A Computational Approach to Analyze the Mechanism of Action of the Kinase Inhibitor Bafetinib

Prediction of drug action in human cells is a major challenge in biomedical research. Additionally, there is strong interest in finding new applications for approved drugs and identifying potential side effects. We present a computational strategy to predict mechanisms, risks and potential new domains of drug treatment on the basis of target profiles acquired through chemical proteomics. Functional protein-protein interaction networks that share one biological function are constructed and their crosstalk with the drug is scored regarding function disruption. We apply this procedure to the target profile of the second-generation BCR-ABL inhibitor bafetinib which is in development for the treatment of imatinib-resistant chronic myeloid leukemia. Beside the well known effect on apoptosis, we propose potential treatment of lung cancer and IGF1R expressing blast crisis

Design and Synthesis of Trisubstituted Pyrimidine Derivatives as Coactivator Binding Inhibitors (CBIs) of Estrogen Receptor Signaling

Breast cancer (BC) is one of the most commonly diagnosed cancers in women. Estrogen signaling and the estrogen receptors (ERα, ERβ) are implicated in breast cancer progression. Majority of breast cancers start out as estrogen dependent as a result of overexpression of ER-regulated genes: Estrogen deprivation therapy using anti-estrogens (AEs) and aromatase inhibitors (AIs) to block ER activity and arrest the estrogen-dependent growth of BC still represents the primary treatment for breast cancer patients. This approach, however, frequently fails and patients develop resistant breast cancer, which is almost untreatable. In this project we focused on synthesizing a potent coactivator binding inhibitors (CBIs) molecules that block ER activity through a different mechanism that may arrest the estrogen-dependent growth of BC and offer a solution to the existing resistance. Coactivator binding inhibitors (CBIs) act by blocking the conformational change needed for DNA binding and gene expression. In this project set of compounds have been designed, synthesized through sequential substitution of the chlorine atoms of 2,4,6-trichloropyrimidine with amines or other nucleophiles. The synthesized compounds were purified using chromatography techniques, and characterized by (1H-NMR, 13C-NMR, FT-IR and MS (ESI)) spectroscopy. Some of these compounds were screened for their inhibitory activity against the acute myeloid leukemia cells (Molm-13) cells. Two forms of Molm-13 had been used to evaluate the role of p53. In one case cells were transfected with empty vector and in the other the cells were tranfected with sh-p53 RNA(sh-p53). The viability of cells was determined using WST-1 assay. Initial results of the tested compounds demonstrated that MY12 (35) and MY3 (26) exhibited potent activities against the two forms of Molm-13 cell lines and have a dose dependent effect while the compound MY2 (25) showed no significant inhibitory action on the same cell lines

Markers and mechanisms of resistance to Toceranib phosphate (Palladia®) in canine cutaneous mast cell tumor

2014 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Aberrant kinase activation as a therapy target : Chronic myeloid leukemia as a model disease

Chronic myeloid leukemia (CML) is a hematologic malignancy that originates from pluripotent hematopoietic stem cells. The genetic abnormality underlying this disease, as well as a subtype of acute lymphoblastic leukemia (ALL), is a translocation between chromosomes 9 and 22, which leads to the formation of the Philadelphia (Ph) chromosome and the oncogenic BCR-ABL1 fusion protein. Targeted inhibition of the BCR-ABL1 protein with imatinib and other specific tyrosine kinase inhibitors (TKIs) has revolutionized CML treatment and is currently regarded as the gold standard of targeted cancer therapy. In this doctoral thesis, different methodological approaches were used to characterize aberrant signaling activities in leukemic and normal cells with the ultimate aim of isolating novel prognostic markers for predicting TKI therapy outcome. First, the molecular mechanism of the disease was investigated in an ALL patient with a translocation between chromosomes 1 and 9. Cytogenetic characterization showed involvement of the ABL1 gene fused to an unknown gene. The RCSD1 gene was selected as a candidate and subsequent molecular dissection confirmed a RCSD1-ABL1 fusion. This novel fusion gene predicts sensitivity to TKI therapy in ALL patients. Second, several phosphoproteins related to immune cell function were analyzed both from the diagnostic phase CML patients and from patients under TKI therapy in order to reveal distinct signaling patterns. A single cell phosphoprotein method based on multiparameter flow cytometry was used to analyze phosphoprotein levels in different cell populations. The responsiveness of myeloid cells to ex vivo cytokine stimulation in diagnostic-phase patients was low, but was normalized in TKI-treated patients. In general, the blood leukocyte subsets responded normally to various cytokine stimulations, which indicated a non-immunosuppressive role for TKIs. This project also led to the development of a software assisted analysis program, which can conduct the whole range of flow cytometry data analysis, thereby diminishing the effort needed for manual cell population gating and interpretation. Lastly, biomarkers for therapy response were identified by analyzing aberrant signaling activities in leukemic cells collected at the time of diagnosis. The analysis was based on a phosphoproteomic array measuring phosphorylation of 46 different kinase targets. Several proteins had aberrant phosphorylation levels in patients with a poor therapy outcome − pSTAT5b being the most prominent. These biomarkers could be useful in guiding therapy selection at the time of diagnosis. In conclusion, the findings imply that TKI therapy responses can be linked to certain biological markers, which can possibly be utilized in clinical applications in the future.Krooninen myelooinen leukemia (KML) on pahanlaatuinen verisairaus, joka saa alkunsa luuytimen kantasolun perimän muutoksesta. Muutoksen seurauksena syntyy Philadelphia-kromosomi (Ph), jonka ohjaamana syntyy aktiivisuudeltaan epänormaalia BCR-ABL1 tyrosiinikinaasia, mikä johtaa myelooisen sarjan verisolujen hallitsemattomaan lisääntymiseen. Ph-kromosomi on havaittavissa myös osalla akuuttia lymfaattista leukemiaa (ALL) sairastavilla potilailla. Nykyään KML:n hoitona käytetään tyrosiinikinaasiestäjiä (TKE), kuten imatinibia, joiden vaikutus kohdentuu spesifisesti BCR-ABL1-proteiiniin. Kyseiset lääkkeet saavat aikaan hyvän hoitovasteen suurimmalla osalla potilaista. Osalla potilaista ongelmana on kuitenkin vasteen puuttuminen tai resistenssin kehittyminen. Tutkimuksen tarkoituksena oli määrittää biologisia mittavälineitä, joiden avulla voitaisiin ennustaa hoitovastetta TKE-lääkitykselle sekä ohjata sopivan lääkityksen valitsemista. Tutkimuksen ensimmäisessä osassa selvitettiin Ph-negatiivisen ALL potilaan taudin molekulaarista mekanismia, koska potilas oli saanut hoitovasteen TKE-lääkitykselle. Taudin taustalta löytyi uudenlainen RCSD1-ABL1 geeni, jota voidaan tulevaisuudessa hyödyntää ALL-potilaiden taudin karakterisoinnissa ja oikeanlaisen lääkityksen valitsemisessa. Toisessa osatyössä tutkittiin TKE-lääkkeiden vaikutusta KML-potilaiden immuunijärjestelmän toimintaan määrittämällä solunsisäiseen viestintään osallistuvien signaalimolekyylien aktiivisuuksia virtaussytometrisellä menetelmällä. Havaintona oli, että diagnoosivaiheessa potilaiden tietyt valkosolut eivät reagoi kasvutekijään, mutta TKE-lääkityksen aikana reagointi oli normaalia, mistä voidaan päätellä etteivät TKE-lääkkeet haittaa potilaiden immuniteettia. Tämän työn pohjalta kehitettiin myös automaattinen tietokoneohjelmisto virtaussytometriadatan käsittelyyn. Viimeisessä osassa määritettiin KML-potilaiden luuytimistä solunsisäisten signaalimolekyylien aktiivisuuksia, joista osan todettiin korreloivan potilaiden hoitovasteiden kanssa. Näiden molekyylien aktiivisuuksia voidaan mahdollisesti hyödyntää oikeanlaisen TKE-lääkityksen valitsemisessa KML-potilaille. Yhteenvetona voidaan todeta, että TKE-lääkitykseen liittyvät hoitovasteet voidaan yhdistää tietynlaisiin biologisiin mittareihin, jotka antavat mahdollisuuden potilaiden lääkityksen suunnitteluun ja hoitovasteen ennustamiseen

Morphological profiling of small molecules for mode-of-action studies using the Cell Painting Assay

The Cell Painting Assay (CPA) is an unbiased morphological profiling approach that generates a holistic view of the bioactivity space. Therefore, it is a powerful tool to deconvolute targets or mode-of-action (MoA) of small molecules, which is especially important for non-protein targets as they are difficult to identify with commonly applied methods. In order to examine the applicability of the CPA to identify non-proteins targets, the morphological fingerprint of the iron chelator Deferoxamine (DFO), as well as references and so far uncharacterized compounds with similar fingerprints, were investigated. Reference compounds, biosimilar to DFO, possess different annotated targets and activities but share a common MoA of cell cycle arrest. This was experimentally confirmed for a representative selection of references. The cluster analysis enabled the identification of novel and so far uncharacterized chelating agents and DNA synthesis modulators. Furthermore, hierarchical clustering, using the CPA fingerprints, revealed a first insight into the different mechanisms of action. To investigate the bioactivity of a small tetrahydroindolo[2,3-a]quinolizine compound class, a combination of morphological profiling using the CPA and proteome profiling was pursued. The results revealed an altered cholesterol homeostasis induced by the compound’s physicochemical properties that led to an accumulation and an increased pH in lysosomes. More than 400 reference compounds and well-characterized drugs with different annotated targets and activities share high biosimilarity to the most active derivative. The majority of the compounds in this cluster also possess physicochemical properties, that are predictive for the accumulation in lysosomes. Modulation of cholesterol homeostasis was experimentally confirmed for a representative selection of references. Therefore, this cluster can be used to identify novel modulators of cholesterol homeostasis but also to associate the regulation of corresponding genes or proteins to an effect induced by the physicochemical properties of the compounds. The findings presented in this thesis emphasize the power of the CPA to evaluate bioactive small molecules and to predict diverse MoA for uncharacterized compounds as well as to uncover and expand so far unknown activity for already characterized small molecules and drugs

Multiscale modeling of the causal functional roles of nsSNPs in a genome-wide association study: application to hypoxia

Background It is a great challenge of modern biology to determine the functional roles of non-synonymous Single Nucleotide Polymorphisms (nsSNPs) on complex phenotypes. Statistical and machine learning techniques establish correlations between genotype and phenotype, but may fail to infer the biologically relevant mechanisms. The emerging paradigm of Network-based Association Studies aims to address this problem of statistical analysis. However, a mechanistic understanding of how individual molecular components work together in a system requires knowledge of molecular structures, and their interactions. Results To address the challenge of understanding the genetic, molecular, and cellular basis of complex phenotypes, we have, for the first time, developed a structural systems biology approach for genome-wide multiscale modeling of nsSNPs - from the atomic details of molecular interactions to the emergent properties of biological networks. We apply our approach to determine the functional roles of nsSNPs associated with hypoxia tolerance in Drosophila melanogaster. The integrated view of the functional roles of nsSNP at both molecular and network levels allows us to identify driver mutations and their interactions (epistasis) in H, Rad51D, Ulp1, Wnt5, HDAC4, Sol, Dys, GalNAc-T2, and CG33714 genes, all of which are involved in the up-regulation of Notch and Gurken/EGFR signaling pathways. Moreover, we find that a large fraction of the driver mutations are neither located in conserved functional sites, nor responsible for structural stability, but rather regulate protein activity through allosteric transitions, protein-protein interactions, or protein-nucleic acid interactions. This finding should impact future Genome-Wide Association Studies. Conclusions Our studies demonstrate that the consolidation of statistical, structural, and network views of biomolecules and their interactions can provide new insight into the functional role of nsSNPs in Genome-Wide Association Studies, in a way that neither the knowledge of molecular structures nor biological networks alone could achieve. Thus, multiscale modeling of nsSNPs may prove to be a powerful tool for establishing the functional roles of sequence variants in a wide array of applications

Ecological and Chemical Studies on the Gq-protein Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, displays a strong and selective inhibition of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as corroborated by in vivo experiments on mice, affinity to insect Gq proteins and insect toxicity studies. The uncultured endosymbiont of A. crenata, 'Candidatus Burkholderia crenata' was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. In this study we provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time, paving the way for a biotechnological production of FR independent from time- and work-intensive plant cultivation, harvesting and extraction. Direct targeting of intracellular Gα subunits is a challenging task in pursuit of chemical tools for pharmacological studies and for developing novel therapeutic approaches. We isolated four new FR analogs (1-4) from A. crenata and elucidated their structures by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. Next, we analyzed all currently known inhibitors of Gq protein including YM-254890, FR900359, above mentioned novel FR-derivatives from A. crenata, and synthetic cyclic peptides to devise a strategy for the elucidation of characteristics that determine interaction with Gq. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocycles that govern specific binding to and inhibition of Gq. While all novel compounds were devoid of effects on Gi and Gs proteins, no inhibitor surpassed biological activity of FR or YM. This raises the question of whether nature has optimized these depsipeptides for specific inhibition of Gq. Thus, rather than attempting to enhance Gq activity of newly synthesized inhibitors, future synthetic efforts on FR/YM-analogs should target Gα subunits other than Gq. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated A. lucida as well as from a soil bacterium, implicating a much broader distribution of FR as originally anticipated. Furthermore the first reported fluorescent FR analogs were synthesized, biologically evaluated and applied to study the mechanism of cellular uptake of FR