein-observed NMR. As fragments identified by such screening approaches typically exhibit low binding affinities usually in µM to mM range, structure-based fragment optimisation based on a fragment merging and growing approach was performed. In total, ten compounds were synthesised and subjected to protein observed NMR and X-ray structural analysis. Strikingly, a fragment merger based on T. brucei and T. cruzi active-site binders bound in a new binding site close to the SARM instead to the active site. Taken together, this work presents high-resolution structures of T. brucei FPPS and identified 19 compounds binding to seven different sites thereby paving the way for future studies aiming to identify high-affinity non-bisphosphonate inhibitors for T. brucei FPPS with pharmacokinetic properties that are suitable for parasitic indications

Chemoinformatische Entwicklung von Naturstofffragmenten, sowie deren strukturbiologische und biochemische Evaluierung zur fragmentbasierten Inhibitorentwicklung

Die Fragmentbasierte Wirkstoffentwicklung (FBDD) hat sich als wertvolle Methode zur Detektion neuer Moleküle mit inhibitorischen Eigenschaften etabliert. Allerdings bestehen die derzeit üblicherweise verwendeten Fragment-Bibliotheken aus sp2-reichen Molekülen und es werden daher verstärkt Wege gesucht den erforschbaren chemischen Strukturraum zu erweitern.1-4 Biologisch validierte Naturstoffe sind reich an Stereozentren und decken einen Bereich des chemischen Strukturraums ab, der von im Allgemeinen genutzten synthetischen Molekülen nicht abgebildet wird.5 Daher wurde im Rahmen dieser Arbeit erfolgreich ein Algorithmus entwickelt, der in der Lage ist Naturstoffe zu Naturstofffragmeten zu reduzieren und dabei die wichtigen funktionellen Gruppen, sowie Anknüpf- bzw. Modifikationspunkte an ihrer natürlichen Position zu erhalten. Es wurde ein Datensatz mit über 180,000 annotierten Naturstoffen analysiert und eine repräsentative Bibliothek aus 2,000 von Naturstoffen abgeleiteten Fragmenten zusammengestellt, die die Eigenschaften aller generierten ~110,000 Naturstofffragmente und der ursprünglichen Naturstoffe widerspiegeln und reich sind an sp3-konfigurierten Stereozentren. Diese Strukturen unterscheiden sich erwartungsgemäß vom bisher untersuchbaren chemischen Strukturraum. Dennoch sind für ungefähr die Hälfte der virtuellen Bibliothek repräsentative Fragmente kommerziell verfügbar. Mit Hilfe dieser Naturstofffragmente konnten, auch bei etablierten Zielproteinen, bisher unbekannte inhibitorische Fragmente gefunden werden. Unter anderem wurde das Konzept durch die Identifizierung von neuen Inhibitoren der aktiven Form und neuartigen Stabilisatoren der inaktiven Form von p38a MAP Kinase validiert. Zudem konnten insgesamt 52 Fragmente als neuartige Phosphataseinhibitoren gefunden werden.Fragment-based drug discovery (FBDD) has proven to be a valuable method to find new inhibitory small molecules, but it mostly employs sp2-rich compounds and the community is intensively looking for new fragment collections to expand the explorable chemical space.1-4 Biologically validated natural products (NPs) are rich in stereogenic centers and populate areas of chemical space not occupied by average synthetic molecules.5 Therefore, a new algorithm was developed that successively degenerates natural products to natural product-derived fragments keeping their attachment points at natural positions. In this work a large set (> 180,000) of natural product structures was analyzed to arrive at ca. 2,000 natural product derived fragments which are structurally highly diverse, resemble the properties of NP scaffolds and NPs themselves and are rich in sp3-configured centers. The structures of these cluster centers differ from previously explored fragment libraries, but for nearly half of the clusters representative members are commercially available. We validate their usefulness for the discovery of novel ligand and inhibitor types by identification of novel fragments stabilizing the inactive conformations of p38a MAP kinase and inhibitors of several phosphatases

Design, synthesis, and biological evaluation of PqsR antagonists guided by classic hit-to- lead optimisation process and fragment- based methods for the treatment of Pseudomonas aeruginosa infections

Pseudomonas aeruginosa (P. aeruginosa) a nosocomial pathogen, has become a serious public health threat due to its high mortality rates and serious antibiotic resistance issue. The Pseudomonas quinolone signal (pqs) system of P. aeruginosa is essential in regulating the biosynthesis of virulence factors. The transcriptional regulator of pqs system PqsR has been regarded as an interesting research topic for the treatment of P. aeruginosa infections. This thesis is focused on using multiple hit-to-lead optimization methods to find novel PqsR antagonists to overcome P. aeruginosa infections. Chapter 1 provides background information about P. aeruginosa pathogenicity, the pqs system and current progress towards finding PqsR antagonists. An overview of fragment-based lead discovery (FBLD) including hit identification, fragment library construction, biophysical methods and hit-to-lead evolution methods is also provided. Chapter 2 describes a classic hit-to-lead optimisation process starting from the virtual screening of an in-house compound library against PqsR protein to obtain 19. Compound 19 displayed good hit likeness and was subjected to hit-to-lead optimization to achieve a potent drug sized PqsR antagonist 69 with IC50 values of 0.25 μM and 0.34 μM in PAO1-LmCTX::PpqsA-lux and PA14mCTX::PpqsA-lux reporter assays respectively. The X-ray crystal structure of the 69-PqsR LBD complex was also obtained, which provides insights into specific ligand-target interactions. Chapter 3 focuses on fragment-based methods in the discovery of PqsR antagonists. Assisted by in silico methods, five fragment libraries were screened against PqsR protein and the high scoring fragments were subjected to a thermal shift assay (TSA) to give fragment hits 106, 107. Through hit exploration study, fragments 106, 107 were optimised and led to the identification of fragments 145a, 145c and 146b displaying improved biophysical profiles and these fragments can act as good starting points for the identification drug-sized PqsR antagonists (350 < MWt < 500). Chapter 4 demonstrates the evolution of fragment hits 106, 149, 145a, 145c and 146b to drug-sized molecules through fragment linking, merging, and growing methods. Applying a fragment growing method on 106 led to the discovery of 148b and 148c displaying pqs inhibition observed as remaining activity (RA%) values of 60% and 63% at 50 μM screening concentration in PAO1-LmCTX::PpqsA-lux reporter assays, respectively. Linking fragment 146b and 152a led to the discovery of compound 154b showing a RA% value of 34% at 10 μM screening concentration. It was hypothesized that two fragments bound to the PqsR LBD in different sub-pockets can functionalize as synergistic combinations observed as the fragment cocktails displaying a greater effect in bioreporter assay and biophysical experiments than the single fragments. A synergistic exploration experiment was designed assisted by TSA and mCTX::PpqsA-lux based bioreporter assay and led to the identification of two pairs of synergistic combinations (81 and 108, 81 and 105) showing improved in vitro or biophysical profiles in combination than in single fragments

Design, synthesis, and biological evaluation of PqsR antagonists guided by classic hit-to- lead optimisation process and fragment- based methods for the treatment of Pseudomonas aeruginosa infections

Pseudomonas aeruginosa (P. aeruginosa) a nosocomial pathogen, has become a serious public health threat due to its high mortality rates and serious antibiotic resistance issue. The Pseudomonas quinolone signal (pqs) system of P. aeruginosa is essential in regulating the biosynthesis of virulence factors. The transcriptional regulator of pqs system PqsR has been regarded as an interesting research topic for the treatment of P. aeruginosa infections. This thesis is focused on using multiple hit-to-lead optimization methods to find novel PqsR antagonists to overcome P. aeruginosa infections. Chapter 1 provides background information about P. aeruginosa pathogenicity, the pqs system and current progress towards finding PqsR antagonists. An overview of fragment-based lead discovery (FBLD) including hit identification, fragment library construction, biophysical methods and hit-to-lead evolution methods is also provided. Chapter 2 describes a classic hit-to-lead optimisation process starting from the virtual screening of an in-house compound library against PqsR protein to obtain 19. Compound 19 displayed good hit likeness and was subjected to hit-to-lead optimization to achieve a potent drug sized PqsR antagonist 69 with IC50 values of 0.25 μM and 0.34 μM in PAO1-LmCTX::PpqsA-lux and PA14mCTX::PpqsA-lux reporter assays respectively. The X-ray crystal structure of the 69-PqsR LBD complex was also obtained, which provides insights into specific ligand-target interactions. Chapter 3 focuses on fragment-based methods in the discovery of PqsR antagonists. Assisted by in silico methods, five fragment libraries were screened against PqsR protein and the high scoring fragments were subjected to a thermal shift assay (TSA) to give fragment hits 106, 107. Through hit exploration study, fragments 106, 107 were optimised and led to the identification of fragments 145a, 145c and 146b displaying improved biophysical profiles and these fragments can act as good starting points for the identification drug-sized PqsR antagonists (350 < MWt < 500). Chapter 4 demonstrates the evolution of fragment hits 106, 149, 145a, 145c and 146b to drug-sized molecules through fragment linking, merging, and growing methods. Applying a fragment growing method on 106 led to the discovery of 148b and 148c displaying pqs inhibition observed as remaining activity (RA%) values of 60% and 63% at 50 μM screening concentration in PAO1-LmCTX::PpqsA-lux reporter assays, respectively. Linking fragment 146b and 152a led to the discovery of compound 154b showing a RA% value of 34% at 10 μM screening concentration. It was hypothesized that two fragments bound to the PqsR LBD in different sub-pockets can functionalize as synergistic combinations observed as the fragment cocktails displaying a greater effect in bioreporter assay and biophysical experiments than the single fragments. A synergistic exploration experiment was designed assisted by TSA and mCTX::PpqsA-lux based bioreporter assay and led to the identification of two pairs of synergistic combinations (81 and 108, 81 and 105) showing improved in vitro or biophysical profiles in combination than in single fragments