QSAR Modelling to Identify LRRK2 Inhibitors for Parkinson's Disease

Parkinson's disease is one of the most common neurodegenerative illnesses in older persons and the leucine-rich repeat kinase 2 (LRRK2) is an auspicious target for its pharmacological treatment. In this work, quantitative structure-activity relationship (QSAR) models for identification of putative inhibitors of LRRK2 protein are developed by using an in-house chemical library and several machine learning techniques. The methodology applied in this paper has two steps: first, alternative subsets of molecular descriptors useful for characterizing LRRK2 inhibitors are chosen by a multi-objective feature selection method; secondly, QSAR models are learned by using these subsets and three different strategies for supervised learning. The qualities of all these QSAR models are compared by classical metrics and the best models are discussed in statistical and physicochemical terms.Fil: Sebastián Pérez, Víctor. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Martínez, María Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias e Ingeniería de la Computación. Universidad Nacional del Sur. Departamento de Ciencias e Ingeniería de la Computación. Instituto de Ciencias e Ingeniería de la Computación; ArgentinaFil: Gil, Carmen. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Campillo Martín, Nuria Eugenia. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Martínez, Ana. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Ponzoni, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias e Ingeniería de la Computación. Universidad Nacional del Sur. Departamento de Ciencias e Ingeniería de la Computación. Instituto de Ciencias e Ingeniería de la Computación; Argentin

Aktiivsete ühendite disain neurodegeneratiivsete haiguste raviks

Väitekirja elektrooniline versioon ei sisalda publikatsiooneNeurodegeneratiivsed haigused on tänapäeval kujunenud keskseks meditsiiniliseks ja sotsiaalseks probleemiks. Ühest küljest on selle põhjuseks haigustega kaasnevad rasked füüsilised ja vaimsed puuded ning mõjusate ravimeetodite puudumine. Teisalt on valdav enamus neurodegeneratiivseid haigusi seotud vananemisega. Oodatava eluea märkimisväärne pikenemine on põhjustanud patsientide arvu olulist kasvu. Üks peamisi takistusi neurodegeneratiivsete häirete jaoks radikaalsete ravimeetodite leidmisel on uute ravimite väljatöötamise protsessi pikkus ja kulukus. Viimase kümnendi jooksul on aga molekulaarse modelleerimise ja tehisintellekti arvutusmeetodite kaasamine võimaldanud märkimisväärselt lühendada nii uute ravimite väljatöötamiseks kuluvat aega kui ka maksumust. Käesolevas väitekirjas rakendati mitmesuguseid arvutipõhiseid ravimite otsimise meetodeid uute potentsiaalsete aktiivsete keemiliste ühendite väljatöötamiseks neurodegeneratiivsete haiguste raviks. Kaasaegsete arvutikeemia molekulaarsildamise ja molekulaardünaamika meetodite abil sõeluti virtuaalselt suuri keemiliste ühendite andmebaase, leidmaks neurodegeneratiivsete haigustega seotud valkudele toimivaid aineid. Nii tehti kindlaks rida uusi looduslikke ühendeid, mis toimivad erinevate ensüümvalkude inhibiitoritena, aga ka uudne ühend, mis toimib efektiivselt samaaegselt kahele Alzheimeri tõvega seotud valgule. Üks peamisi neurodegeneratiivsete haiguste tekke põhjusi on nn närvikasvufaktorite puudulikkus neuronites. Seetõttu on väga huvitavaks ja perspektiivseks suunaks keemiliste ühendite leidmine, mis käituksid analoogselt nende faktoritega ning kaitseks närvirakke suremise eest. Käesoleva töö käigus uuriti erinevate arvutusmeetodite abil põhjalikult ühe taolise närvikasvufaktori (gliia närvikasvufaktor GDNF) toimemehhanismi ning ennustati seda faktorit imiteeriv aktiivne ühend. Kuigi selle eksperimentaalselt mõõdetud neuroneid kaitsev toime ei vasta veel ravimitele esitatavatele nõutele, on siiski tegemist esimese sellelaadse ühendiga maailmas, mille alusel oleks võimalik välja arendada täiesti uut tüüpi ravimeid nii Parkinsoni kui ka Huntingtoni tõve raviksToday, neurodegenerative diseases are one of the most acute medical and social problems. This is due to both severe physical and mental disabilities resulting from the constant progression of the process, and the age-dependent nature of the vast majority of neurodegenerative diseases. The current accelerating increase in life expectancy inevitably leads to a significant increase in the number of such patients. There is currently no radical treatment for neurodegenerative disorders. One of the main obstacles to finding effective drugs is the length and cost of the process of developing a new drug. However, the development of modern molecular modelling and artificial intelligence methods has substantially shortened the time to dispense new medicines and reduced their cost. This dissertation provides examples of the use of various methods of computer-aided drug design such as molecular docking and molecular dynamics to develop new potential candidates against neurodegenerative diseases. The high-throughput virtual screening of large molecular libraries enabled to identify effective compounds against target proteins related to neurodegenerative diseases. In result, a series of natural compounds acting as inhibitors to enzymes related to different diseases was established. Notably, a fully novel compound acting against two proteins related to Alzheimer’s disease was predicted and experimentally verified. One of the main causes of the neurodegeneration is the mostly age-related deficiency of so called neurotrophic factors. Small molecules that can mimic the activity of these factors in cells would be thus very attractive novel drug candidates. In the present thesis, the computational modelling was used for detailed study of the mechanism of action of one of the most important neurotrophic factors (glial cell-derived neurotrophic factor GDNF). The results of this study enabled to develop first time a compound that acted similarly to this factor itself. Whereas the experimentally measured activity of this compound was moderate, it creates a basis for the development of fully new type of drugs against Parkinson’s and Huntington’s diseases.https://www.ester.ee/record=b535990

Amelioration of Neuroprotective Effect of Semisynthetic Derivatives of Piperine in Rotenone induced Rat Model of Parkinson’s Disease

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder and defined as a complex disorder with multifactorial pathogenesis. The systemic rotenone model accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. Piperine is an alkaloid isolated from black pepper and possess antioxidant, antidepressant, anti-inflammatory and neuroprotective effects. AIM OF THE STUDY: The present study has been designed to evaluate the amelioration of neuroprotective effect of semisynthetic derivatives of piperine in rotenone- induced rat model of Parkinson’s disease. METHODS: In the present study, about 113 semisynthetic piperine derivatives (SDP) were subjected to ADMET properties, molecular docking studies using AutoDock 4.2 against target enzymes COMT (3A7D) and MAO-B (3PO7). Black pepper was used for the isolation of piperine. Based on in silico study, 6 compounds were selected and synthesized by three schemes with various substitutions. Analytical studies (UV, IR, NMR and Mass spectra) were done to confirm its structure and in vitro MAO-B inhibitory activity was performed. The compound (SDP 20) offered better IC50 value and hence it was selected for in vivo study. Acute toxicity was performed following OECD TG 423. About 24 Male Wistar rats were divided into 4 groups (n=6). Animals in all the groups received rotenone in the dose of 2.5 mg/kg i.p daily for 28 days. Group I served as negative control. The animals in group II and III received SDP 20 (100 and 200 mg/kg respectively) p.o. The animals in group IV received levodopa (9mg/kg) and carbidopa(2.25mg/kg) p.o. Behavioral parameters (Rota rod, grip strength, locomotion and narrow beam walk test) were performed on weekly intervals throughout the study. Animals were sacrificed on 29th day for neurotransmitter and biochemical estimation and histopathological changes. RESULTS: The compounds were selected based on BBB barrier penetration and docking scores. A total of 6 compounds were synthesized and analytical studies were carried out. The in vitro MAO-B inhibitory activity was performed for the compounds and SDP 20 was selected for in vivo study. In in vivo study it was demonstrated that significant alteration in behavioral parameters, neurochemical changes, oxidative and histopathological changes were observed in rotenone- treated animals. Administration of SDP 20 prevented alterations induced by rotenone. CONCLUSION: These findings supports the PD treatment by lowering the impairments in motor activity. The results also suggest that SDP 20 may offer a promising and new therapeutic lead for the treatment of PD which needs further research

Small molecular inhibitors of Amyloid β and α Synuclein amyloidogenic aggregation, toxicity and in silico design of amyloid-binding ligands

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common forms of dementia and a leading cause of death amongst the older population worldwide. Pathologically, both AD and PD are characterized as progressive neurodegenerative disorders, in which there is a progressive loss of neuronal structure and function leading to loss of neurons in parts of the brain associated with cognition, memory and movement. Amyloidogenic protein misfolding and aggregation is at the centre of the neurodegenerative processes associated with AD and PD. In the case of AD, the amyloidogenic protein is amyloid β (Aβ) and in PD, it is the α Synuclein (αS) protein, or its mutant forms such as αSA53T found in familial PD which aggregates and exerts toxicity. In both AD and PD, we lack truly disease-modifying drug treatments, with current medications largely providing only modest and transient symptomatic improvement. Therefore, the aim of this project was to identify a diverse set of new molecules from both natural and synthetic origin, that can alter Aβ and αSA53T aggregation and fibril formation. Mostly small molecule binding has been studied by molecular docking and effects on Aβ and αSA53T aggregation investigated using a ligand-binding fluorescence kinetic assay (Thioflavin T) and transmission electron microscopy (TEM). For neuroprotection studies, a mitochondrial viability (MTT) assay of neuronal cells (PC-12) was used. Finally, using molecular docking and density functional theory (DFT) approaches, a set of novel amyloid-binding ligands was designed in silico through ligand-based drug design. In the first study, four polyphenolic bioactives including a neolignan, ellagitannin and two flavonoid class of compounds have been comparatively studied for their binding interactions with Aβ, effects on fibril and aggregate formation and neuroprotection. In the second study, a structure-based virtual screening method based on molecular docking has been implemented to identify new small molecule inhibitors of Aβ aggregation and neurotoxicity. Five heterocyclic compounds were selected and tested using the anti-aggregation and neuroprotection methods. Of these, the two best ‘hits’ bearing a novel molecular scaffold have been used in further studies. In the third study, the two virtual screening hits, polyphenolic bioactives from first study and two structurally related flavonoids were tested for inhibition of amyloidogenic aggregation and neuroprotection of the pathological αS mutant, αSA53T. Seven molecules have been compared as per the first and second study. Additionally, the impact on native αSA53T protein conformation was investigated by ion mobility mass spectrometry (IM-MS). In the fourth study, a diverse set of natural bioactives including a neolignan, flavonoid, chalcone, diterpene and alkaloid class of compound were tested for anti-aggregative effects on both αSA53T and Aβ proteins. Additionally, their direct interactions with these amyloidogenic protein targets were studied by molecular docking. The final study employed molecular docking and small molecular structure optimization using DFT method for rational design of a set of novel amyloid ligands. The favourable molecular attributes gleaned from both the bioactive neolignan in the first study and a favourable molecular scaffold in the second study were used to optimise binding for potential anti-aggregation. These ligands were predicted to have improved binding to Aβ and αSA53T in silico. Since there is an urgent need of disease-modifying therapies in both AD and PD, identification of anti-amyloidogenic and neuroprotective molecules would facilitate future drug discovery efforts. Design of a novel amyloid binder would be valuable for research and development of a therapeutic candidate or may assist in further design of an amyloid tracer molecule for diagnostic imaging.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

DISCOVERY OF INDOLYL PIPERAZINYLPYRIMIDINES WITH DUAL-TARGET PROFILES ADENOSINE A2¿ AND DOPAMINE D2 RECEPTORS FOR PD TREATMENT

Ph.DDOCTOR OF PHILOSOPH

Développement de nouvelles approches protéo-chimiométriques appliquées à l'étude des interactions et de la sélectivité des inhibiteurs de kinases

The human kinome contains 518 proteins. They share a common mechanism of protein phosphorylation known to play an important role in cellular signaling pathways. Impaired kinase function is recognized to be involved in severe diseases like cancer. Due to high structural similarity between protein kinases, development of potent and selective kinase inhibitors is a challenging task. The selectivity of kinase inhibitors may lead to side effects potentially harmful. In this thesis, we first developed new selectivity metrics to determine inhibitor selectivity directly from biological inhibition data. Compared to existing metrics, the new selectivity scores can be applied on diverse inhibition data types. Second, we developed a proteometric approach in order to understand why some protein kinases are never inhibited by Type II inhibitors. The statistical model built for this purpose allowed us to identify several discriminant residues of which few of them correspond to experimentally described residues of interest. Third, using a new 3D protein kinase descriptor, we developed and validated novel proteo-chemometrics approaches to study and discover new kinase inhibitors.Le kinome humain comprend 518 protéines. Elles participent au processus de phosphorylation des protéines qui joue un rôle important dans les voies de signalisation cellulaire. Leur dérégulation est connue comme étant une cause de nombreuses maladies graves telle que les cancers. Du fait de leur grande similarité structurale des protéines kinases, il est difficile de développer des inhibiteurs qui soient à la fois efficaces et sélectifs. L’absence de sélectivité conduit le plus souvent à des effets secondaires particulièrement néfastes pour l’organisme. Au cours de cette thèse, nous avons d’abord développé de nouvelles métriques dont le but est de déterminer la sélectivité d’inhibiteurs à partir de données d’inhibition. Elles présentent l’avantage, comparées à d’autres métriques, d’être applicables sur n’importe quel type de données. Dans un deuxième temps, nous avons développé une approche protéométrique dans le but de comprendre pourquoi certaines protéines kinases ne sont jamais inhibées par des inhibiteurs de Type II. Le modèle statistique mis en place nous a permis d’identifier plusieurs résidus discriminants dont certains déjà décrits expérimentalement dans la littérature. Dans un troisième temps, nous avons développé un nouveau descripteur 3D de protéines kinases avec lequel nous avons mis en place et validé des modèles protéo-chimiométriques visant à étudier et découvrir de nouveaux inhibiteurs

Biochemical mechanisms and target drugs in neurodegenerative diseases

Neurodegenerative diseases, namely Alzheimer’s and Parkinson’s diseases are a major challenge for medicine and public health due to their prevalence in developed countries. Thus, the research for therapies for neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, should be based on understanding their molecular and biochemical pathogenesis. The research conducted in this thesis involves screening of different families of compounds (isoquinolinones, azepanones, indolinones, diether-esters, chromanones, chromanols and rivastigmine derivatives) based on their ability to inhibit the activity of the therapeutic targets acetylcholinesterase, butyrylcholinesterase and monoamine oxidase B. These targets were chosen for their importance in the neuropathology of Alzheimer’s and Parkinson’s diseases. The most promising compounds were then selected, and the determination of their action at the molecular level was studied via STD-NMR. These studies allow us to understand the importance of different functionalities within the inhibitor molecule on the inhibition of the selected targets, and thus direct the investigation in the sense of developing compounds that can be better inhibitors. Toxicological and pharmacological evaluation of the most promising synthesized compounds was performed using two different biological models, A. salina and Swiss mouse model. Compound 4-[(3-hydroxy-2-oxo-3-phenylindolin-1-yl)methyl]piperidin-1-ium chloride was tested ex vivo against hepatic AChE and BuChE, showing IC50 values of 594.64 μM and 434.51 μM, respectively. This compound was also assayed in vivo after intraperitoneal administration of 3 mg kg-1 and 6 mg kg-1 in Swiss mice, using donepezil (3 mg kg-1) as a benchmark. This synthetic compound gave better brain AChE inhibition than donepezil, indicating that this compound might have a similar brain uptake mechanism to that of donepezil; Resumo: Mecanismos bioquímicos e alvos terapêuticos em patologias neurodegenerativas As doenças neurodegenerativas, nomeadamente as doenças de Alzheimer e Parkinson são um enorme desafio quer para a medicina quer em termos de saúde pública devido à sua prevalência nos países desenvolvidos. Assim, a pesquisa de terapias para doenças neurodegenerativas, como o Parkinson e o Alzheimer, deve ser baseada na compreensão da sua patogénese molecular e bioquímica. Este trabalho envolve screening de diferentes famílias de compostos; derivados de isoquinolinonas, azepanonas, indolinonas, dieter-ester, cromanonas, cromanois e de rivastigmina, baseado na sua capacidade de inibir a atividade de alvos terapêuticos de algumas doenças neurodegenerativas como Alzheimer e Parkinson, nomeadamente: acetilcolinesterase, butirilcolinesterase e monoamino oxidase B. Os compostos promissores foram selecionados para determinação da sua ação a nível molecular por STD-NMR. Este estudo permite compreender a importância dos diferentes grupos funcionais na inibição dos alvos selecionados, e desta forma, direcionar a investigação no sentido de desenvolver compostos com maior atividade inibitória. A avaliação toxicológica e farmacológica dos compostos sintetizados mais promissores foi efetuada utilizando dois modelos biológicos diferentes, A. salina e ratinho Swiss. O composto cloreto de 4-[(3-hidroxi-2-oxo-3-fenilindolin-1-il) metil] piperidin-1-ium foi testado ex vivo em homogenatos de hepatócito de murganho Swiss, tendo apresentado valores de IC50 de 594.64 μM e de 434.51 μM para as atividades de AChE e BuChE, respetivamente. A capacidade inibitória deste composto foi também avaliada in vivo após administração interperitoneal de 3 mg kg-1 e 6 mg kg-1 em ratinhos Swiss, utilizando donepezilo (3 mg kg-1) como padrão. Os resultados mostraram que o composto sintetizado apresentou valores de inibição de atividade de AChE no cérebro superiores aos observados para o donepezilo, podendo indicar que a sua captação pelos tecidos cerebrais poderá ser efetuada de modo semelhante à do donepezilo

Approaches to target WD40 proteins and synthesis and evaluation of chemical tools for on-bead screening

A database consisting of information on human WD40 domains was compiled from literature sources. Data collected included information on function, structure, links to disease and information on molecules known to bind to WD40 domain containing proteins. Curation of the data collected suggested that 21% of WD40 domain containing proteins are linked to cancer, and that only 6% had known small molecule binders. From the database a shortlist of WD40 domain containing proteins that were considered of interest as research targets was produced. It was determined that WD40 Domain Containing Protein 5 (WDR5) was a potential cancer target open to several targeting methods. WDR5 normally plays a structural roll in the formation of a complex containing WDR5, RbBP5, ASH2L, and DPY-30. This complex is required for methylation of H3K4, when MLL1 joins the complex it is able to methylate H3K4me2. It was also recently determined that WDR5 complexes with MYC, another protein with roles in transcriptional control. Both MLL1 and MYC are known to be prominent cancer targets. His-tagged WDR5 was successfully expressed in BL21 (DE3) cell line and purified by a 2 step method. First the protein was purified via His tag - Ni-NTA agarose affinity chromatography, the eluted protein was then further purified via Size Exclusion Chromatography. The first approach targeting WDR5 consisted of a combination of an in-silico approach and a small molecule binder screen. Two in-silico methods, Q-mol and USRCAT, were used to determine small molecules that would potentially bind to WDR5. From this suggested set, 81 compounds were screened against WDR5. Thermal denaturation fluorescence (TDF) was chosen as assay technique. A single compound increased the thermal stability of WDR5 in repeated experiments. This compound, NCI292249, was further characterised in microdialysis experiments where it was determined to have a low affinity of 564 μM to WDR5. In a second approach, in order to target the MYC-WDR5 interaction, a series of truncated peptides derived from the WDR5 binding motif from MYC were produced testing a variation of the One-Bead One-Compound (OBOC) synthesis method derived in this work. These peptide fragments were synthesised using Fmoc solid phase peptide synthesis on TentaGel micobeads experimenting with the SOBOC technique (Scanning OBOC) adapted to produce all possible fragments of a peptide in parallel. The peptide fragments were fluorescently labelled with tetramethylrhodamine and screened against 6XHis-WDR5 isolated on Ni-NTA functionalised agarose beads. The peptide fragments were ranked based on their affinity to the WDR5-coated micro-beads assayed by Confocal Scanning (CONA). The highest affinity peptide found in the CONA screen was further tested for WDR5 binding in solution by fluorescence anisotropy which resulted in an affinity of 96 μM to WDR5. This 7-mer truncate of the MYC peptide was used as input for an in-silico method of peptide optimisation named MorPH. MorPH is a technique developed in the Auer Lab in which amino acids in a peptide are systematically replaced by all commercially available non-natural amino acids in a sequential manner. Each of ~ 1000 modified peptidomimetics are docked in-silico to the target structure. The suggestions from MorPH for the MYC peptide truncate were analysed and the potential replacements discussed in order to plan a possible future synthesis. The MorPH technique was tested experimentally in this thesis in a second example, targeting of Survivin. Survivin is followed as cancer target in the Auer lab and it is found in significantly high concentrations in cancer cell lines and in stem cells. Increased Survivin expression has also been linked to a poor prognosis and reduced patient survivability in the clinic. Several suggestions resulting from the MorPH in-silico screen were synthesised and screened against Survivin. The best-in-series peptide was shown to have a Kd of 2.5 μM, with significantly increased plasma stability. Several chemical tools were developed and characterised for use with on-bead synthesis methods. A contribution was made to a novel synthetic method for isomerically pure rhodamine dyes and their functionalisation[1] (Tetramethylrhodamine was azide-functionalised for use in peptide labelling, based on the azide-alkyne Huisgen cycloaddition reaction). The Auer lab synthesises many of its compounds and libraries using solid phase synthesis techniques. Several compounds exist in literature for the linking of chemicals to a solid-support, all of which are stable to different chemistries and require different conditions to cleave the reaction product from the solid phase. The use of methionine as a linker is described in literature as being highly specific in its cleavage conditions. A series of literature cleavage conditions were tested, the method that offered the highest purity was selected to be improved through further testing. The improved cleavage method was then characterised by cleaving the 20 natural amino acids from the methionine linker. In this experiment it was determined that all amino acids tolerated the new conditions with the exception of methionine, cysteine and tryptophan, which were expected to react poorly to the harsh conditions. This verified that methionine was a suitable alternative to current lab standards for bead linkage