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Background: the study aims to evaluate whether high plasma trough levels of the kinase inhibitors (K.I.s) crizotinib, alectinib, osimertinib, dabrafenib, and trametinib were associated with a higher risk of toxicity in non–small-cell lung cancer patients. Methods: In this retrospective cohort study, patients with non–small-cell lung cancer treated with the selected K.I.s were included if at least one plasma trough level at steady state (Cmin,ss) was available. Data were extracted from electronic medical records and laboratory databases. The high group for each K.I. was defined as 10% of patients with the highest first Cmin,ss. The remaining patients were placed in the non-high group. The frequency of dose-limiting toxicities (DLTs), defined as adverse events leading to dose reduction, dose interruption, or permanent discontinuation, was compared between the 2 groups. Results: A total of 542 patients were included in the different K.I. groups. A high Cmin,ss of crizotinib (n = 96), alectinib (n = 105), osimertinib (n = 227), dabrafenib (n = 52), and trametinib (n = 62) correlated with a Cmin,ss ≥490, ≥870, ≥405, ≥150, and ≥25 ng/mL, respectively. DLTs were more common in the alectinib high group than in the alectinib non-high group (64% vs. 29%, P = 0.036). Liver toxicity was observed in 4 (36%) patients in the high group and 5 (5%) patients in the non-high group (P = 0.007). For other K.I.s, no significant differences were observed in the frequency of DLTs between the high and non-high groups. Conclusions: For alectinib, high Cmin,ss was correlated with a higher risk of DLT. No differences in the frequency of DLTs were observed between the high and non-high groups for crizotinib, osimertinib, dabrafenib, and trametinib.

Colorectal cancer (CRC) is the second leading cancer death cause worldwide after lung cancer and is rising in young adults. A typical APC gene mutation, followed by subsequent mutations, is the cancer driving change in individuals evolving CRC. Current used pan-targeting treatments often show poor or short-term response in CRC patients, caused by resistance mechanisms, resulting in disease relapse. Novel therapies such as targeted drug treatment by kinase inhibitors could help overcome these problems by targeting the cancer cells and inducing immunogenic cell death (ICD), a special form of cell death recognized by the adaptive immune system, resulting in long-term effects and heal cancer patients, going with less to milder side effects. Here we investigate 6 FDA approved kinase inhibitors and their ICD inducing potential on 6 patient derived CRC organoids from one MSI, four MSS patients and one liver metastasis derived organoid. We selected two ICD markers, Calreticulin and HMGB1, which are analyzed by immunofluorescence staining and laser microscopy. We demonstrate that the individual CRC organoids respond heterogenous and show first signs of ICD after targeted drug treatment by the selected kinase inhibitors. Therefore, we affirm an ICD inducing potential of the 6 selected kinase inhibitors, but further investigation of additional ICD markers and their behavior while undergoing ICD, is needed. After establishment of these assays, we suggest combination therapy and coculture of patient derived immune cells and CRC organoids in the future, to uncover the signaling crosstalk and interaction of the adaptive immune system and the cancer organoids., Masterarbeit Universität Innsbruck 2023

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.

Due to the importance of RTKs in AML leukemogenesis and the current challenges in improving AML patient survival with KI treatment, phosphoproteomics may provide an additional layer of information of functional relevance to bridge the gap between a patient’s mutational status and their variable response to KI treatment. Therefore, the aim of this thesis is to explore the potential of pY phosphoproteomics in guiding AML patient selection for treatment with KIs. Chapter 2 focusses on the evaluation of the Integrated Inferred Kinase Activity Analysis, INKA. The utility of INKA is shown in multiple settings relevant for cancer research; (i) cancer cell lines with known oncogenes, (ii) cell lines in a differential setting (wild-type versus mutant, +/- drug), (iii) pre- and on-treatment tumor needle biopsies, (iv) cancer cell panel with available drug sensitivity data, and (v) patient-derived tumor xenografts with INKA-guided drug selection and testing. To expand on previous pY phosphoproteomics experiments, chapter 3 investigates tyrosine kinase signaling in 16 AML cell lines, using INKA in combination with functional testing, to evaluate the potential for individualized identification of signaling pathways and active driver kinases in the context of AML. Validation using KIs is further correlated with target kinase INKA activation metrics. In chapter 4 we explore the impact of delay of mononuclear cell isolation on the phosphorylation profiles of four clinical AML samples. Here we provide data for a range of time points and evaluate their effect on the stability of the phosphoproteome. Based on the results we formulate a recommendation as to the time frame within which samples should be processed. In chapter 5 we explore the AML phosphoproteome in 35 clinical samples, including both FLT3-ITD mutant- and FLT3WT samples in relation to their in-vitro response to two FLT3 inhibitors, midostaurin and gilteritinib. Data is provided on differential phosphorylation between FLT3

Masterarbeit Universität Innsbruck 2022

Cancers and neurological disorders are two major types of diseases. We previously developed a new concept termed "Aberrant Cell Cycle Diseases" (ACCD), revealing that these two diseases share a common mechanism of aberrant cell cycle re-entry. The aberrant cell cycle re-entry is manifested as kinase/oncogene activation and tumor suppressor inactivation, which are hallmarks of both tumor growth in cancers and neuronal death in neurological disorders. Therefore, some cancer therapies (e.g., kinase inhibition, tumor suppressor elevation) can be leveraged for neurological treatments. The United States Food and Drug Administration (US FDA) has so far approved 74 kinase inhibitors, with numerous other kinase inhibitors in clinical trials, mostly for the treatment of cancers. In contrast, there are dire unmet needs of FDA-approved drugs for neurological treatments, such as Alzheimer's disease (AD), intracerebral hemorrhage (ICH), ischemic stroke (IS), traumatic brain injury (TBI), and others. In this review, we list these 74 FDA-approved kinase-targeted drugs and identify those that have been reported in preclinical and/or clinical trials for neurological disorders, with a purpose of discussing the feasibility and applicability of leveraging these cancer drugs (FDA-approved kinase inhibitors) for neurological treatments.

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.

Melanoma is a cancer of the pigment-producing cells of the body and its incidence is rising. Targeted inhibitors that act against kinases in the MAPK pathway are approved for BRAF-mutant metastatic cutaneous melanoma and increase patients' survival. Response to these therapies is limited by drug resistance and is less durable than with immune checkpoint inhibition. Conversely, rare melanoma subtypes have few therapeutic options for advanced disease and MAPK pathway targeting agents show minimal anti-tumor effects. Nevertheless, there is a future for targeted kinase inhibitors in melanoma: in new applications such as adjuvant or neoadjuvant therapy and in novel combinations with immunotherapies or other targeted therapies. Pre-clinical studies continue to identify tumor dependencies and their corresponding actionable drug targets, paving the way for rational targeted kinase inhibitor combinations as a personalized medicine approach for melanoma.

The dopamine D2 receptor (D2R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D2R is expressed as two isoforms: long (D2LR) and short (D2SR) differing by a stretch of 29 residues in the third intracellular loop, with D2SR being the predominant presynaptic isoform. Recent reports described a Ca2+ sensitivity of the desensitization time course of potassium currents elicited via D2SR, but not via D2LR, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca2+ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D2LR and D2SR using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of β-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D2R desensitization in native cells. We found that co-expression of both GRK2 and β-arrestin2 was necessary for reconstitution of the Ca2+ sensitivity of D2SR desensitization, while D2LR did not display Ca2+ sensitivity under these conditions. The effect of Ca2+ chelation by BAPTA-AM to slow the rate of D2SR desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca2+-sensitive desensitization of D2SR appears to be mediated via a GRK2 phosphorylation-dependent mechanism.

A liquid chromatography-tandem mass spectrometry method was developed and validated to quantify alectinib, crizotinib, erlotinib and gefitinib. This assay can be combined with our method for osimertinib, allowing quantification of the most used ALK- and EGFR-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer with a single-assay setup. Chromatographic separation was performed on a HyPurity (R) C-18 analytical column using an elution gradient of ammonium acetate in water and in methanol, both acidified with formic acid 0.1%. Detection and quantification were performed using a triple quad mass spectrometer with an electrospray ionization interface. This method led to robust results, as the selectivity, carryover, precision and accuracy met all pre-specified requirements. The assay was validated over a linear range of 100-2,000 ng/ml for alectinib and erlotinib and 50-1,000 ng/ml for crizotinib and gefitinib. Alectinib, crizotinib, erlotinib and gefitinib were all stable for at least 4 h in whole blood (at room temperature and at 4 degrees C) and for at least 1 month in EDTA plasma when stored at -80 degrees C, while osimertinib proved to be unstable at room temperature. Although high-performance liquid chromatography was used, the run time was short and comparable with other methods using ultra-high performance liquid chromatography.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This unprecedented mechanism of action resulted in highly potent and selective pathway inhibition, in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this novel mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders.

BACKGROUND: For oral anticancer drugs, trough concentration (Cmin) is usually used as a target in therapeutic drug monitoring (TDM). Recording of Cmin is highly challenging in outpatients, in whom there is typically a variability in sample collection time after dosing. Various methods are used to estimate Cmin from the collected samples. This simulation study aimed to evaluate the performance of 3 different methods in estimating the Cmin of 4 oral anticancer drugs for which TDM is regularly performed.METHODS: Plasma concentrations of abiraterone, dabrafenib, imatinib, and pazopanib at a random time (Ct,sim) and at the end of the dosing interval (Cmin,sim) were simulated from population pharmacokinetic models including 1000 patients, and the values were converted into simulated observed concentrations (Ct,sim,obs and Cmin,sim,obs) by adding a residual error. From Ct, sim,obs, Cmin was predicted (Cmin,pred) by the Bayesian estimation (method 1), taking the ratio of the Ct,sim,obs and typical population concentration and multiplying this ratio with the typical population value of Cmin,sim (method 2), and log-linear extrapolation (method 3). Target attainment was assessed by comparing Cmin,pred with the proposed pharmacokinetic targets related to efficacy and calculating the positive predictive and negative predictive values.RESULTS: The mean relative prediction error and root mean squared relative prediction error results showed that method 3 was out-performed by method 1 and 2. Target attainment was adequately predicted by all 3 methods (the respective positive predictive value of method 1, 2, and 3 was 92.1%, 92.5%, and 93.1% for abiraterone; 87.3%, 86.9%, and 99.1% for dabrafenib; 79.3%, 79.3%, and 75.9% for imatinib; and 72.5%, 73.5%, and 67.6% for pazopanib), indicating that dose adjustments were correctly predicted.CONCLUSIONS: Both method 1 and 2 provided accurate and precise individual Cmin,pred values. However, method 2 was easier to im

Atropisomerism is a dynamic type of axial chirality that is ubiquitous in medicinal chemistry. Majority of drugs (83% of FDA-approved kinase inhibitors) possess an unstable atropisomeric axis, allowing these compounds to access the majority of dihedral conformations around the bond axis. While they engage their targeted protein within a narrow range of these conformations, the remaining available conformations can interact with other proteins leading to off-target inhibition causing unwanted side-effects. The Gustafson lab hypothesized that the selectivity of an atropisomerically unstable, promiscuous inhibitor could be increased by preorganizing it into the conformation preferred by oncogenic targets and preventing bond rotation to bind off-target proteins. Chapter 1 reviews examples of atropisomerism in drug discovery including my colleague’s proof-of-concept paper on leveraging atropisomerism to increase selectivity of pyrrolopyrimidine-based inhibitors (PPYs). My PhD research begins in Chapter 2 where we optimized the atropisomeric PPYs for potency and selectivity, resulting in a therapeutically relevant inhibitor towards RET and mutant EGFR kinases. Along the way, we discovered the increases in inhibitor selectivity was due to the narrowing of accessible low-energy conformations. This led to the development of conformational binding maps that were used to predict a kinase’s preferred binding conformation and guide the design of new covalent inhibitors for BTK kinase (Chapter 3). During this work we encountered unintended structural perturbations when incorporating an atropisomeric locking substituent, which led to the design of a stereochemically unstable, yet conformationally-tuned, selective BTK inhibitor. Chapter 4 presents other PPY preorganization strategies to target unique ligand binding regions as well as the development of conformational binding maps for pyridones and diaryl amines drug scaffolds. Chapter 5 discusses various catalytic atropisomeric met

The research of this thesis describes the development of small molecule scaffolds for use as potent inhibitors of CLK1, DYRK1a and Pim-1 kinases.Chapter 1 details the motivations of this research, outlines the importance of kinase inhibitors in medicinal chemistry and introduces the CLK and DYRK kinases. Previous research towards the development of CLK1 and DYRK1a kinase inhibitors is discussed, while some emerging strategies for the development of new kinase inhibitor scaffolds are introduced.Chapter 2 describes the scaffold hopping experiments conducted on two promising, yet flawed CLK1 and DYRK1a inhibitor scaffolds. In this work, a new quinazolin-4(H)-one scaffold was identified, which displayed a high conservation of the known binding mode of the parent, Leucettamine 41 molecule. Further lead optimisation is described, in which a novel substitution of the quinazolin-4(3H)-one scaffold was identified.Chapter 3 describes the synthesis of the previously identified quinazolin-4(3H)-one scaffold. In this work, several C7 headgroup analogues were explored, with furan and pyrazole heterocycles showing potent inhibition of CLK1 and DYRK1a. X-ray crystallography was performed by a collaborator (Martin Schröder and Prof. Stefan Knapp), with a binding mode in CLK1 being established for the scaffold. Following this work, additional C2 analogues were synthesised, producing highly potent CLK1 and DYRK1a inhibitors. Chapter 4 introduces another key kinase, Pim-1, and using findings of the previous chapter, describes the development of selective single digit nanomolar Pim-1 inhibitors through modifications to the previously identified quinazolin-4(3H)-one scaffold. Later, a Pim-1 inhibitor pharmacophore was modelled and another pyrimidin-4(3H)-one scaffold was identified. The synthesis of this pyrimidin-4(3H)-one scaffold is described, and its kinase inhibitory activity determined. Interestingly, although a serendipitous discovery, two CLK1 selective inhibitors were identified

Rational drug design implies usage of molecular modeling techniques such as pharmacophore modeling, molecular dynamics, virtual screening, and molecular docking to explain the activity of biomolecules, define molecular determinants for interaction with the drug target, and design more efficient drug candidates. Kinases play an essential role in cell function and therefore are extensively studied targets in drug design and discovery. Kinase inhibitors are clinically very important and widely used antineoplastic drugs. In this review, computational methods used in rational drug design of kinase inhibitors are discussed and compared, considering some representative case studies.

A new method for quantification of osimertinib (OSIM) in human plasma using a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated. Methanol was used for protein precipitation and pazopanib as internal standard. Separation was performed on a HyPURITY (R) C-18 analytical column (50 x 2.1 mm; 3 mu m) using a gradient elution of ammonium acetate in water and ammonium acetate in methanol, both acidified with formic acid 0.1%. Detection and quantification of OSIM and pazopanib was performed using a triple quadruple mass spectrometer after electrospray ionization. This method led to robust results, as the selectivity, carryover, precision and accuracy all met pre-specified requirements. OSIM was stable in human serum when stored at -80 degrees C. Reduced stability was found when stored at 2-4 degrees C or room temperature. Degradation of OSIM slowed down in EDTA-plasma and acidified human serum. The limited stability of OSIM at room temperature should be considered for transport and sample preparation. Plasma samples should be frozen as soon as possible and sample preparation should be performed on dry-ice. In the future, EDTA-plasma and sample acidification may be used to improve OSIM stability at room temperature. However, more research and validation of such an approach are required.

Cutaneous melanoma is an aggressive skin cancer that emerges from the unrestrained proliferation of melanocytes, which are the pigment producing cells in the basal layer of the epidermis. Despite the fact that it only accounts for approximately 5% of all skin cancers, melanoma is responsible for the vast majority of skin cancer-related deaths. As more than half of the patients with sporadic melanoma harbour activating mutations in the protein kinase BRAF, the development of small kinase inhibitors targeting mutated BRAF led to an increased overall survival of patients with metastatic melanoma. Despite the initially promising results, the rapidly emerging resistance to these targeted therapies remains a serious clinical issue. To investigate the mechanisms underlying resistance to targeted therapies, we used in vitro BRAF-mutant drug-sensitive and drug-resistant melanoma cell models that were generated in our laboratory. First, we performed a kinase inhibitor library screening with the aim to identify novel kinase inhibitor combinations to circumvent or delay BRAF inhibitor-induced resistance. We have characterised synergistic kinase inhibitors targeting the MAPK pathway and the cell cycle showing promising effects in BRAF-mutant drug-sensitive and -resistant cells, which could be used as an effective sequential or alternative treatment option for late-stage melanoma patients. Additionally, we investigated the impact of BRAF inhibitors at the transcriptional level by comparing miRNome and transcriptome changes in drug-sensitive and -resistant melanoma cells. We identified miRNAs (e.g. miR-509, miR-708) and genes (e.g. PCSK2, AXL) that were distinctly differentially expressed in resistant compared to sensitive cells. Subsequent co-expression analyses revealed a low MITF/AXL ratio in a subset of resistant cell lines, suggesting that miRNAs might be involved in the switch from one molecular phenotype to another, thus conferring tolerance to targeted therapies. Finally

Background: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. Methods: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. Findings: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. Interpretation: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients.

Předložená práce se zabývá syntézou 3-[amino(aryl)methyliden]indol-2-onů s terciární aminoskupinou s využitím Eschenmoserovy reakce. Bylo připraveno a charakterizováno 7 nových dosud nepopsaných látek, nesoucích substituenty na obou aromatických jádrech a methylové skupiny na dusíku aminoskupiny. Výtěžky těchto látek jsou v rozmezí od 19 do 83 % a jsou ovlivněny jak substitucí, tak i podmínkami separace. Klíčová je i přítomnost thiofilu (trimethyl-fosfitu). Dále byl připraven a charakterizován i jeden derivát nesoucí na dusíku dvě ethylskupiny a to ve výtěžku 57 %. Byl učiněn i pokus o přípravu N-methyl-N-fenyl derivátu 3-[amino(aryl)methyliden]indol-2-onu, který však díky nízké rozpustnosti nebylo možné řádně vyčistit a charakterizovat., The present work deals with the synthesis of 3-[amino(aryl)methylidene]indol-2-ones with a tertiary aminogroup using the Eschenmoser reaction. Prepared and characterized were 7 new hitherto undescribed substances bearing substituents on both aromatic rings and methyl substituents on the nitrogen of the aminogroup. Yields of these substances are in the range from 19 to 83% and are affected by both substitution and separation conditions. The presence of thiophile (trimethyl-phosphite) is also crucial. Furthermore, one derivative bearing two ethyl groups on nitrogen was prepared and characterized in a yield of 57%. An attempt was also made to prepare the N-methyl-N-phenyl derivative of 3-[amino(aryl)methylidene]indol-2-one, which could not be properly purified and characterized due to its low solubility., Fakulta chemicko-technologická, Student prezentoval svoji bakalářskou práci a vyjádřil se k připomínkám., Dokončená práce s úspěšnou obhajobou

Eph receptor tyrosine kinases, particulary EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm).These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labeled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabeling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labeling procedure to insert fluorine-18. After radiolabeling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq/µmol. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4 overexpressing A375 melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors by, e.g., PET.

FMS-like tyrosine kinase 3 (FLT3) has been found to be mutated in 30% of acute myeloid leukaemia patients. Small-molecule inhibitors targeting FLT3 that are currently approved or still undergoing clinical trials are subject to drug resistance due to FLT3 mutations. How these mutations lead to drug resistance is hitherto poorly understood. Herein, we studied the molecular mechanism of the drug resistance mutations D835N, Y842S and M664I, which confer resistance against the most advanced inhibitors, quizartinib and PLX3397 (pexidartinib), using enzyme kinetics and computer simulations. In vitro kinase assays were performed to measure the comparative catalytic activity of the native protein and the mutants, using a bacterial expression system developed to this aim. Our results reveal that the differential drug sensitivity profiles can be rationalised by the dynamics of the protein-drug interactions and perturbation of the intraprotein contacts upon mutations. Drug binding induced a single conformation in the native protein, whereas multiple conformations were observed otherwise (in the mutants or in the absence of drugs). The end-point kinetics measurements indicated that the three resistant mutants conferred catalytic activity that is at least as high as that of the reference without such mutations. Overall, our calculations and measurements suggest that the structural dynamics of the drug-resistant mutants that affect the active state and the increased conformational freedom of the remaining inactive drug-bound population are the two major factors that contribute to drug resistance in FLT3 harbouring cancer cells. Our results explain the mechanism of drug resistance mutations and can aid to the design of more effective tyrosine kinase inhibitors., Epub 2020

Introduction: Autophagy, a basic cellular degradation pathway essential for survival, is altered both in aging and in many chronic human diseases, including infections, cancer, heart disease, and neurodegeneration. Identifying new therapeutic targets for the control and modulation of autophagy events is therefore of utmost importance in drug discovery. Serum and glucocorticoid activated kinase 1 (SGK1), known for decades for its role in ion channel modulation, is now known to act as a switch for autophagy homeostasis, and has emerged as a novel and important therapeutic target likely to attract considerable research attention in the coming years., Areas covered: In this general review of SGK1 we describe the kinase’s structure and its roles in physiological and pathological contexts. We also discuss small-molecule modulators of SGK1 activity. These modulators are of particular interest to medicinal chemists and pharmacists seeking to develop more potent and selective drug candidates for SGK1, which, despite its key role in autophagy, remains relatively understudied., Expert opinion: The main future challenges in this area are (i) deciphering the role of SGK1 in selective autophagy processes (e.g. mitophagy, lipophagy, and aggrephagy); (ii) identifying selective allosteric modulators of SGK1 with specific biological functions; and (iii) conducting first-in-man clinical studies.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and, SCOPUS: ar.j, info:eu-repo/semantics/published

Polymeric micelles are attractive nanocarriers for hydrophobic drug molecules such as the kinase inhibitor dactolisib. Two different poly(ethylene glycol)–poly(acrylic acid) (PEG-b-PAA) block-copolymers are synthesized, PEG(5400)-b-PAA(2000) and PEG(10000)-b-PAA(3700), respectively. Polymeric micelles are formed by self-assembly once dactolisib is conjugated via the ethylenediamine platinum(II) linker (Lx) to the PAA block of the block copolymers. Dactolisib micelles with dactolisib loading content of 17% w/w show good colloidal stability and display sustained release of Lx-dactolisib over 96 h in PBS at 37 °C, while media containing reagents that compete for platinum coordination (e.g., glutathione (GSH) or dithiothreitol (DTT)) effectuate release of the parent inhibitor dactolisib at similar release rates. Dactolisib/lissamine-loaded micelles are internalized by human breast adenocarcinoma cells (MCF-7) in a dose and time-dependent manner as demonstrated by confocal microscopy. Dactolisib-loaded micelles inhibit the PI3K/mTOR signaling pathway at low concentrations (400 × 10−9 m) and exhibit potent cytotoxicity against MCF-7 cells with IC50 values of 462 ± 46 and 755 ± 75 × 10−9 m for micelles with either short or longer PEG-b-PAA block lengths. In conclusion, dactolisib loaded PEG-b-PAA micelles are successfully prepared and hold potential for nanomedicine-based tumor delivery of dactolisib.

A liquid chromatography-tandem mass spectrometry assay was developed and validated for the nine oral anticancer agents alectinib, cobimetinib, lenvatinib, nintedanib, osimertinib, palbociclib, ribociclib, vismodegib and vorinostat in order to support therapeutic drug monitoring (TDM). The assay was based on reversed-phase chromatography coupled with tandem mass spectrometry operating in the positive ion mode. The assay was validated based on the guidelines on bioanalytical methods by the US Food and Drug Administration and European Medicines Agency. The method was validated over a linear range of 10-200 ng/mL for alectinib, lenvatinib, nintedanib and vismodegib; 50-1000 ng/mL for cobimetinib and palbociclib; 100-2000 ng/mL for osimertinib; 5.00-100 ng/mL for ribociclib; 25-500 ng/mL for vorinostat. Intra-assay and inter-assay bias was within ±20% for all analytes at the lower limit of quantification and within ±15% at remaining concentrations. Stability experiments showed that osimertinib is unstable in the biomatrix and should be shipped on dry-ice and stored at -20 °C until analysis. All other compounds were stable in the biomatrix. The described TDM method was successfully validated and applied for TDM in patients treated with these KIs.

A variety of diseases is associated with dysregulation of kinase signaling pathways and kinase inhibitors are hence being considered as potent drugs for the treatment of cancer and inherited diseases such as polycystic kidney disease. Although kinase inhibitors are considered as quite specific drugs that are molecularly targeted, side effects significantly limit their therapeutic applications. Moreover, their specific action also infers that the efficacy of kinase inhibitors is limited by drug resistance. In this thesis, we have explored the development of drug delivery systems such as small molecule-drug conjugates (SMDCs) and polymeric micelles for kinase inhibitors, which eventually can be used to overcome these problems. An SMDC typically is composed of a targeting ligand, a drug payload and a spacer which infers a cleavable linkage with the drug (see Figure 3, Chapter 1). SMDCs have attracted tremendous interest in the pharmaceutical field because they have several advantageous features. Firstly, the small size (as can be deduced from their low molecular weight) of SMDCs enables them to accumulate and penetrate in solid tumors very efficiently. Secondly, the synthesis strategy of SMDCs is versatile and controllable. Polymeric micelles are colloidal nanoparticles with a diameter size around 10 - 200 nm and have a core-shell structure that is formed by self-assembling of amphiphilic block copolymers in aqueous media. The hydrophilic block of the copolymers forms the shell of polymeric micelles to ensure their colloidal stability while the hydrophobic block forms the core of polymeric micelles. Hydrophobic drugs can be loaded in the core by physical entrapment or -preferably, in view of stable drug retention- by chemical crosslinking to the core. The physicochemical properties of polymeric micelles can be controlled by adjusting the components of polymers and drugs. The relatively small size of polymeric micelles -although much larger than SMDCs- enables them to p

Tofacitinib is an oral Janus kinase inhibitor approved for the treatment of psoriatic arthritis (PsA). It provides an alternative option for patients who have had an inadequate response and tolerance to other disease modifying antirheumatic drugs (DMARDs). It has demonstrated comparable efficacy to biologics, is effective in the management of treatment resistant disease, and is reported to improve enthesitis, dactylitis, and radiographic progression. Tofacitinib is also associated with an increased risk of serious infections, malignancy, and laboratory abnormalities. There is currently a large armamentarium of therapies for psoriatic arthritis, and choosing among treatments can be challenging. Due to this wide selection, a thorough assessment of psoriatic disease phenotype, patient preference, disease presentation, and comorbidities is critical. This review addresses key considerations in patient selection for the treatment of PsA with tofacitinib.

EGFR mutations display striking organ-site asymmetry and heterogeneity. We have shown that structurally diverse extracellular mutations, typical of glioblastomas, converge to a similar intermediate conformation, which can be synergistically targeted extra- and intracelullarly by antibody mAb806 and type-II kinase inhibitors. Our findings reveal convergence behind heterogeneity, paving the way for allostery-based co-targeting.

The review summarizes and systematizes data of the last twenty years on the synthetic methods and biological properties of pyrido[2,3-d]pyrimidin-7-ones, promising objects of organic and pharmaceutical chemistry. Two main approaches to the formation of the pyrido[2,3-d]pyrimidine system are considered. The first of them involves the cyclization of substituted pyridines containing functional groups in positions 2 and 3 of the heterocyclic ring. The second approach is based on the formation of a bicyclic system by adding a pyridone moiety to the pyrimidine ring. The methods developed allow to introduce various functional groups and aromatic substituents into the pyrido[2,3-d]pyrimidine system, as well as to obtain most of the target products with high yields. The effective three-component one-pot synthetic approaches to the formation of pyridine ring with the participation of functionalized pyrimidines and compounds with an active methylene group have been proposed. The analysis of the literature has shown that functionalized pyrimidines are the most common starting reagents, which structural modification is useful for the further annelation of the pyridine cycle. Much attention is paid to the biological properties of pyrido[2,3-d]pyrimidin-7-ones. The prospect of using pyrido[2,3-d]pyrimidin-7-one derivatives as tyrosine kinase inhibitors has been shown. Data on the biological effects of pyrido[2,3-d]pyrimidin-7-one derivatives indicate the possibility of detecting new biologically active compounds among pyridopyrimidines., В обзоре обобщены и систематизированы данные последних двадцати лет по методам получения и биологическим свойствам пиридо[2,3-d]пиримидин-7-онов – перспективных объектов органической и фармацевтической химии. Рассмотрены два основных подхода к формированию пиридо[2,3-d]пиримидиновой системы. К первому из них относятся циклизации замещенных пиридинов, содержащих функциональные группы в положениях 2 и 3 гетероциклического кольца. Второй подход основывается на формировании бициклической системы за счет достраивания пиридинового фрагмента к пиримидиновому кольцу. Разработанные методы позволяют вводить в состав пиридо[2,3-d]пиримидиновой системы различные функциональные группы и ароматические заместители, а также получать большинство целевых продуктов с высокими выходами. Предложены эффективные трехкомпонентные однореакторные синтетические подходы к формированию пиридинового кольца с участием функционализированных пиримидинов и соединений с активной метиленовой группой. Анализ литературных данных показал, что к числу наиболее распространенных исходных реагентов следует отнести функционально замещенные пиримидины, структурная модификация которых перспективна для дальнейшего аннелирования пиридинового цикла. Значительное внимание уделено биологическим свойствам пиридо[2,3-d]пиримидин-7-онов. Показана перспективность использования производных пиридо[2,3-d]пиримидин-7-она как ингибиторов тирозинкиназ. Данные о биологической активности производных пиридо[2,3-d]пиримидин-7-она свидетельствуют о целесообразности поиска новых биологически активных соединений в ряду пиридопиримидинов., В огляді узагальнено та систематизовано дані останніх двадцяти років щодо методів одержання та біологічних властивостей піридо[2,3-d]піримідин-7-онів – перспективних об’єктів органічної та фармацевтичної хімії. Розглянуто два основних підходи до формування піридо[2,3-d]піримідинової системи. До першого з них відносяться циклізації заміщених піридинів, що містять функціональні групи в положеннях 2 та 3 гетероциклічного кільця. Другий підхід базується на формуванні біциклічної системи за рахунок добудови піридинового фрагменту до піримідинового кільця. Розроблені методи дозволяють вводити до складу піридо[2,3-d]піримідинової системи різні функціональні групи та ароматичні замісники, а також одержувати більшість цільових продуктів з високими виходами. Запропоновано ефективні трикомпонентні однореакторні синтетичні підходи до формування піридинового кільця за участі функціоналізованих піримідинів та сполук з активною метиленовою групою. Аналіз літературних даних показав, що до числа найбільш поширених вихідних реагентів слід віднести функціонально заміщені піримідини, структурна модифікація яких є корисною для подальшого анелювання піридинового циклу. Значну увагу приділено біологічним властивостям піридо[2,3-d]піримідин-7-онів. Показано перспективність використання похідних піридо[2,3-d]піримідин-7-ону як інгібіторів тирозинкіназ. Дані біологічної дії похідних піридо[2,3-d]піримідин-7-ону свідчать про доцільність пошуку нових біологічно активних сполук в ряду піридопіримідинів.

Oncogenic BRAF mutations contribute to the development of a number of cancers, and small-molecule BRAF inhibitors have been approved by the Food and Drug Administration (FDA) for anticancer therapy. In this study, we employed two targeted quantitative proteomics approaches for monitoring separately the alterations in protein expression and ATP binding affinities of kinases in cultured human melanoma cells elicited by two FDA-approved small-molecule BRAF inhibitors, dabrafenib and vemurafenib. Our results showed that treatment with the two inhibitors led to markedly different reprograming of the human kinome. Furthermore, we confirmed that vemurafenib could compromise the ATP binding capacity of MAP2K5 in vitro and inhibit its kinase activity in cells. Together, our targeted quantitative proteomic methods revealed profound changes in expression levels of kinase proteins in cultured melanoma cells upon treatment with clinically used BRAF inhibitors and led to the discovery of novel putative target kinases for these inhibitors.

Prostate cancer (PCa) is the most common cause of cancer-related death in men after lung cancer. Annually, more than 9000 new cases are diagnosed in Sweden and 2500 of them die each year. Metastatic prostate cancer can be treated with castration. However, the disease eventually recurs within 2 years in more aggressive form and becomes insensitive to castration which is termed as castration resistant prostate cancer (CRPC). Currently, effective treatment for CRPC is lacking. Numerous studies have shown that even under castrated level of androgen, the androgen receptor (AR) signaling pathway is constitutively active, which promotes the growth of CRPC cells.Alterations such as AR gene amplification, AR mutation and expression of truncated variants of AR (ARVs) is commonly observed in CRPC patients and through these alterations, CRPC cells become hypersensitive to extremely low level of androgen or become independent of androgens. Furthermore, hyperactivation of the phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently observed in metastatic CRPC patients. The PI3K/Akt pathway can enable the cells to survive in the absence of androgen. In the studies presented in my thesis, I have revealed the importance of a kinase called the phosphatidylinositol 4-phosphate 5-kinase type І alpha (PIP5K1α) incancer progression and therapeutic resistance. The PIP5K1α kinase acts as the upstream activator of the PI3K/AKT pathway by producing the substrate phosphatidylinositol-4,5-bisphosphate (PIP2) necessary for the activation of thePI3K/AKT signaling pathway. We, for the first time have shown that high level of PIP5K1α and its product PIP2 is a common event in cancer patients and is negatively associated with survival of prostate cancer and breast cancer patients. Furthermore, higher expression of PIP5K1α is associated with the expression of AR variant-7 (AR-V7) which is responsible for enzalutamide resistance in CRPC. We have discovered an inhibitor of PIP5K1α, ISA-2011B through

Atropisomerism represents a form of chirality that is often swept under the rug in medicinal chemistry and drug development. Nonetheless, our lab has demonstrated that atropisomerism can in fact be exploited to increase the target selectivity of kinase inhibitors, with subsequent perspectives and reports leveraging our findings as a “call to arms” to invoke atropisomerism as a design element in drug discovery efforts. As atroposelective methodology remains a relatively underdeveloped niche in chemistry, we focused our efforts to develop new chemical methodology to move the field of atropisomer synthesis forward. Rigidifying the atropisomeric axis of known kinase inhibitor scaffolds remained a challenging task until we developed a Lewis base catalyzed chlorination of arenes and heterocycles, which is discussed in Chapter 2. We then extended this mode of catalysis towards a regiodivergent chlorination of phenols presented in Chapter 3. The ability to control the site of electrophilic aromatic chlorination by choice of catalyst has been utilized in the synthesis of several natural products and drug-like molecules. Lastly, Chapter 4 discloses a proof of concept towards a first-in-class enantioselective synthesis of diverse atropisomeric scaffolds with the potential for unparalleled access to several different functional groups proximal to the atropisomeric axis. We expect these developed methodologies to make an impact beyond our own research program, hopefully with implications in drug discovery efforts.

BACKGROUND: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients. METHODS: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays. RESULTS: Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance. CONCLUSIONS: To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.

Kinase inhibitors have dramatically increased patient survival in a multitude of cancers, including hematological malignancies. However, kinase inhibitors have not yet been integrated into current clinical trials for patients with T-cell-lineage acute lymphoblastic leukemia (T-ALL). In this study, we used a high-throughput flow cytometry (HTFC) approach to test a collection of small-molecule inhibitors, including 26 FDA-approved tyrosine kinase inhibitors in a panel of T-ALL cell lines and patient-derived xenografts. Because hypoxia is known to cause resistance to chemotherapy, we developed a synthetic niche that mimics the low oxygen levels found in leukemic bone marrow to evaluate the effects of hypoxia on the tested inhibitors. Drug sensitivity screening was performed using the Agilent BioCel automated liquid handling system integrated with the HyperCyt HT flow cytometry platform, and the uptake of propidium iodide was used as an indication of cell viability. The HTFC dose-response testing identified several compounds that were efficacious in both normal and hypoxic conditions. This study shows that some clinically approved kinase inhibitors target T-ALL in the hypoxic niche of the bone marrow.

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.

In my thesis, I elucidated several aspects of melanoma biology, all related to the influence of targeted therapies in both responding and resistant melanoma cells. To better understand the MAPK signalling pathway, the impact of BRAF inhibitors on metabolic alterations as well as the connection between BRAF inhibitors and the onset of drug resistance was investigated. This introduction is focused on four topics: i) melanoma, ii) cancer metabolism, iii) miRNAs and iv) extracellular vesicles. First, melanoma biology including incidence rates, etiology, canonical and altered signalling pathways, therapies and resistance mechanisms will be introduced. The second part of the introduction will concentrate on metabolic alterations in the context of cancer and their implication on proliferation and survival. Thirdly, miRNAs and extracellular vesicles will be illustrated providing insights into their role in cancer development and especially drug resistance

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.

[no abstract]

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity. Munk et al. use mass spectrometry-based proteomics to analyze the interplay between SUMOylation and phosphorylation in replication stress. They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage.

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT-quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein-drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations., CAN 2015/387, SNIC 2016/1–55, SNIC 2016/1–222, SNIC 2017/1–23

Tématem této bakalářské práce je literární zpracování možností radiosenzibilizace nádorových buněk pankreatu. Práce popisuje anatomii a histologii pankreatu, onemocnění pankreatu se zaměřením na nádory pankreatu. Dále se práce věnuje současným metodám terapie karcinomu pankreatu a zaměřuje se na cílenou léčbu, specificky na možnosti využití inhibitorů tyrozin kináz a možnosti radiosenzibilizace a chemosenzibilizace nádorových buněk pankreatu pomocí inhibitorů ATM a ATR kináz., The topic of this bachelor thesis is the literal processing of possibilities of radiosensitization of pancreatic tumor cells. This bachelor thesis describes anatomy and histology of pancreas, pancreatic diseases with focus on pancreatic tumors. Further the thesis focuses on current methods of pancreatic cancer therapy and on targeted therapy, specifically on the use of tyrosine kinase inhibitors and the possibility of radiosensitization and chemosensitization of pancreatic tumor cells by ATM and ATR kinase inhibitors., Fakulta zdravotnických studií, Hodnocení vedoucího: výborně Hodnocení oponenta: výborně minus Doplňující otázky k obhajobě: 1. Jaké jsou vhodné vyšetřovací metody ke zjištění karcinomu pankreatu? 2. Užívá se nebo se užívala nějaká radiosenzibilizující látka v klinické praxi? 3. Jaká jsou stádia dle TNM a k čemu je dobré je určovat? Obhajoba bakalářské práce s prezentací výborná 4. Jaké jsou výhody a nevýhody léčby protony?, Dokončená práce s úspěšnou obhajobou

The survival of advanced metastatic melanoma has been greatly improved within the past few years. New therapeutic strategies like kinase inhibitors for BRAF-mutant melanoma and immune checkpoint blockers proved to prolong survival times within clinical trials, and many of them have already entered routine clinical use. However, these different treatment modalities have not yet been tested against each other, which complicate therapy decisions. We performed an explorative analysis of survival data from recent clinical trials. Thirty-five Kaplan-Meier survival curves from 17 trials were digitised, re-grouped by matching inclusion criteria and treatment line, and averaged by therapy strategy. Notably, the survival curves grouped by therapy strategy revealed a very high concordance, even if different agents were used. The greatest survival improvement was observed with the combination of BRAF plus MEK inhibitors as well as with Programmed-death-1 (PD1) blockers with or without cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) blockers, respectively, with these two treatment strategies showing similar survival outcomes. For first-line therapy, averaged survival proportions of patients alive at 12 months were 74.5% with BRAF plus MEK inhibitor treatment versus 71.9% with PD-1 blockade. This explorative comparison shows the kinase inhibitors as similarly effective as immune checkpoint blockers with regard to survival. However, to confirm these first trends for implementation into an individualised treatment of melanoma patients, data from prospective clinical trials comparing the different treatment strategies head-to-head have to be awaited.

Controlled drug delivery systems have been extensively investigated as means to prolong the action of drugs in the body. In this regard, a drug is incorporated into a carrier (e.g., polymeric material) in such a way that the drug is released from the matrix in a controlled manner for an extended period of time. As of 1970th, the Food and Drug Administration (FDA) has approved >70 new products based on the drug delivery systems. In particular a number of biodegradable polyesters, such as poly(lactic-co-glycolic acid) (PLGA), has been frequently used for the development of controlled drug delivery systems. PLGA-based particulate systems (i.e., nanoparticles and microparticles) are suitable systems for controlled release of small molecules as well as macromolecular biotherapeutics. These drug-loaded particulate systems can be injected in/or adjacent to the targeted organs ensuring relatively high drug concentrations in the targeted organs/tissues and minimizing drug distribution into other untargeted and healthy organs/tissues. Besides different cancer types, ophthalmic diseases affecting both anterior and posterior segments of the eye can greatly benefit from local drug delivery. Due to the presence of many ocular barriers (e.g., tear film, corneal and conjunctival epithelia, efflux pumps, blood retinal and blood aqueous barriers) delivering and maintaining drug concentrations at therapeutic levels in the eye is challenging. Aim of the thesis This thesis focuses on the preparation of particulate systems based on biodegradable aliphatic polyesters and their application for sustained and local delivery of low molecular weight hydrophilic and amphiphilic drugs. Outline of the thesis Chapter 2 gives an overview of strategies for efficient encapsulation of small molecule drugs into polymeric microspheres. This chapter addresses the challenges and formulation-related aspects for efficient encapsulation of small hydrophilic and amphiphilic molecules into PLGA microspheres

Protein kinases play a central role in the oncogenesis of colorectal tumors and are attractive druggable targets. Detection of activated kinases within a tumor could open avenues for drug selection and optimization of new kinase inhibitors. By using a phosphokinase arrays with human colorectal tumors we identified activated kinases, including the Epidermal Growth Factor Receptor (EGFR), components of the PI3K/mTOR pathway (AKT and S6), and STAT, among others. A pharmacological screening with kinase inhibitors against these proteins helped us to identify a new kinase inhibitor, termed EC-70124 that showed the highest anti-proliferative activity in cell lines. EC-70124 also inhibited cell migration and biochemical experiments demonstrated its effect targeting the PI3K/mTOR pathway. This drug also arrested cells at G2/M and induced apoptosis. Experiments in combination with standard chemotherapy used in the clinical setting indicated a synergistic effect. EC-70124 also reduced tumor growth in vivo and inhibited pS6 in the implanted tumors. In conclusion, by studying the kinase profile of colorectal tumors, we identified relevant activated pathways, and a new multi-kinase compound with significant antitumor properties.

Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR), a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr172-AMPK and phospho-Ser79-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI) conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increased Cd36, Acot1, and Ucp3 mRNA levels. AICAR treatment induced the expression of Acadvl and Glut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptation via regulation of transcriptional mechanisms. © 2015 Ricardo Rodríguez-Calvo et al.