The application of molecular modelling in the safety assessment of chemicals: A case study on ligand-dependent PPARγ dysregulation.

The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q(2)cv=0.610, Nopt=7, SEPcv=0.505, r(2)pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

BIOINFORMATICS ANALYSIS OF CYCLIN-DEPENDENT KINASE 5: INSIGHTS TO DRUG DISCOVERY

Ph.DDOCTOR OF PHILOSOPH

Technological developments in Virtual Screening for the discovery of small molecules with novel mechanisms of action

Programa de Doctorat en Recerca, Desenvolupament i Control de Medicaments[eng] Advances in structural and molecular biology have favoured the rational development of novel drugs thru structure-based drug design (SBDD). Particularly, computational tools have proven to be rapid and efficient tools for hit discovery and optimization. The main motivation of this thesis is to improve and develop new methods in the area of computer-based drug discovery in order to study challenging targets. Specifically, this thesis is focused on docking and Virtual Screening (VS) methodologies to be able to exploit non-standard sites, like protein-protein interfaces or allosteric sites, and discover bioactive molecules with novel mechanisms of action. First, I developed an automatic pipeline for binding mode prediction that applies knowledge- based restraints and validated the approach by participating in the CELPP Challenge, a blind pose prediction challenge. The aim of the first VS in this thesis is to find small molecules able to not only disrupt the RANK-RANKL interaction but also inhibit the constitutive activation of the receptor. With a combination of computational, biophysical, and cell-based assays we were able to identify the first small molecule binders for RANK that could be used as a treatment for Triple Negative Breast Cancer. When working with challenging targets, or with non-standard mechanisms of action, the relationship between binding and the biological response is unpredictable, because the biological response (if any) will depend on the biological function of the particular allosteric site, which is generally unknown. For this reason, we then tested the applicability of the combination of ultrahigh-throughput VS with low-throughput high content assay. This allowed us to characterize a novel allosteric pocket in PTEN and also describe the first allosteric modulators for this protein. Finally, as the accessible Chemical Space grows at a rapid pace, we developed an algorithm to efficiently explore ultra-large Chemical Collections using a Bottom-up approach. We prospectively validated the approach in BRD4 and identified novel BRD4 inhibitors with an affinity comparable to advanced drug candidates for this target.[spa] Els avenços en biologia estructural i molecular han afavorit el desenvolupament racional de nous fàrmacs a través del disseny de fàrmacs basat en l'estructura (SBDD). En particular, les eines computacionals han demostrat ser ràpides i eficients per al descobriment i l'optimització de fàrmacs. La principal motivació d'aquesta tesi és millorar i desenvolupar nous mètodes en l'àrea del descobriment de fàrmacs per ordinador per tal d'estudiar proteïnes complexes. Concretament, aquesta tesi se centra en les metodologies d'acoblament i de cribratge virtual (CV) per poder explotar llocs no estàndard, com interfícies proteïna-proteïna o llocs al·lostèrics, i descobrir molècules bioactives amb nous mecanismes d'acció. En primer lloc, vaig desenvolupar un protocol automàtic per a la predicció del mode d’unió aplicant restriccions basades en el coneixement i vaig validar l'enfocament participant en el repte CELPP, un repte de predicció del mode d’unió a cegues. L'objectiu del primer CV d'aquesta tesi és trobar petites molècules capaces no només d'interrompre la interacció RANK-RANKL sinó també d'inhibir l'activació constitutiva del receptor. Amb una combinació d'assajos computacionals, biofísics i basats en cèl·lules, vam poder identificar les primeres molècules petites per a RANK que es podrien utilitzar com a tractament per al càncer de mama triple negatiu. Quan es treballa amb proteïnes complexes, o amb mecanismes d'acció no estàndard, la relació entre la unió i la resposta biològica és impredictible, perquè la resposta biològica (si n'hi ha) dependrà de la funció biològica del lloc al·lostèric particular, que generalment és desconeguda. Per aquest motiu, després vam provar l'aplicabilitat de la combinació de CV d'alt rendiment amb assaig de contingut alt de baix rendiment. Això ens va permetre caracteritzar un nou lloc d’unió al·lostèric en PTEN i també descriure els primers moduladors al·lostèrics d'aquesta proteïna. Finalment, a mesura que l'espai químic accessible creix a un ritme ràpid, hem desenvolupat un algorisme per explorar de manera eficient col·leccions de productes químics molt grans mitjançant un enfocament de baix a dalt. Vam validar aquest enfocament amb BRD4 i vam identificar nous inhibidors de BRD4 amb una afinitat comparable als candidats a fàrmacs més avançats per a aquesta proteïna

Identification by virtual screening of protein tyrosine phosphatase 1B and matrix metalloproteinase 13 inhibitors for the treatment of obesity and obesity-associated disorders

L'obesitat és un dels principals problemes de salut pública del segle XXI. La gran expansió econòmica de les últimes dècades en els països desenvolupats ha contribuit a l’increment del consum d’aliments poc saludables i a l’ús excessiu de tecnologies d’estalvi d’energia. Aquests canvis han generat estils de vida poc saludables i el consegüent augment de la prevalença d'obesitat. Així doncs, l'obesitat sorgeix com una resposta natural a un entorn antinatural. Amb l'augment continu de la població obesa en cada generació, la prevalença de trastorns associats a l'obesitat com la diabetis tipus II i l'artrosi també augmenta, i la perspectiva de desenvolupar una teràpia mèdica específica per a cada pacient va guanyant interès. En aquest sentit, les dianes proteïna tirosina fosfatasa 1B (PTP1B) i la metaloproteasa de la matriu 13 (MMP-13) estan implicades tant en l’obesitat com, respectivament, la diabetis mellitus de tipus II i l’artrosi. La present tesi doctoral es centra en el desenvolupament d'estratègies de cribratge virtual per tal d’identificar compostos que modulin l'activitat d'aquestes dues dianes i puguin influir positivament en l'obesitat i els trastorns associats a l'obesitat.La obesidad es uno de los principales problemas de salud pública del siglo XXI. La gran expansión económica de las últimas décadas en los países desarrollados a contribuido al incremento del consumo de alimentos poco saludables y al uso excesivo de tecnologías de ahorro de energía. Estos cambios han generado estilos de vida poco saludables y el consiguiente aumento de la prevalencia de obesidad. Así pues, la obesidad surge como una respuesta natural a un entorno antinatural. Con el aumento continuo de la población obesa en cada generación, la prevalencia de trastornos asociados a la obesidad como la diabetes tipo II y la artrosis también aumenta, y la perspectiva de desarrollar una terapia médica específica para cada paciente va ganando interés. En este sentido, las dianas proteína tirosina fosfatasa 1B (PTP1B) y la metaloproteasa de la matriz 13 (MMP-13) están implicadas tanto en la obesidad como, respectivamente, la diabetes mellitus de tipo II y la artrosis. La presente tesis doctoral se centra en el desarrollo de estrategias de cribado virtual para identificar compuestos que modulen la actividad de estas dos dianas y puedan influir positivamente en la obesidad y los trastornos asociados a la obesidad.Obesity is one of the major public health problems in the 21st century. The great economic expansion of the last decades in developed countries has contributed to the increased consumption of unhealthy foods and the excessive usage of energy-saving technologies. These have in turn led to the development of unhealthy lifestyles and the consequent increase of obesity prevalence. Thus, obesity has emerged as a natural response to an unnatural environment. With the continuous increase in obese population in each generation, the prevalence of obesity-associated disorders such as type II diabetes and osteoarthritis is also on the rise, and the prospect of developing a medical therapy specific for each patient earns increasing interest. In this regard, the targets protein tyrosine phosphatase 1B (PTP1B) and matrix metalloproteinase 13 (MMP-13) are involved in both obesity and, respectively, type II diabetes mellitus and osteoarthritis. The present doctoral thesis focuses on developing virtual screening strategies to identify compounds that modulate the activity of these two targets which may have a positive influence on both obesity and its associated disorders

Targeting farnesyl pyrophosphate synthase of Trypanosoma cruzi by fragment-based lead discovery

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas disease (CD), which mostly affects underprivileged populations in South and Central America. The current standard of care for this disease are the two empirically discovered drugs benznidazole and nifurtimox. They show low efficacy, difficulties in administration and severe side effects. Moreover, there are T. cruzi strains that have formed resistances. Thus, the development of a safe and efficient drug is urgently needed. T. cruzi is dependent on isoprenoid biosynthesis as ergosterol and other 24 alkylsterols are essential metabolites that cannot be acquired by other mechanisms. Therefore, it was hypothesised that enzymes along this pathway are promising drug targets. A number of compounds targeting these enzymes were tested and have been shown to inhibit parasite growth. Among those enzymes is farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the isoprenoid pathway, which is in the focus of this work. It catalyses the synthesis of farnesyl pyrophosphate (FPP), a C15 building block in sterol biosynthesis and in protein prenylation of signalling proteins. Bisphosphonates (BPs) are known active site directed FPPS inhibitors, which exhibit ideal pharmacokinetics to target bone mineral and are used to treat bone diseases. BPs can also combat T. cruzi flagellates but are not ideal to treat CD due to their pharmacokinetics. In the search for new chemotypes, several non-BP inhibitors that bind to another pocket were found for human FPPS (hFPPS) by fragment based screening (FBS). Recently, it was shown that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and locks the enzyme in an open and inactive state, thus showing the allosteric character of this pocket. The current work aims at the discovery of non-BP inhibitors of T. cruzi FPPS (TcFPPS), which could be starting points for the development of a treatment against CD. Towards this goal, recombinant expression in E. coli cells and purification by means of IMAC and SEC yielded pure und homogenous TcFPPS (chapter 5.1). This includes unlabelled, 13C15N labelled and in vivo biotinylated avi-tagged TcFPPS. Furthermore, a novel, reliable, highly reproducible, and well diffracting crystallization system was established. The system exhibits excellent properties for FBS as it was compatible with different types of 96-well plates. Apo crystals were stable for up to 24 h in 15% DMSO and allowed collection of data sets with a diffraction limit of around 1.6 Å. The best achieved diffraction limit was 1.28 Å for a soaked TcFPPS crystal (PDB ID 6R09). The allosteric region in TcFPPS was investigated by means of sequence analysis and structural superimposition of various orthologous FPPSs (chapter 5.2). This revealed that the allosteric region is less conserved than the active site. Differences among residues in equivalent positions that form the allosteric site were observed, which is surprising if it is assumed that all FPPSs can be product inhibited as hFPPS. A remarkable finding is that residue Phe50 in TcFPPS is an exception in an otherwise highly conserved position. It causes steric hindrance of the pocket in TcFPPS. An attempt to reposition established allosteric inhibitors of hFPPS showed binding affinity to TcFPPS but the two obtained crystal structures demonstrated their binding to sites on the protein surface (sites S1 and S2, PDB IDs 6R08 and 6R07, respectively). The Novartis core and fluorine library (1336 and 482 compounds) were screened on TcFPPS, which resulted in 63 and 45 validated fragment hits, respectively (chapter 5.3). Performing the same screen with T. brucei FPPS (TbFPPS), the causative agent of African sleeping sickness, and counter screening on hFPPS led to unique, pairwise and triple binders demonstrating selectivity at the early stage of FBS. Strikingly, TcFPPS has generally more binders than TbFPPS, and TcFPPS has many unique hits when compared to TbFPPS. Subsequent crystallization experiments with the core library hits resulted in 3D structures of two TcFPPS complexes. One ligand binds to the homodimer interface (site S12) and the other one in the active site. The latter was identified by using the statistical analysis tool Pan-Dataset Density Analysis (PanDDA). FBS by X-ray crystallography at the XChem facility in Harwell, UK, and the HTXlab in Grenoble, France, were conducted (chapter 5.4). The XChem screen identified 35 fragment binders (PDB IDs 5QPD – Z, 5QQ0 – 9, 5QQA – C) in binding sites that were distributed over the entire protein. This includes the active site, the allosteric site, the homodimer interface, sites on the surface and a new site in close proximity to the active site. Strikingly, the first two fragments binding to the allosteric site of TcFPPS in its open state were identified. Rotation of the phenyl side chain of Phe50 led to opening of the former closed pocket. The HTXlab screen identified additional binders for the active and allosteric site. In total 1244 data sets were collected and analysed. This process was accelerated using PanDDA. The first fragment-to-lead optimization by means of virtual screening using the web-based platform ANCHOR.QUERY was based on fragment hit LUY (chapter 5.5). Compounds were synthesised using one-pot one-step multi-component reactions. Synthesis of 11 compounds (MCR 1 – 11) was successful, but poor solubility was detrimental in subsequent testing on TcFPPS and crystallization experiments did not lead to a structural model of a complex. A second fragment to lead optimization using a fragment merging approach for chemical optimization was based on the active site directed binders AWM, LVV, LUY, LDV and AWV (chapter 5.6). A library of 12 compounds (MCN 1 – 12) was synthesised by reductive amination. X-ray structures revealed unexpected binding modes for compounds MCN-1, -4 and -8. Instead of retaining the binding site of the fragment, the merged compounds bind to the surface directed binding site S1 (PDB IDs 6R09, 6R0A, 6R0B). Nevertheless, the 50 new crystal structures of TcFPPS fragment complexes discussed in this work will pave the way for future drug discovery campaigns for CD. The large diversity of the fragments’ scaffolds and different binding sites are potential starting points for inhibitors with different physicochemical properties and a novel mode of action that might help to overcome the limitations related to the BP scaffold