Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.Peer reviewe

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.Peer reviewe

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors

Optimized design and analysis of preclinical intervention studies in vivo

Recent reports have called into question the reproducibility, validity and translatability of the preclinical animal studies due to limitations in their experimental design and statistical analysis. To this end, we implemented a matching-based modelling approach for optimal intervention group allocation, randomization and power calculations, which takes full account of the complex animal characteristics at baseline prior to interventions. In prostate cancer xenograft studies, the method effectively normalized the confounding baseline variability, and resulted in animal allocations which were supported by RNA-seq profiling of the individual tumours. The matching information increased the statistical power to detect true treatment effects at smaller sample sizes in two castration-resistant prostate cancer models, thereby leading to saving of both animal lives and research costs. The novel modelling approach and its open-source and web-based software implementations enable the researchers to conduct adequately-powered and fully-blinded preclinical intervention studies, with the aim to accelerate the discovery of new therapeutic interventions.Peer reviewe

Synthesis, identification and structure-activity relationship analysis of GATA4 and NKX2-5 protein-protein interaction modulators

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.Peer reviewe

Basal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers

INTRODUCTION: Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup. METHODS: IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed. RESULTS: From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification. CONCLUSION: Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors

Suberiinin rasvahappojen hyödyntäminen pinnoite- ja pakkaussovelluksissa

Generally, the literature survey introduces coating and packaging materials and reviews industrial carboxylic acid chemical modifications for polymerization applications like polyesterification, amidification and oxidation methods. Focus is on tungstic acid and phase transfer catalyst catalysed hydrogen peroxide oxidation in a water solution and on deep study of its mechanism. Factors affecting the function of catalyst are acidity of solvent and free ions, which can either improve or weaken the activity of catalyst through ligandation. In addition, biosynthesis of suberin fatty acids main components is reviewed. In the experimental part, a method for extracting suberin fatty acids is shown. In the developed method the extraction solvent was changed from toluene to water, which led to much purer products. In addition, experimental part covers chemical modification of suberin fatty acids for polymer industry applications. Focus was on tungstic acid and phase transfer catalyst catalysed oxidative cleavage of fatty acids by using hydrogen peroxide as oxidant in water. Best results were achieved by using tetrabutylammonium hydrogen sulfate (TBA) as phase transfer catalyst. TBA's counter ion hydrogensulfate doesn't form a complex with tungsten which affects the reaction by lowering catalyst activity. Also reduction, amidification through acid chloride by bubbling ammonia and direct amidification with urea were studied by using azelaic acid as a model substance. Best amidification results were obtained by using direct amidification of carboxylic acids with urea.Kirjallisessa osassa esitellään pinnoitteita ja pakkausmateriaaleja yleisesti sekä teollisia karboksyylihappojen modifiointimenetelmiä polymeerisovelluksiin kuten polyesterointi-, amidointi- sekä hapetusmenetelmiä. Hapetusmenetelmissä keskitytään volframihappokatalysoituun vetyperoksidihapetukseen vesiliuoksessa faasinsiirtokatalyytin avulla ja katalyysimekanismin syvälliseen tarkasteluun. Katalyytin toimintaan vaikuttavia tekijöitä ovat liuoksen happamuus ja liuoksessa olevat ionit, jotka voivat muodostaa joko katalyytin tehoa huonontavia tai parantavia komplekseja. Lisäksi kirjallisessa osassa esitellään suberiinin rasvahappojen pääkomponentteina biosynteesi sekä suberiinin ja sen rasvahappojen nykyisiä sovelluksia. Kokeellinen osa käsittelee suberiinin rasvahappojen eristysmenetelmän kehittämistä ja rasvahappojen analysointia. Kehitetyssä menetelmässä vaihdettiin uuttoliuotin tolueenista veteen, minkä seurauksena saatiin eristettyä huomattavasti vähemmän epäpuhtauksia sisältäviä rasvahappoja. Lisaksi kokeellinen osa käsittelee eristettyjen rasvahappojen kemiallista modifiointia polymeeriteollisuuden sovelluksiin. Rasvahappojen kemiallinen modifiointi keskittyi volframihappo- ja faasinsiirtokatalysoituun hapettavaan katkaisuun vetyperoksidilla. Tutkimusten perusteella havaittiin, että parhaaseen hapetustulokseen päästään käyttämällä faasinsiirtokatalyyttina tetrabutyyliammoniumvetysulfaattia, jonka vastaioni vetysulfaatti ei heikennä volframihappokatalyytin tehoa. Myös rasvahappojen pelkistystä dioleiksi, amidointia happokloridin kautta kuplittamalla ammoniakkikaasulla sekä suoraa amidointia urealla tutkittiin käyttämällä malliaineena atselaiinihappoa. Parhaaseen tulokseen päästiin atselaiinihapon reaktiolla urean kanssa

Synthesis and SAR Analysis of GATA4- NKX2-5 Interaction Inhibitors with Antihypertrophic Activity

Cardiac disease continues to be a leading cause of death and hospitalizations in developed countries. Transcription factors GATA4 and NKX2-5 are master regulators of cardiac gene expression, taking part in multiple processes during heart development, as well as hypertrophy and recovery after e.g. myocardial infarction. Pathological hypertrophy is a homeostatic process, which often leads to cardiac dysfunction in pathological conditions (e.g. hypertension, genetic alterations, and myocardial infarction) further progressing to heart failure. There is an urgent need for treatments that would prevent disease progression at a molecular level. To date, no therapies have directly targeted the transcriptional regulation of cardiac hypertrophy. A novel treatment for this target would be particularly interesting, as current treatments are slowing the disease progression without directly targeting hypertrophic gene expression. The aim of this thesis was to design and synthesize nontoxic GATA4- NKX2-5 interaction inhibitors with antihypertrophic activity and determine their mechanism of action. Furthermore, the generated luciferase and toxicity assay data were analyzed to select compounds for further evaluation. In addition, different cycloaddition methods were explored for a facile synthesis of isoxazole scaffold. Finally, the mechanism of action of the original hit compound 1 was validated with affinity chromatography. In conclusion, novel inhibitors of GATA4-NKX2-5 transcriptional synergy were identified, which inhibit hypertrophic gene expression in rat cardiomyocytes. Remarkably, the immobilized hit compound (1) was shown to bind to GATA4 in the target validation study. In addition, with hierarchical clustering, a group of synergy inhibitors were identified that did not inhibit GATA4 transcriptional activity at 3 μM concentration. Further studies to determine the therapeutic potential of these more selective compounds are clearly needed.Sydänsairaudet ovat merkittävin kuolinsyy ja syy lisääntyneisiin sairaalapäiviin teollisuusmaissa. Transkriptiotekijät GATA4 ja NKX2-5 säätelevät sydängeenien ilmentymistä ja osallistuvat moniin prosesseihin, jotka liittyvät sydämen kehittymiseen, liikakasvuun ja esimerkiksi sydäninfarktin jälkeiseen toipumiseen. Patologinen liikakasvu on homeostaattinen prosessi, joka johtaa usein toimintahäiriöihin sydämen patologisissa tiloissa (esim. verenpainetauti, geneettiset muutokset ja sydäninfarkti) edeten sydämen vajaatoimintaan. Tulevaisuudessa on suuri tarve hoitomuodoille, jotka hidastavat taudin etenemistä molekyylitasolla. Nykyiset sydämen liikakasvun hoitomuodot eivät kohdistu suoraan sydämessä tapahtuvan transkription säätelyyn. Uusi hoitomuoto tähän kohteeseen olisi erityisen mielenkiintoinen, koska nykyiset hoitomuodot hidastavat sairauden etenemistä mutta eivät suoraan vaikuta sydämen liikakasvuun johtavien geenien ilmentymiseen. Tämän väitöskirjatyön tavoitteena oli suunnitella ja valmistaa sydämen liikakasvua vähentäviä GATA4:n ja NKX2-5:n välisen vuorovaikutuksen estäjiä, jotka eivät olisi toksisia, sekä todentaa niiden vaikutusmekanismi. Lusiferaasi- ja toksisuusdata analysoitiin, jotta voitaisiin valita yhdisteitä jatkotutkimuksiin. Tämän lisäksi tutkittiin erilaisia sykloadditioreaktiota isoksatsolirakenteisen molekyylirungon valmistamiseen. Lopuksi alkuperäisen hit-yhdisteen 1 vaikutusmekanismi validoitiin käyttämällä molekyylibiologisia menetelmiä ja affiniteettikromatografiaa. Tässä väitöskirjatyössä tunnistettiin uusia GATA4:n ja NKX2-5:n transkriptiosynergiaan vaikuttavia isoksatsolirakenteisia yhdisteitä, jotka estävät rotan kardiomyosyyttien liikakasvuun johtavien geenien ilmentymistä. Merkittävä löytö oli se, että hit-yhdiste (1) sitoutui GATA4:ään biologisen vaikutuskohteen validointitutkimuksessa. Hyödyntäen hierarkista klusterointia voitiin lisäksi tunnistaa joukko synergiaestäjiä, jotka eivät estäneet GATA4:n transkriptioaktiivisuutta, kun yhdisteiden pitoisuus oli 3 μM. Lisätutkimuksia tarvitaan, jotta voitaisiin arvioida näiden aiempaa selektiivisempien yhdisteiden toimivuutta mahdollisissa lääkehoidoissa