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Die Beteiligung an Schlüsselfunktionen in zellulären Signalwegen macht Kinasen zu einem vielversprechenden Ansatzpunkt in der Wirkstoffentwicklung bei verschiedenen menschlichen Erkrankungen wie z.B. Krebs oder auch Autoimmun- und Entzündungskrankheiten. Die Prävention von post-translationalen Modifikationen durch Phosphorylierung und somit die Regulierung der nachgeschalteten Signalwege ist das Ziel von Kinaseinhibitoren. Die katalytische Aktivität von Kinasen ist abhängig von ATP, welches im hochkonservierten aktiven Zentrum bindet. Bedingt durch diese kinomweite hohe Konservierung stellt die Entwicklung von hoch selektiven ATP-mimetischen Inhibitoren eine Herausforderung dar. Typische ATP-Mimetika sind flach und die oft hydrophoben Moleküle weisen meist eine große Zahl an frei rotierbaren Bindungen auf. Um das aus dieser Flexibilität hervorgehende Problem der teils mangelnden Selektivität zu umgehen, kann eine bioaktive Konformation des Inhibitors durch Makrozyklisierung fixiert werden. Als Konsequenz dieser konformationellen Einschränkung können die entropischen Kosten während des Bindens reduziert werden und folglich zu einer gesteigerten Affinität gegenüber der Kinase führen.
Der Grundstein dieser Arbeit war der makrozyklische Pyrazolo[1,5-a]pyrimidin basierte FLT3 Kinaseinhibitor ODS2004070 (37). Im Rahmen eines kinomweiten Screenings konnten hohe Affinitäten zu verschiedensten Kinasen detektiert werden, was 37 zu einer guten Leitstruktur für das Design von potenten und selektiven Kinaseinhibitoren machte. Im Rahmen dieser Arbeit blieb das literaturbekannte Pyrazolo[1,5-a]pyrimidin basierte ATP-mimetische Bindemotiv sowie das makrozyklische Grundgerüst 37 bis auf einige wenige Variation unverändert.
Strukturelle Optimierungen zur Fokussierung der Selektivität wurden am sekundären Amin zwischen Bindemotiv und Linker als auch über die freie Carbonsäure durchgeführt. Mit einer Anzahl von mehr als 430 identifizierten Phosphorylierungsstellen ist die pleiotropisch und konstitutiv aktive Casein Kinase 2 (CK2) an verschiedensten zellulären Prozessen wie dem Verlauf des Zellzyklus, der Apoptose oder der Transkription regulatorisch beteiligt. Die Fehlregulation von CK2 wird häufig mit der Pathologie von Krankheiten wie zum Beispiel Krebs assoziiert, was CK2 zu einem vielversprechenden Ziel klinischer Untersuchungen macht.
Im Rahmen des CK2-Projekts war es möglich, durch spezifische Modifikationen an 37, die hoch selektiven und potenten CK2-Inhibitoren 47 und 60 zu entwickeln. Ebenfalls gezeigt wurde, dass kleine strukturelle Veränderungen, wie z.B. Makrozyklisierung, einen signifikanten Effekt auf Selektivität und Potenz des Inhibitors haben kann.
Weiter Untersuchungen der Verbindungen lenkten den Fokus weiterer Arbeiten u.a. auf die Serin/Threonin Kinase 17A (STK17A) oder auch death-associated protein kinase-related apoptosis-inducing protein kinase 1 (DRAK1) genannt. Sie ist Teil der DAPK Familie und gehört zusammen mit anderen Kinasen zu den weniger erforschten Kinasen. Bis heute ist nicht viel über ihre zellulären Funktionen und die Beteiligung an pathophysiologischen Prozessen bekannt. Berichtet wurde jedoch eine Überexpression in verschiedenen Formen von Hirntumoren des zentralen Nervensystems (Gliom). Strukturelle Modifikationen, unter Erhalt des makrozyklischen Grundgerüsts 37, führten zu dem hoch selektiven und potenten DRAK1 Inhibitor 121, der alle Kriterien für eine chemical probe Verbindung erfüllt.
Ein weiteres Ziel dieser Arbeit war die AP-2-assoziierte Protein Kinase 1 (AAK1) aus der NAK Familie, bestehend aus AAK1, BIKE und GAK. Sie ist als potenzielles therapeutisches Ziel für viele verschieden Krankheiten wie z.B. neuropathische Schmerzen, Schizophrenie und Parkinson identifiziert. Durch die Regulierung der Clathrin-mediierten Endozytose ist AAK1 an intrazellulären Bewegungen verschiedener nicht zusammenhängenden RNS- und DNSViren, wie beispielsweise HCV, DENV oder EBOV, beteiligt. Ebenfalls berichtet wurde eine mögliche Assoziation mit dem SARS-CoV-2 Virus, was das Interesse an neuen selektiven AAK1 Inhibitoren verstärkte. Die Entwicklung der hochpotenten und selektiven AAK1 Inhibitoren 61 und 63 basierte ebenfalls auf dem makrozyklischen Grundgerüst 37, das bereits im CK2- und DRAK1-Projekt verwendet wurde.
Zusammenfassend lässt sich sagen, dass es im Rahmen dieser Arbeit gelungen ist, ausgehend von einem höchst unselektiven makrozyklischen Grundgerüst, hochpotente und selektive Kinaseinhibitoren für CK2, DRAK1 und AAK1 zu entwickeln und zu charakterisieren. Im Zuge von Untersuchungen verschiedener Struktur-Wirkungsbeziehungen wurde gezeigt, dass es durch geringfügige strukturelle Modifikationen möglich ist, die kinomweite Selektivität zu variieren und auf eine Kinase zu fokussieren. Diese Arbeit brachte nicht nur die erwähnten Inhibitoren hervor, sondern bildet auch die Grundlage für weitere Projekte zur Entwicklung von hoch potenten und selektiven Verbindungen als potenzielle chemische Werkzeuge für den Einsatz in der Forschung.
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
Polyketides are highly valuable natural products, which are widely used as pharmaceuticals due to their beneficial characteristics, comprising antibacterial, antifungal, immunosuppressive, and antitumor properties, among others. Their biosynthesis is performed by large and complex multiproteins, the polyketide synthases (PKSs). This study solely focuses on the class of type I PKSs, which arrange all their enzymatic domains on one or more polypeptides. Despite their high medical value, little is known about mechanistic details in PKSs.
One central domain is the acyl transferase (AT), which is present in all PKSs and channels small acyl substrates into the enzyme. More precisely, the AT loads the substrates onto the essential acyl carrier protein (ACP), which subsequently shuttles the substrates and all intermediates for condensation and modification to additional domains to build the final polyketide.
Some PKSs use their domains several times during biosynthesis and work iteratively – these are called iterative PKSs. Others feature several sets of domains, each being used only once during biosynthesis – these PKSs are called modular PKSs. All PKSs or PKS modules consist of minimum three essential domains to connect the acyl substrates. Three modifying domains are optional and can enlarge the minimal set. According to the domain composition, the acyl substrate is fully reduced, partly reduced, or not reduced at all. This variation of modifying domains accounts for the huge structural and therefore functional variety of polyketides.
Even though the structure of fatty acids is not exactly reminiscent of polyketides, their biosynthetic pathways are closely related. Fatty acid biosynthesis is carried out by fatty acid synthases (FASs), which share many similarities with PKSs. Both megasynthases feature the same domains, performing the same reactions to connect and modify small acyl substrates. In contrast to PKSs, FASs always contain one full set of modifying domains which is used iteratively, leading to fully reduced fatty acids.
The present thesis extensively analyzes the AT of different PKSs in its substrate selectivity, AT-ACP domain-domain interaction, and enzymatic kinetic properties. The following key findings are revealed through comparison: 1.) ATs of PKSs appear slower than the ones of FASs, which may reflect the different scopes of biosynthetic pathways. Fatty acids as essential compounds in all organisms are needed in high amounts for physiological functions, whereas polyketides as secondary metabolites only require basal concentrations to take effect. 2.) The slower ATs from modular PKSs do not load non-native substrates even in absence of the native substrates. This is different to the faster ATs from iterative PKSs and FASs, which indicates high substrate specificity solely for the ATs from modular PKSs and emphasizes their role as gatekeepers in polyketide synthesis. 3.) The substrate selectivity can emerge in either the first or the second step of the AT-mediated ACP loading and is not assured by a hydrolytic proofreading function.
Moreover, a mutational study on the AT-ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) shows that single surface point mutations can influence AT-mediated reactions in a complex manner. Data reveals high enzyme kinetic plasticity of the AT-ACP interaction, which was also recently demonstrated for the interaction in a type II FAS.
Based on these findings, the mammalian FAS is engineered towards a modular PKS-like as- sembly line with the long-term goal to rationally synthesize new products. Basically, three important aspects need to be considered: 1.) AT’s loading needs to be splitted in specific loading of a priming substrate by a priming AT and in specific loading of an elongation substrate by an elongation AT. 2.) FAS-based elongation modules need to be designed with varying domain compositions for introducing functional groups in the product. 3.) Covalent and non-covalent linkers need to be designed for connection of priming and elongation modules.
This study focuses on the first aspect, splitting loading of priming and elongation substrates. An elongation substrate-specific AT is installed in the mammalian FAS via domain swapping. Since ATs from modular PKSs were proven to be substrate specific, these are used to exchange the mammalian FAS AT. This work demonstrates that it is extremely challenging to create stable and functional chimeras, but first essential steps are taken. Proper domain boundaries for AT swapping are established and a stable chimera with 70 % wild type AT activity is created. However, this chimera is only of limited value for application in an elongation module due to the intrinsic slow turnover rate of the wild type AT. Using another PKS AT, a stable elongation module is designed and analyzed in its activity in combination with a priming module. These experiments demonstrate that the loading of priming substrates are successfully suppressed in the elongation module, but nonetheless only minor turnover rates are detected in the assembly line.
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The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications
(2020)
Biological Function of Bacterial Dodecins
In this thesis, the dodecins of Mycobacterium tuberculosis (MtDod), Streptomyces coelicolor (ScDod) and Streptomyces davaonensis (SdDod) were studied. Kinetic measurements of the flavin binding of MtDod revealed that the dodecin binding pocket is filled in two distinct steps, for which a kinetic model then was established and verified by experimental data. The analysis with the two-step model showed that the unique binding pocket of dodecins allows them to bind excessive amounts of flavins, while at low flavin concentrations, flavin is released and only weakly bound. This function of flavin buffering prevents accumulation of free oxidised flavins and therefore helps to keep the redox balance of the cell and prevents potential cell damage caused by excessive free flavins. To further gain insights into the role of bacterial dodecins, the effect of knocking out the dodecin encoding gene in S. davaonensis was analysed. The knockout strain showed increased concentrations of various stress related metabolites, indicating that without dodecin the cellular balance is disrupted, which supports the role of dodecins as a flavin homeostasis factor.
With a self-designed affinity measurement method based on the temperature dependent dissociation of the dodecin:flavin complex, which allowed parallel screening of multiple conditions, it was shown that MtDod, ScDod and SdDod have much higher affinities towards FMN and FAD under acidic conditions. Under these conditions, the three dodecins might function as a FMN storage. M. tuberculosis encounters multiple acidic environments during its infection cycle of humans and can adopt a state of dormancy. During recovery from the dormant state, a flavin storage might be beneficial. For some Streptomyces species it was reported that the formed spores are slightly acidic and therefore ScDod and SdDod could function as flavin storages for the spores. Further details on the flavin binding mechanism of MtDod were revealed by a mutagenesis study, identifying the importance of a histidine residue at the fourth position of the protein sequence for flavin binding, but contrary to expectations, this residue seems only to be partly involved in the pH related affinity shift.
The data, reported in this thesis, demonstrates that bacterial dodecins likely function as flavin homeostasis factors, which allow overall higher flavin pools in the cell without disrupting the cellular balance. Further, the reported acid-dependent increase in binding affinity suggests that under certain conditions bacterial dodecins can also function as a flavin storage system.
Application of the Dodecin of M. tuberculosis
In this thesis, the stability of MtDod, ScDod SdDod and HsDod was analysed to find a suitable dodecin for the use as a carrier/scaffold. Therefore, a method to easily measure the stability of dodecins was designed, which measures the ability of the dodecamer to rebind flavins after a heating phase with stepwise increasing temperatures. Using this assay and testing the stability against detergents by SDS PAGE, showed that the dodecamer of MtDod possesses an excellent stability against a vast array of conditions, like temperatures above 95 °C, low pH and about 2% SDS. By solving the crystal structure of ScDod and SdDod, the latter forming a less stable dodecamer, combined with a mutagenesis study, the importance of a specific salt bridge for dodecamer stability was revealed and might be helpful to find further highly stable dodecins.
In addition to the intrinsic high stability of the MtDod dodecamer, also the robustness of the fold was tested by creating diverse MtDod fusion constructs and producing them in Escherichia coli. Here it was shown that MtDod easily tolerates the attachment of proteins up to 4-times of its own size and that both termini can be modified without affecting the dodecamer noticeably. Further, it was shown that MtDod and many MtDod fusion constructs could be purified in high yields via a protocol based on the removal of E. coli proteins through heat denaturation and subsequent centrifugation. In a case study, by fusing diverse antigens from mostly human proteins to MtDod and using these constructs to produce antibodies in rabbits, it was demonstrated that MtDod is immunogenic and presents the attached antigens to the immune system.
The here reported properties of MtDod and to a lesser degree of other bacterial dodecins, show that bacterial dodecins are a valuable addition to the pool of scaffold and carrier proteins and have great potential as antigen carriers.
The aim of this work was to establish a new way of predicting novel dual active compounds by combining classical fingerprint representation with state-of-the-art machine learning algorithms. Advantages and disadvantages of the applied 2D- and 3D-fingerprints were investigated. Further, the impact of various machine learning algorithms was analyzed. The new method developed in this work was used to predict compounds, which inhibit two different targets (LTA4H and sEH) involved in the same disease pattern (inflammation). The development of multitarget drugs has become more important in recent years. Many widespread diseases like metabolic syndrome, or cancer are of a multifactorial nature, which makes them hard to be treated effectively with a single drug. The new in silico method presented in this work can help to accelerate the design and development of multitarget drugs, saving time and efforts.
The nowadays readily available access to a large number of 3D-structures of biological targets and published activity data of millions of synthesized compounds enabled this study and was used as a starting point for this work. Four different data sets were compiled (crystalized ligands from the PDB, active and inactive compounds from ChEMBL23, newly designed compounds using a combinatorial library). Those data sets were collected and processed using an automated KNIME workflow. This automation has the advantage of allowing easy change and update of compound sources and adapted processing ways.
In a next step, the compounds from the compiled data sets were represented using a variety of well-established 2D- and 3D-fingerprints (PLIF, AtomPair, Morgan, FeatMorgan, MACCS). All those fingerprints share the same underlying bit string scheme but vary in the way they describe the molecular structure. Especially the difference between 2D- and 3D-fingerprints was investigated. 2D-fingerprints are solely based on ligand information. 3D-fingerprints, on the other hand, are based on X-ray structure information of protein-ligand complexes. One major difference between 2D- and 3D-fingerprints usage is the need for a 3D-conformation (pose) of the compound in the targets of interest when using 3D-fingerprints. This additional step is time-consuming and brings further uncertainties to the method.
Based on the calculated fingerprints state-of-the-art machine learning algorithms (SVC, RF, XGB and ADA) were used to predict novel dual active compounds. The models were evaluated by 10-fold cross validation and accuracy as the primary measure of model performance was maximized. Second, individual parameters of the four machine learning algorithms were optimized in a grid search to achieve maximal accuracy using the optimized partitioning scheme. Overall accuracies, regardless of fingerprint and machine learning algorithm, are slightly better for LTA4H than for sEH.
The goal to predict dual active compounds was realized by comparing the set of predicted to be active compounds for LTA4H and sEH. For the 3D-fingerprint PLIF the machine learning algorithm Random Forest was chosen, from which compounds for synthesis and testing were selected. Of 115 predicted to be active compounds, six compounds were cherry picked. Two compounds showed very good/moderate dual inhibitory activity. Of the 2D-fingerprints, the AtomPair fingerprint in combination with the machine learning algorithm Random Forest was chosen from which compounds were selected for synthesis and testing. 116 compounds were predicted to be dual active against LTA4H and sEH. One of those compounds showed good dual inhibitory activity.
In this work it was possible to show advantages and disadvantages of using 2D- and 3D-fingerprints in combination with machine learning algorithms. Both strategies (2D: ligand-based, 3D: structure-based) lead to the prediction of novel dual active compounds with moderate to very good inhibitory activity. The method developed in this work is able to predict dual active compounds with very good inhibitory activity and novel (previously unknown) scaffolds inhibiting the targets LTA4H and sEH. This contribution to in silico drug design is promising and can be used for the prediction of novel dual active compounds. Those compounds can further be optimized regarding binding affinity, solubility and further pharmacological and physicochemical properties.
Leitstrukturoptimierung mit Hilfe von Matched Molecular Paris im Kontext der Rezeptorumgebung
(2015)
In der hier vorgestellten Arbeit wurde ein strukturbasierter Ansatz zur gezielten Leitstrukturoptimierung entwickelt. Die Grundlage dafür bildeten die sogenannten Matched Molecular Pairs (MMPs). Dabei handelt es sich um Paare von Molekülen, welche sich lediglich in einer wohldefinierten Modifikation (Transformation) unterscheiden und sich in einer Datenbank mit gemessenen Moleküleigenschaften befinden. Diese Transformationen wurden im Kontext ihrer Targetumgebung untersucht und eine mathematische Beziehung zwischen Transformation und dem Effekt auf die Bindungsaffinität (Transformationseffekt) hergestellt. Auf Basis der generierten Datengrundlage wurde anschließend ein Webserver zur gezielten Leitstrukturoptimierung implementiert und zur freien Nutzung zur Verfügung gestellt.
The p38α mitogen-activated protein kinase (MAPK) is activated through stress stimuli such as heat shock or hypoxia. In the nucleus, p38α modulates the activity of other kinases and transcription factors, a process that regulates the expression of specific target genes, most importantly pro-inflammatory cytokines. Dysregulation of p38α therefore plays a major role in the development of inflammatory diseases such as rheumatoid arthritis. Despite many years of intensive research, no p38 small-molecule inhibitors have been approved yet. Several inhibitor design strategies have been reported, leading to >100-fold selective compounds for α/β over the γ and δ isoforms. Achieving such a selectivity among the two structurally most related α and β isoforms, however, remains a challenging task. Targeting an inactive DFG-out conformation offers another strategy for the development of potent kinase inhibitors (type-II), exemplified by the BCR/ABL-inhibitor Imatinib. Achieving selectivity with type-II binders is challenging, because many kinases can adopt an inactive DFG-out conformation. This is exemplified by the p38 type-II inhibitor BIRB-796, which exhibits picomolar on-target affinity but only a poor kinome-wide selectivity. A potent and selective type-II chemical probe for p38α/β was still lacking at the start of this thesis.
The promising hit VPC-00628, was chosen for a combinatorial synthetic approach to develop a type-II chemical probe. The studies covered the optimization of the hinge-binding head group, the hydrophobic region I and the DFG-out deep pocket of the lead compound VPC-00628. Selectivity for the p38α and p38β isoforms was monitored during the optimization process, which identified several inhibitors with favorable isoform selectivity, providing valuable insights into the potential of isoform-selective inhibitor design for p38. A potent and highly selective p38 MAPK probe (SR-318) was discovered, which showed IC50 values in the low nanomolar range in HEK293T cells. An unusual P-loop conformation induced upon binding of SR-318 to p38α contributed most likely to the impressive selectivity profile within the kinome that surpassed both the parent compound and BIRB-796. A negative control compound, SR-321, was developed, to distinguish between on-target effects and non-specific effects due to cross-reactivity with other cellular proteins. Studies of the metabolic stability in human liver microsomes revealed a high stability of the compounds, with only a small amount of metabolites formed over several hours. Compound SR-318 also exhibited a good in vitro efficacy, quantitatively reducing the LPS-stimulated TNF-α release in whole blood. Taken together, SR-318 is a highly potent and selective type-II p38α/β chemical probe, which will help to gain a better understanding of the catalytic and non-catalytic functions of these key signaling kinases in physiology and pathology.
The next studies focused on the exploration of the highly dynamic allosteric back pocket of p38 MAPK, and allosteric BIRB-796 derived compounds for targeting the αC- and DFG-out pockets were synthesized. Kinase activities of allosteric pyrazole-urea fragments were analyzed against a comprehensive set of 47 diverse kinases by differential scanning fluorimetry (DSF), revealing that BIRB-796 off-targets remain a problem when targeting this back-pocket binding motif. Revisiting the recently published compound MCP-081, which combines the allosteric part of BIRB-796 with the active-site directed part of VPC-00628, showed that it displays a clean selectivity profile in our kinase panel. Because the potency of MCP-081 was slightly reduced compared with VPC-00628 and the allosteric tert-butyl pyrazole moiety seemed suboptimal, a set of VPC-00628 derivatives for targeting the αC-out pocket region was synthesized. Through structure-guided extension of the terminal amide of VPC-00628 toward this allosteric site, the potent and selective compound SR-43 was developed, which showed excellent cellular activity on p38 MAPK in NanoBRETTM assays (IC50 [p38α/β] = 14.0 ± 0.1/ 16.8 ± 0.1 nM). SR-43 showed a dose-dependent inhibition of activating phosphorylation of p38 in HCT-15 cells as well as inhibition of phosphorylation of p38 downstream substrates MK2 and Hsp27. In addition, SR-43 induced an anti-inflammatory response by blocking TNF-α release in whole blood and displayed a high metabolic stability. Selectivity profiling of SR-43 revealed a narrow selectivity for additional targets such as the discoidin domain receptor kinases (DDR1/2). DDR kinases play a central role in fibrotic disorders, such as renal and pulmonale fibrosis, atherosclerosis and different forms of cancer. Since selective and potent inhibitors for these important therapeutic targets are largely lacking and the existing inhibitors are of low scaffold diversity, the next study focused on the optimization of SR-43 toward DDR1/2 kinase inhibition. The synthetic work covered the optimization of the hinge-binding head group and the allosteric part of SR-43 toward DDR1/2 kinase inhibition. These studies provided novel insights into the P-loop folding process of p38 MAPK and how targeting of non-conserved amino acids affects inhibitor selectivity. Importantly, they led to the development of a selective dual DDR/p38 inhibitor probe, SR-302, with picomolar affinity for DDR2. SR-302 was efficient in vitro and showed a destabilizing effect on the surface adhesion protein E-cadherin in epithelial cells. In summary, SR-302 and its negative control SR-301 provide a valuable tool set for studying the phenotypic effects of DDR1/2 signaling, e.g., in cancer cell lines.
A necessary requirement for a pharmacological effect is that a drug molecule tightly interacts with its disease relevant target molecule in the patient. Kinases are regulatory, signal transmitting enzymes and are a large protein family that belongs to the most frequent targets of pharmaceutical industry, as deregulation of kinases has been associated with the development of a variety of diseases, including cancer. In drug discovery, equilibrium binding metrics such as the affinity (Ki, KD) or potency (IC50, EC50) are usually applied for the systematic profiling for potent and selective drug candidates. In recent years, dynamic binding parameters, the drugs association (kon) and dissociation (koff) rates for desired primary-targets and undesired off-targets, were discussed to be better predictors than steady-state affinity per se (KD = koff / kon) for the onset and duration of the drug-target complex in the open in vivo environment and thereby for the therapeutic effect and safety of the drug. It is yet unclear whether and when the binding kinetics parameters can influence drug action in the complex context of pharmacokinetics and pharmacodynamics and how the kinetic rate constants can be optimized rationally. One major obstacle for providing proof for the hypothesis that drug binding kinetics is of importance for drug action is the generation of large and comparable binding kinetic datasets.
The aim of this thesis was the comprehensive analysis of the binding kinetic and affinity parameters of a diverse spectrum of 270 small-molecule kinase inhibitors against a panel of pharmacologically relevant kinases to study the role played by binding kinetics for drug discovery: The generated dataset was utilized to assess the effect of chemical properties on drug binding kinetics, and to evaluate the impact of kinetic rate constants on the success of compounds in the drug discovery pipeline.
Large scale profiling was made possible by a recently developed “kinetic Probe Competition Assay” (kPCA), whose evaluation is based on Motulsky’s and Mahan’s “kinetics of competitive binding” theory. Monte Carlo analyses performed in this dissertation widened the theoretical knowledge of this theory, provided new insights into its limitations and allowed to derive recommendations about how to best design assays. It was demonstrated that kPCA is indeed high-throughput compatible and that it is comparable to other biochemical and biophysical assay formats in terms of precision and accuracy.
Multivariable linear regression for the description of the determined kinase inhibitors’ target binding characteristics (kon or koff or KD) using molecular properties and/or particular kinase-inhibitor interactions as descriptors supported the assumption that molecular properties of compounds might affect binding kinetics, generated new hypothesis about molecular determinants influencing binding kinetic parameters and provided a rational basis for following structure-kinetic relationship studies. Remarkably, the binding kinetic rate constants were better described by the established models than binding affinities.
Interestingly, the systematic, quantitative analysis of kinase inhibitors’ target binding kinetics indicated that a slow dissociation rate for the main target is a feature which is more frequently observed in inhibitors that reached approval or late stage clinical testing than in earlier phases of clinical development. In addition, it was demonstrated that binding kinetics of kinase inhibitors is a better predictor for the time course of target engagement in cells as compared to affinity per se. Furthermore, in some study cases simulations using a standard pharmacokinetics model and a modified model considering the inhibitors binding kinetics lead to different in vivo kinase occupancy time profiles. It was illustrated by simulations how the concept of kinetic selectivity can be applied to turn an unselective compound in equilibrium conditions into a more selective compound in the open in vivo situation, where the thermodynamic equilibrium of drug-target binding is not necessarily reached.
Thus the generated data and models provide evidence for the importance of binding kinetics in drug discovery and represent a valuable resource for future studies in this field.
Standard cancer therapy research targets tumor cells while not considering the damage on the tumor microenvironment (TME) and its associated implications in impairing therapy response. Employing patients-derived organoids (PDOs) and matched stroma cells or a novel murine preclinical rectal cancer model of local radiotherapy, it was demonstrated that tumor cells-derived IL-1α polarizes cancer-associated fibroblasts towards an inflammatory (iCAFs) phenotype. While numerous studies in different tumor entities highlighted the molecular heterogeneity of CAFs, so far there are no clear findings on their functional heterogeneity and relevance in therapy resistance and response. The present study molecularly characterized iCAFs subpopulation among RCA patients as well as the preclinical mouse model and importantly unraveled the detailed molecular mechanism underlying their contribution to impair therapy response. Mechanistically, iCAFs were demonstrated to be characterized by an upregulation of nitric oxide synthase (iNOS) which triggered accumulation of reactive nitrogen species (RNS) and subsequently an oxidative DNA damage response (DDR). Such a baseline IL-1α-driven DNA damage further sensitized iCAFs to a p53-mediated therapy induced senescence (TIS) causing extensive extracellular matrix (ECM) changes and induction of senescence associated secretory phenotype (SASP) that favored tumor progression and hindered tumor cell death. Moreover, iCAFs reversibility and repolarization into more quiescent like phenotype was demonstrated upon IL-1 signaling inhibition by anakinra, a recombinant IL-1 receptor antagonist (IL1RA). Accordingly, treating mice with anakinra or specific deletion of Il1r1 in CAFs sensitized stroma-rich resistant tumors to chemoradiotherapy (CRT). Similarly, targeting CAFs senescence by senotherapy (venetoclax chemical) or employing Trp53 deficient mice reverted therapy resistance among non-responsive tumors in vivo by reducing ECM deposition and consequently favoring CD8+ T cells intratumoral infiltration posttherapy. Importantly, rectal cancer patients that do not completely respond to neoadjuvant therapy displayed an iCAFs senescence program post-CRT. Moreover, these patients presented a baseline increased CAFs content, a dominant iCAFs signature that correlated with poorer disease-free survival (DFS) and a significantly reduced circulating IL1RA serum levels. While reduced pretherapeutic IL1RN gene expression predicted poor prognosis among RCA patients, IL1RA serum levels were associated with rs4251961 (T/C) single nucleotide polymorphism (SNP) in the IL1RN gene. Finally, functional validation assays revealed that conditioned media of PDOs drove inflammatory polarization of fibroblasts and consequently rendered them sensitive to RNS-mediated DNA damage and TIS. Collectively, the study highlighted a crucial and novel role of a CAFs subset, iCAFs, in therapy resistance among RCA patients, shedding light on their functional relevance by identifying IL-1 signaling as an appealing target for their repolarization and successful targeting. Therefore, it makes sense to combine the newly demonstrated and thoroughly proven therapeutic approach of targeting IL-1 signaling in combination with conventional CRT and possibly immunotherapy. This might have a major impact on RCA therapy and be of immense relevance for other stroma-rich tumors.
Epigenetic mechanisms largely influence how genetic information on DNA level is translated into different phenotypes. DNA methylations and histone post-translational modifications make up what is referred to as "epigenetic landscape", an interconnected pattern that regulates access to genes and serves as platform for specific binding partners. The epigenetic landscape is maintained by "writers", which add the modifications, "erasers", which delete the modifications and "readers" which specifically bind modifications and mediate their location to other proteins connected to transcription. In the context of acetylations, which are the focus of this thesis, the writers are called histone acetyl transferases (HATs), the erasers are called histone deacetylases (HDACs) and the readers comprise Bromodomains (BRDs) as well as Yaf9, ENL, AF9, Taf14, Sas5 (YEATS) domains. An aberrant epigenetic landscape and mutated forms of epigenetic readers can lead to diseases including cancer and inflammatory diseases, making epigenetic reader domains attractive drug targets.
The focus of this thesis were YEATS domains and the development of inhibitors for this new class of epigenetic readers. Eleven-nineteen-leukemia protein (ENL) and ALL1-fused gene from chromosome 9 protein (AF9) are also part of the super elongation complex and are common fusion partners of mixed lineage leukemia protein (MLL) in acute myeloid leukemia (AML) (Wan et al., 2017, Erb et al., 2017). In this thesis, the first ligand-free crystal structure of ENL YEATS revealed an inherent flexibility of the Y78 side chain in the aromatic triad and two conserved water molecules. Soaking experiments led to the first co-crystal structures between a YEATS domain and small molecule inhibitors and defined prerequisites for ENL YEATS inhibitor scaffolds. The discovered inhibitory fragments had a central amide bond in common, which replaced one of the two conserved water molecules to form beta-sheet-like hydrogen bonds between the loop 6 backbone and the S58 side chain. The amide bond was flanked by two aromatic moieties, of which one stacks with H56 in the front pocket and the other interacts with the aromatic triad in the rear pocket. The development of the first chemical probe for ENL/AF9, SGC-iMLLT, show that the affinity is increased to low nanomolar levels if the rear flanking aromatic moiety forms additional hydrogen bonds with loop 6 and the side chain of E75 (Moustakim et al., 2018). In case of the probe, this is achieved with a 2-methyl-pyrrolidine-benzimidazole moiety. The probe binds with high affinity to ENL (129 nM) and AF9 (77 nM) and shows no significant affinity towards other human YEATS domains or BRDs. Target engagement was shown by fluorescence recovery after photobleaching (FRAP), cellular thermal shift assay (CETSA) and in case of AF9 also with NanoBRET. The probe changed the expression of three AML-related genes (MYC, dendrin and CD86) in MV4;11 cells, encouraging application of this probe in more AML cell lines.
Human protein kinases play essential roles in cellular signaling pathways and - if deregulated - are linked to a large diversity of diseases such as cancer and inflammation or to metabolic diseases. Because of their key role in disease development or progression, kinases have developed into major drug targets resulting in the approval of 52 kinase inhibitors by the Food and Drug Administration (FDA) so far.
Within the drug discovery process, the affinity of the inhibitors is the parameter that is used most often to predict the later efficacy in humans. However, the kinetics of binding have recently emerged as an important but largely neglected factor of kinase inhibitor efficacy. To efficiently suppress a signaling pathway, the targeted kinase needs to be continuously inhibited. Thus, it has been hypothesized that fast binding on-rates and slow off-rates would be the preferred property of an efficacious inhibitor. Despite optimizing the potency of kinase inhibitors, in the past decade optimization of kinetic selectivity has therefore gained interest as a molecule cannot be active unless it is bound, as Paul Ehrlich once stated. There is increasing evidence of correlations between prolonged drug-target residence time and increased drug efficacy, and that inhibitor selectivity in cellular contexts can be modulated by altered residence times. In order to contribute to the understanding of the effect of long residence times on cellular targets we initiated two projects.
The first of these projects is related to the STE20 kinase Serine/threonine kinase 10 (STK10) and its close relative STE20 like kinase (SLK) which have been reported to be frequent off-targets for kinase inhibitors used in the clinics. Also, an inhibition of STK10 and SLK has been linked to a common side-effect of severe skin rash developed upon treatment with the EGFR inhibitor erlotinib, but not gefitinib and the severity of this rash correlated with the treatment outcome, which fits the known biology of STK10 and SLK to be regulators of lymphocyte migration and PLK kinases. However, there are yet no explanations why these two proteins show such high hit-rates across the kinome among the kinase inhibitors. Using structural analysis, we identified the flexibility of STK10 to be the main reason for this hit-rate. The observed strong in vitro potencies did however not translate to the cellular system which is why we investigated the inhibitors residence time on STK10. We found the same flexibility to be the main reason for slow residence times among several inhibitors. We observed large rearrangements in the hydrophobic backpocket of STK10 including the αC, the P-loop enclosing the inhibitor like a lid and strong π-π-stackings to be the main reasons for prolonged residence times on STK10. Interestingly, we observed an increased residence time for erlotinib, which showed skin-related side-effects, giving rise whether the binding kinetics should be investigated for weak cellular off-target effects in future drug discovery efforts.
In the second project we initiated, we illuminate a structural mechanism that allows kinetic selection between two closely related kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). Using an inhibitor series designed to probe the mechanism, residence times measured in vitro and in cells showed a strong correlation. Crystal structures and mutagenesis identified hydrophobic interactions with L567, adjacent to the DFG-motif, as being crucial to kinetic selectivity of FAK over PYK2. This specific interaction was observed only when the DFG-motif was stabilized into a helical conformation upon ligand binding to FAK. The interplay between the protein structural mobility and ligand-induced effect was found to be the key regulator of kinetic inhibitor selectivity for FAK over PYK2.
These two projects showed that the parameter residence time should be considered for different problems among the drug discovery process. First, in an open in vivo system not only the potency of a drug alone, but as well its residence time might be of importance. Here we showed that the weak cellular potency translated to prolonged residence times for several inhibitors in cells and established a link between the phenotypic outcome of skin rash after erlotinib treatment and the residence time of this inhibitor on STK10 in cells. On the other hand, medicinal chemistry efforts should consider structure kinetic relationships (SKR) in the optimization process and aim to understand the molecular basis for prolonged target residence times. Here, we showed that a hydrophobic interaction that is enforced upon inhibitor binding is crucial for an unusual helical DFG conformation which arrests the inhibitor and prolongs its residence time providing the molecular basis for understanding the kinetic selectivity of two closely related protein kinases. Establishing the SKRs will help medicinal chemists to kinetically optimize their drug candidates to select a suitable molecule to proceed into further optimization programs. Hence, the projects showed that the target residence time parameter needs to be considered both as a molecular optimization parameter to improve compound potency and binding behavior as well as a parameter to be understood for proceeding to the open system of in vivo models to later modulate the in vivo efficacy of protein kinase targeting drugs.
Die Autophagie ist ein in Eukaryonten evolutionär konservierter Prozess, bei dem es zu einem lysosomalen Abbau von cytosolischen Bestandteilen kommt. Die dabei entstehenden biochemischen Bausteine stehen anschließend erneut zum Aufbau benötigter Strukturen zur Verfügung. Verschiedene Stimuli, wie beispielsweise Nährstoffmangel, können die Aktivität der Autophagie erhöhen und ermöglicht Zellen dadurch die Aufrechterhaltung der Zellhomöostase, selbst unter Stressbedingungen. Im Verlauf der Autophagie bildet sich eine tassenförmige Doppelmembran-Struktur, das sogenannte Phagophor. Dieses wächst, um das abzubauende Material zu umschließen und wird dabei von sogenannten Atg-Proteinen (autophagy-related genes) prozessiert. Nach der Schließung spricht man vom Autophagosom, welches letztlich mit einem Lysosom verschmilzt und das Autophagolysosom bildet, welches wiederum die eingeschlossenen Bestandteile zerlegt und die recycelten Bausteine freigibt. Die einzelnen Schritte während der Autophagie sind hochgradig durch die Atg-Proteine reguliert. Eines dieser Atg-Proteine, das Atg8, ist an einigen entscheidenden Schritten wie dem Phagophor-Wachstum, der Autophagosom-Reifung sowie der Schließung beteiligt. Während es in Hefen nur ein einziges Atg8-Protein gibt, so zeigt sich in höheren Eukaryonten meist eine gewisse Diversität. So codiert beispielsweise das humane Genom mindestens sechs Atg8-Homologe. Neben den drei Proteinen der LC3-Familie (A, B, C) zählen auch GABARAP, GABARAPL1 und GABARAPL2 dazu. Die Gründe für diese Diversität sind noch nicht vollständig aufgeklärt, weshalb es wichtig ist, möglichst selektive Modulatoren zu entwickeln, um so die Aufgaben der einzelnen Homologen entschlüsseln zu können. Eine weitere wichtige Aufgabe übernimmt Atg8 beim Binden des abzubauenden Materials über sogenannte Autophagie-Rezeptoren, wie beispielsweise p62. Der Bindevorgang beruht dabei auf der Interaktion von p62 mit ubiquitinierten Zellbestandteilen auf der einen Seite und der Interaktion zwischen p62 und LC3 auf der anderen Seite. Letztgenannte beruht auf dem Binden des LIR-Motivs (LC3-interagierende Region) von p62 an die LDS (LIR-docking site) des LC3-Proteins. Das LIR-Motiv zeichnet sich durch Aminosäure-Sequenz D-D-D-W/F/Y-X1-X2-L/I/V aus. Währende die aromatische Seitenkette (W/F/Y) die hydrophobe Tasche 1 (HP1) der LDS besetzt, ragt die aliphatische Seitenkette (L/I/V) in die HP2 hinein. Damit sollte es möglich sein, die LIR-LC3-Interaktion, durch das Besetzen der LDS zu stören bzw. zu inhibieren. Solche Inhibitoren könnten zum einen der weiteren Aufklärung der Prozesse, an denen die Autophagie beteiligt ist, dienen, zum anderen jedoch auch die Untersuchung fehlerhafter Autophagie ermöglichen. Ausgangspunkt für diese Arbeit stellt die Verbindung Novobiocin dar, die im Rahmen eines Mitteldurchsatz-Screenings als potenzieller Inhibitor der LIR-LC3-Interaktion identifiziert und mittels ITC, TSA und 1H-15N-HSQC verifiziert werden konnte. Die Struktur des Novobiocins setzt sich aus dem 3-Amino-4-hydroxy-8-methylcoumarin-Kern, der über eine Amidbindung an 3-iso-Prenyl-4-Hydroxybenzoesäure gebunden ist, sowie einer O-glykosidischen Bindung in Position C7 des Coumarins mit L-Noviose zusammen. Da es sich bei Novobiocin (XL6) um ein verhältnismäßig komplexes Molekül handelt, wurde der Einfluss einzelner funktionellen Gruppen des Moleküls auf die Bindungsaffinität hin untersucht. Hierfür wurden Synthesestrategien sowohl für die Coumarin-Gerüste als auch verschiedene Benzoesäuren entwickelt. Die erhaltenen Verbindungen wurden mittels ITC und TSA untersucht. Dabei wurde die Verbindung MH507 als geeigneter Ausgangspunkt für die Untersuchung der Struktur-Aktivitätsbeziehungen (SAR) bezüglich der Benzamid-Seite identifiziert. Im Rahmen einer ersten SAR-Untersuchung wurden neben verschiedenen 3-Alkyl-benzoesäuren, auch verschiedene divalente Isostere (-O-, -S-, -NHSO2-) der benzylischen Methylengruppe synthetisiert. Diese, sowie kommerzielle Aminosäuren, wurden mit 3-Amino-4,7-dihydroxycoumarin zu den entsprechenden Endverbindungen gekuppelt. Ergänzend dazu wurden auch eine Verbindung mit umgekehrter Konstitution der Amidbindung dargestellt, um den Einfluss der Reihenfolge zu verifizieren. In einer weiteren SAR-Studie wurden Derivate synthetisiert, die zusätzlich eine Funktionalisierung am C7 des Coumarin-Gerüstes über Amidkupplung, Sulfonamid-Bildung bzw. Suzuki-Reaktion erlauben und somit eine Interaktion mit der HP1 ermöglichen könnten. Dafür wurde eine weitere Synthesestrategie zur Darstellung von 7-Nitro- bzw. 7-Brom-3-amino-4-hydroxycoumarinen ausgearbeitet und eine Reihe von Endverbindungen dargestellt. Neben den Coumarin-Derivaten wurden auch vier Peptidomimetika synthetisierten. Hierfür wurde, basierend auf den Interaktionen zwischen dem LIR-Motiv und der LC3 Proteinoberfläche, ein Pharmakophor-Modell erstellt. Neben einem Pentapeptid wurden auch drei Verbindungen dargestellt, die ein 5-Amino 2-methoxybenzohydrazid-Gerüst besitzen. Um die synthetisierten Verbindungen auf ihre inhibitorische Aktivität auf LC3A bzw. LC3B gegenüber dem LIR-Motiv von p62 hin untersuchen zu können, wurde ein HTRF-basierter Verdrängungsassay entwickelt. Dabei diente ein mit dem LIR-Motiv modifiziertes sGFP als FRET-Akzeptor, während das jeweilige Terbium-Kryptat-gelabelte SNAP-LC3-Fusionsprotein als FRET-Donor fungierte. Neben den Titrationsexperimenten zur Bestimmung der IC50-Werte wurden auch die jeweiligen Dissoziationskonstanten (Kd) von LC3A und LC3B gegenüber dem LIR-sGFP-Fusionsprotein bestimmt, um die IC50-Werte in inhibitorische Konstanten (Ki) zu überführen, da diese untereinander besser vergleichbar sind.
Die Verbindung MH209 zeigte die höchste Aktivität auf LC3A bzw. LC3B und besitzt aufgrund der Noviose-Einheit eine gute Wasserlöslichkeit, weshalb sie für die weiteren Untersuchungen ausgewählt wurde. Im Zuge von Kristallisationsexperimenten gelang die Isolierung und Vermessung eines Co-Kristalls von LC3A mit Verbindung MH209. Durch die Kristallstruktur wurden wichtige Einblicke in die intermolekularen Wechselwirkungen der 4-Hydroxycoumarine mit der LC3A- bzw. LC3B-Proteinoberfläche gewonnen und die Bindungsmode aufgeklärt. Diese Erkenntnisse passen gut zu den Ergebnissen aus den durchgeführten TSA-, ITC- und HTRF-Assays, wie beispielsweise der korrekten Konstitution der Amidbindung am C3 des Coumarin-Gerüstes. Mittels ITC wurde die Verbindung MH209 auf ihre Bindungsaffinität gegenüber den anderen humanen Homologen der Atg8-Proteinfamilie hin untersucht. Dabei zeigte sich, dass MH209 abgesehen von LC3A und LC3B keinerlei Aktivität auf den humanen Atg8-Homologen besitzt. Diese Selektivität ist nützlich, um die biologische Bedeutung der Diversität von Atg8-Homologen in höheren Eukaryonten zu untersuchen und Prozesse, in die diese involviert sind, aufzuklären.
Für jeden Betroffenen ist die Diagnose Krebs ein schwerwiegender Einschnitt in der Lebensqualität und -führung, da die Behandlung oftmals mit langen Chemotherapien einhergeht. Moderne Durchbrüche in der Krebsbehandlung stammen aus dem Forschungsbereich der zielgerichteten Molekulartherapie oder aus dem Gebiet der Immuntherapien, die zu beachtlichen Erfolgen bei der Behandlung von Krebspatienten führten. Trotzdem bleiben auf dem Gebiet der Onkologie weiterhin Fragen zu den grundlegenden biologischen Prozessen unbeantwortet.
Zu den Onkoproteinen, die das Tumorwachstum in Leukemiezellen stark beeinflussen, gehören die Proteine der Klasse der mixed lineage leukemia (MLL) Histonmethyltransferasen. Genetische Fusionen des mll Gens, sogenannte Rearragments, führen zu MLL-fusion Produkten, die erheblich zum Verlauf der aggressiven akuten myeloischen Leukämie (AML) beitragen. Ein weiteres Onkoprotein, das für den Krankheitsverlauf vieler Krebsarten relevant ist, ist die Transkriptionsfaktorfamilie MYC. Überexprimierung von MYC wurde in einem Drittel aller humanen Tumore beobachtet. Zahlreiche Studien belegen, dass hohe MYC Level die Expression von Genen regulieren, die essentiell für den Transformationsprozess und somit das Tumorwachstum sind. Da der Transkriptionsfaktor weder eine sabile tertiäre Proteinstruktur noch eine für Inhibitoren adressierbare Bindetasche aufweist, gilt MYC bis heute als undruggable.
Sowohl die Histonmethyltransferase MLL1, als auch der Transkriptionsfaktor MYC interagieren mit einem ca. 37 kDa Protein namens WD40-repeat containing Protein 5 (WDR5), das durch seine propellerförmige Struktur eine Oberfläche mit insgesamt zwei Bindestellen aufweist. Mehrere Studien zeigten, dass WDR5 die Stabilität und somit die Funktion epigenetischer Proteinkomplexe wie SET/ MLL und NSL gewährleistet. In diesem Kontext wurde WDR5 als relevantes Target für die MLL-rearragend akute lymphatische Leukämie (ALL) postuliert. Weitere Studien zeigten zusätzliche Rollen von WDR5, wie die Interaktion zwischen WDR5 und dem Onkoprotein MYC sowie dessen Rekrutierung zum Chromatin. Seit 2015 wurden erfolgreich mehrere niedermolekulare Wirkstoffe für die Inhibierung von WDR5 entwickelt. Dabei zielten die meisten der literaturbekannten Inhibitoren auf die Argininmotiv-erkennende WDR5-interacting (Win) Bindestelle, eine große, hydrophobe Bindetasche im Zentrum des WDR5-Propellers. Die Resultate der besser erforschten Win Inhibitoren zeigten, dass WDR5 ein erfolgsversprechendes Target zur Inhibierung von leukämischen (MLL-r-abhängigen) und neuroblastomatischen (MYC-abhängigen) Zellwachstum ist.
Da beide Bindestellen des WDR5 Proteins Interaktionen mit onkologisch bedeutsamen Faktoren eingehen, würde eine einseitige Inhibierung nur die Effekte der jeweiligen Bindestelle aufzeigen. Diese Limitierung könnte jedoch durch die Entwicklung von WDR5 PROTACs (Proteolysis targeting chimeras) aufgehoben werden, da alle Gerüstfunktionen des Proteins und Protein-Protein-Interaktionen durch die Degradierung von WDR5 entfernt werden würden. Dabei induzieren die heterobifunktionellen Moleküle den Abbau des Zielproteins über das zelleigene Ubiquitin-Proteasom-System, statt die Enzymfunktion zu inhibieren. Nach dem zelleigenen Abbau des Zielproteins wird der PROTAC freigesetzt und kann einen neuen Zyklus der Proteindegradation einleiten, was die erforderliche Menge an Wirkstoff verringert.
Diese Dissertation beschäftigte sich mit dem Design, der Synthese sowie der biophysikalischen und biologischen Evaluierung von WDR5 PROTACs. Ausgehend von literaturbekannten WDR5 Liganden wurden zwei verschiedene PROTAC Typen entworfen. Diese beiden Molekültypen besitzen einen unterschiedlichen geometrischen Austrittswinkel, wodurch die Chance auf eine erfolgreiche Komplexbildung zwischen WDR5, PROTAC und E3 Ligase erhöht wird. Als Leitstruktur fungierten die Verbindungen OICR-9429 sowie DDO-2117 und ausgehend von Ligand (6d) wurden heterobifunktionelle Moleküle mit verschiedenen Linkersystemen ([PEG]- und alkyl-basiert, sowie aromatisch verbrückt) und verschiedenen E3 Ligase Liganden (Cereblon, VHL und MDM2) synthetisiert. Die anschließenden biochemischen und biophysikalischen Evaluierungen der verschiedenen PROTACs durch Thermofluor (DSF) und ITC zeigten eine hohe in vitro Affinität einiger Moleküle. Die zelluläre Permeabilität der großen Moleküle wurde in einem hier etablierten BRET Assay untersucht. Zur Assay-Etablierung wurden drei Tracer (21a-c), basierend auf BODIPY Konjugaten, synthetisiert und getestet, bevor die PROTACs in intakten und lysierten Zellen vermessen wurden. Während die zellulären Affinitäten von Cereblon- und VHL-adressierenden PROTACs sich im niedrigen μM Bereich bewegten, wurden die nicht zellgängigen MDM2 PROTACs von weiteren Experimenten ausgeschlossen.
Die Degradierungeffizienz der WDR5 PROTACs (7a-e) und (8a-j) wurden in der Leukämie Zellinie MV4-11 untersucht, da diese die am meisten auftretende MLL fusion Mutation AF4 birgt. Dabei wurde der Proteinabbau von WDR5 über den HiBiT Assay sowie Western Blots nachgewiesen. ...
Bezüglich der Arzneimittelforschung galt für sehr lange Zeit das Paradigma "ein Gen, ein Medikament, eine Krankheit". In jüngerer Zeit ändert sich dieses Paradigma jedoch auf Grund von redundanten Funktionen und alternativen sich kompensierenden Signalmustern, die insbesondere bei Krebserkrankungen vorherrschend sind. Daher kann die logische Konsequenz nur sein, Multi-Target-Strategien gegenüber Single-Target-Ansätzen in Betracht zu ziehen. Auf Grund der Schwierigkeit, mit einer Kombination von zwei Einzelwirkstoffen, in diesem Fall BET- und HDAC-Inhibitoren eine konsistente Biodistribution und Pharmakokinetik zu erreichen, wurde nach Einzelmolekülen gesucht, die mehrere inhibitorische Aktivitäten aufweisen. Dies wurde hier zunächst durch die einfache Konjugation von zwei unterschiedlichen Pharmakophoren erreicht.
Insgesamt wurden vier verschiedene Liganden dieses Typs synthetisiert und einer von ihnen, Verbindung 14, zeigte sehr vielversprechende Ergebnisse. 14 vereint den BET Inhibitor JQ1- mit dem HDAC Inhibitor CI994 und hat eine hemmende Wirkung sowohl gegen BRD4- als auch HDAC-Proteine wie durch DSF- und nanoBRET-Assay gezeigt werden konnte. Außerdem zeigten in vitro Assays in PDAC-Zellen, dass 14 ein noch potenterer dualer BET/HDAC-Inhibitor ist als die Kombination aus JQ1 und CI994. Während die Effekte von 14 auf das BETi-Antwortgen MYC denen von JQ1 ziemlich ähnlich sind, sind insbesondere die HDAC-inhibitorischen Effekte nachhaltiger und verstärkt, wahrscheinlich aufgrund einer längeren Verweildauer von 14 auf HDAC als dies bei CI994 der Fall ist. Dies ist durch das hohe Niveau der acetylierten Lysine von Histon H3 im Western Blot erkennbar. Dieses veränderte Expressionsverhalten hatte einen großen Einfluss auf das Zellwachstum und überleben in allen getesteten PDAC-Zelllinien. Hier wurde die Überlegenheit von 14 gegenüber der gleichzeitigen Behandlung der Zellen mit JQ1 und CI994 sehr deutlich. Wurden PDAC-Zellen mit dem dualen Inhibitor 14 behandelt, hatte dies ein geringeres Wachstum und Überleben der Krebszellen zur Folge als mit beiden ursprünglichen Molekülen, unabhängig davon, ob diese einzeln oder simultan verabreicht wurden. Außerdem wurde 14 mit Gemcitabin, einem gut verträglichen Chemotherapeutikum, kombiniert, dass bei PDAC allein nur eine begrenzte Aktivität aufweist. Es stellte sich heraus, dass die Reihenfolge, in der die Medikamente verabreicht werden, einen großen Einfluss auf die Effektivität hatte. Der durch 14 induzierte Stopp des Zellzyklus verhindert den Einbau von Gemcitabin in die DNA, wenn 14 vor oder gleichzeitig mit Gemcitabin verabreicht wird. Wenn jedoch die Behandlung mit 14 nach der Verabreichung von Gemcitabin folgt, wird der durch Gemcitabin induzierte S-Phasen-Arrest und Replikationsstress aufrechterhalten. Im Vergleich zu den meisten früheren Studien, die sich mit dualen BET/HDAC-Inhibitoren beschäftigten, ist dies eine große Verbesserung, da es bisher keinen signifikanten Unterschied zwischen der Verwendung eines dualen BET/HDAC-Inhibitors und der Kombination von zwei Einzelinhibitoren gab.
Als Proof of Concept unterstützten die Daten weitere Bemühungen zur Entwicklung zusätzlicher dualer BET/HDAC-Inhibitoren. Daher wurden zwei weitere Generationen dualer BET/HDAC Inhibitoren entwickelt, die jedoch bisher nicht an die Eigenschaften von 14 anknüpfen konnten. Vor allem die 3. Generation bietet jedoch Raum für Optimierungen, so dass hier möglicherweise noch ein potenter dualer Inhibitor zu finden ist. Sollte es in Zukunft einen zugelassenen dualen BET/HDAC-Inhibitor geben, ist es jedoch nicht unwahrscheinlich, dass keine der hier verwendet BET inhibierenden Strukturen verwendet werden, aber Struktur des HDAC inhibierenden Teils immer noch vergleichbar ist. Der Grund dafür ist, dass die HDAC Inhibitoren größtenteils relativ einfach aufgebaut. So lange das wichtigste, die zinkbindende Gruppe vorhanden ist, scheint der Linker sowie die Capping-Gruppe zweitranging zu sein. Die größere Herausforderung wird vermutlich die Suche nach dem passenden BET Inhibitor sein und die Wahlmöglichkeiten sind schon jetzt vielfältig.
Generell lässt sich sagen, dass die Idee der dualen BET/HDAC-Inhibitoren äußerst vielversprechend und es wert ist, weiter verfolgt zu werden. Dies liegt vor allem an den guten Testergebnissen, die mit Verbindung 14 erzielt wurden. Mit Hilfe dieser Art von Inhibitoren könnte es in Zukunft möglich sein, die Überlebensrate von PDAC-Patienten zu erhöhen, wenn nicht als alleiniges Medikament, so vielleicht als Zusatz zur Chemotherapie. Darüber hinaus scheint der Einsatz von dualen BET/HDAC-Inhibitoren nicht nur auf die Behandlung von PDAC beschränkt zu sein und kann auch bei anderen Krebsarten angewendet werden. NMC zum Beispiel ist ein ebenso seltener wie tödlicher Subtyp des schlecht differenzierten Plattenepithelkarzinoms und zeichnet sich durch eine Fusion des NUT-Gens mit BRD4 aus, wodurch es potenziell anfällig für eine BET-Inhibition ist. Tatsächlich zeigte 14 auch hier einen größeren positiven Effekt auf die getesteten NMC-Zellen als JQ1 oder CI994 und veranlasste die Zellen unter anderem zur Differenzierung. ...
This thesis comprises the usage of two commonly known hinge-binding moieties in drug discovery. First, the quinazoline scaffold of gefitinib (5) was utilized in a macrocyclization strategy to introduce selectivity. In general, the quinazoline hinge-binding moiety is a commonly used scaffold which can be found in 14% of approved kinase inhibitors. The most familiar applications are EGFR inhibitors such as gefitinib (5), erlotinib (6), afatinib, or dacomitinib for the treatment of NSCLC. But other kinases like CDK2, CDK4, or p38 are reported targets as well.
The N-phenylquinazolin-4-amine moiety of gefitinib (5) was conserved however, the residues at the aromatic ring in the linker were modified, the residue targeting the solvent-exposed region was varied, and the linker at the C6 position of the quinazoline was adjusted to enable the macrocyclization. An overview of the structural modifications is shown in Figure 35A.
Kinome-wide screening of gefitinib (5) revealed several off-targets besides EGFR (Figure 35B), making it an excellent starting point for a macrocyclization strategy. Introducing a linker to the N phenylquinazoline-4-amine scaffold and retaining the residues on the aromatic ring as well as the methoxy group targeting the solvent-exposed region improved the selectivity profile and the efficacy towards EGFR WT and its mutants. Truncation of the linker moiety led to the mutant selective macrocycle 26f with an excellent kinome-wide selectivity profile (Figure 35B). An inhibitor that is effective on EGFR mutations while ineffective on the EGFR WT could represent an enhancement of patient treatment, as it potentially causes less side effects. Further studies could determine the effect of the most promising macrocycles in lung cancer cell lines. Additionally, the pharmacokinetic properties could be optimized, e.g. by introducing solubilizing groups, targeting the solvent-exposed region.
The second scaffold comprises the 3-aminopyrazole-based hinge-binding moiety. It is a privileged scaffold in medicinal chemistry for the development of kinase inhibitors. Previous publications report the anti-proliferative and anti-cancer potential of pyrazole-based molecules. They play a crucial role in the treatment of various diseases and cancer types like inflammation disorders, lymphoma, or breast cancer. This scaffold can be found e.g. in the aurora kinase inhibitor tozasertib or in the promiscuous kinase inhibitor 23, published by Statsuk et. al. Rescreening compound 23 in a comprehensive kinase panel against 468 human protein kinases confirmed the unselective behavior with a selectivity score of S35 = 0.56 (Figure 36B), making it a great starting point for further optimizations. The N-(1H-pyrazol-3-yl)pyrimidin-4-amine scaffold was conserved however, the residues targeting the solvent-exposed region were varied and different linkers were attached.
The introduction of different residues at the pyrazole dramatically influenced the selectivity profile of the desired kinases. Ester moieties caused to a favorable combination of selectivity and potency towards the kinase of interest CDK16. The removal of additional residues at the pyrimidine, targeting the solvent-exposed region, increased the efficiency towards CDK16. Further optimization led to the highly potent and selective CDK16 inhibitor 98d (IC50 = 33 nM). NanoBRETTM screening against the complete CDK family revealed a preferred inhibition of the PCTAIRE and PFTAIRE subfamily with cellular IC50 values of 20 nM – 120 nM and 50 nM – 180 nM, respectively. A FUCCI cell cycle assay and viability assessment of 98d confirmed previously published results, reporting a G2/M cell cycle arrest followed by apoptosis and accumulation of p27 through knockout of CDK16 in SCC cells. Consequently, further studies could evaluate the anti-tumor activity of 98d in SCC and NSCLC or elucidate the effect of 98d in AMPK-related macroautophagy. 98d represents a novel tool compound to investigate the understudied kinases of the PCTAIRE family and enable to enlighten the biological role of those kinases.
Macrocyclization of the N-(1H-pyrazol-3-yl)pyrimidin-4-amine core resulted in the selective BMPR2 inhibitor 110a. It showed a good binding affinity towards BMPR2 with a KD value of 205 nM as well as a good potency with an IC50 value of 506 nM. A comprehensive selectivity screen against 468 kinases revealed an excellent selectivity profile with S35 = 0.01. As no BMPR2 inhibitors have been published so far, 110a represents a novel compound that may provide further insights into the canonical BMP pathway, noncanonical signaling, or its impact on BMPR2-associated diseases like PAH.
The introduction of additional residues targeting the solvent-exposed region shifted the selectivity towards the MST kinases. The exchange from the pyrimidine to a quinazoline moiety resulted in the highly potent and selective macrocyclic MST3 inhibitor 113c. NanoBRETTM measurements demonstrated the preferred inhibition of MST3 with IC50 values of 210 nM and 30 nM for intact and lysed cells, respectively. A weaker activity could be seen for MST4 with 1.8 µM and 510 nM, while MST1 and MST2 were not affected. To date, no selective MST3 inhibitors have been published, making 113c a valuable tool compound for further functional studies. As MST3 is influencing the cell cycle progression, 113c could be tested in a further cell cycle assay to elucidate the inhibitory effect of 113c on MST3 and consequently on the cell cycle. Furthermore, the anti-tumor activity of 113c in breast cancer could be determined, as Madsen et. al. reported a high MST3 and MST4 activity triggered by FAM40B mutations.
Protein kinases are key signalling molecules and transduce intracellular signals via the post-translational phosphorylation of substrate proteins, often other protein kinases. Dysregulation of this protein family has been linked to many diseases including neurodegenerative diseases, inflammation and cancer and amplifications of kinases play important roles as diagnostic biomarkers in a variety of cancers. Various strategies have been developed to treat dysregulated protein kinases. Most commonly, chemical small molecule inhibitors are used to modulate protein kinase activity in cancer cells. Many inhibitor and general research efforts have focused only on a small subset of protein kinases, resulting in a large portion of the kinome, the so-called “dark” kinome, remaining largely unexplored. As part of the strategy to develop inhibitors, it is crucial to understand the structure-activity-relationships (SAR) of small molecules to the activity towards the targets based on understanding small molecule-target affinities as determined by biophysical, biochemical, and cellular methods. However, not always do in vitro determined affinities, which are frequently used as basis for SAR considerations, correlate with the cellular affinity. For protein kinases in particular, it has been shown that the cellular concentration of the natural substrate adenosine-triphosphate (ATP) plays a critical role for the resulting small molecule affinity, as substrate and inhibitor frequently compete for the same binding site of the protein kinase. The cellular target engagement assay NanoBRET is a versatile assay that overcomes this problem and can be used to assess binding of a compound to the full-length protein kinase, in the presence of natural binding partners. Another important factor in inhibitor optimization is the selectivity of the molecule within the family of protein kinases. When comparing the selectivity profiles of small molecule kinase inhibitors in vitro and in cells, different profiles can be observed. Frequently, a compound, binds fewer protein kinases with high affinity in cells, indicating that cellular profiling of protein kinase inhibitors is necessary to understand the selectivity profile of an inhibitor.
The goal of this work was to understand cellular SARs of inhibitors for kinases and dark kinases in medicinal chemistry projects, and to understand the selectivity profiles of existing small molecules in cells, including already approved drugs and clinically used kinases inhibitors. The cellular potency and selectivity aspects guided optimization of the inhibitors towards selective small molecules ‘chemical probes’ or highly validated inhibitors with a narrow selectivity profile as part of ‘chemogenomic libraries’. One strategy to improve selectivity has been to use sterically restricted cyclic small molecules, called macrocycles, that allow fewer conformations of the molecule than their non-cyclic parent compound. In this thesis the dark kinase STK17A was investigated. Macrocyclization was used to develop a selective chemical probe molecule that is also selective in the cellular context. For another kinase, SIK2, a rational design approach was used to exclude off-targets bound by the lead structure, resulting in a chemical probe that selectively targets the SIK1/2/3 proteins. Assessing cellular potency of another series of inhibitors, a probe was developed for the PCTAIRE subfamily of the CDK kinases. This required co-expression of the binding partners of CDKs, the cyclins, in cells to obtain a functional assay. To identify new candidates for the neglected family of splicing kinases comprising the CLK, SRPK, DYRK and HIPK protein kinase subfamilies, a literature review was conducted, and the best small molecule candidates were compared for their target engagement in cells. This led to a series of small molecule inhibitors that may be used as a set or single agents to target the CLK proteins and SRPK proteins or in combination to target the remaining proteins. In search of new starting points for this subfamily of kinases, an initial screen with NanoBRET technology was performed using a library of over 2000 inhibitors, and new starting points were identified. Additionally, a set of clinical and approved small molecule kinase inhibitors was assessed for their selectivity in cells. Several highly selective molecules were identified that were much less selective in in vitro approaches. The set of data allowed for a comprehensive comparison of cellular potencies with published data using in vitro binding, in vitro activity and data obtained from cell lysates and identified several protein kinases that would need to be investigated in cells...
Nukleäre Rezeptoren (NRs) sind ligandengesteuerte Transkriptionsfaktoren, die sich aus einer Superfamilie von 48 humanen Mitgliedern zusammensetzt. Seit vielen Jahrzehnten stellen sie ein attraktives Forschungsgebiet für die Arzneistoffentwicklung dar, da sie eine bedeutende Rolle in zahlreichen Prozessen unseres Körpers spielen. Das Ziel dieser Forschungsarbeit bestand darin, neue innovative Liganden für den Peroxisomen-Proliferator-aktivierter-Rezeptor γ (PPARγ) sowie die Waisenrezeptoren Nervenwachtumsfaktor induzierter Klon B (Nur77) und Neuronen-abgeleiteter Waisenrezeptor (NOR-1) zu identifizieren.
Bei den Rezeptoren Nur77 und NOR-1 handelt es sich um noch unzureichend erforschte NRs der NR4A-Familie. Es fehlt insbesondere an Modulatoren dieser Rezeptoren als Werkzeuge, um ihr zum Teil noch unentdecktes Potential zu erforschen. Um diese Lücke zu schließen, wurde ein in vitro Screening durchgeführt und eine Arzneistoff-Fragment-Bibliothek mit 480 Fragmenten, die aus bekannten strukturellen Motiven zugelassener Arzneimittel stammen, auf ihre modulatorische Aktivität an Nur77 und NOR-1 gescreent. Durch das Screening und weitere Testungen konnten jeweils für Nur77 und für NOR-1 drei Verbindungen als Liganden identifiziert werden. Bei der weiteren Charakterisierung stellte sich insbesondere 41 als besonders vielversprechenden Ausgangspunkt für die Entwicklung von Liganden für Nur77 und NOR-1 heraus, der ein besseres Verständnis für die invers agonistische Aktivität lieferte und die Möglichkeit für eine agonistische Modulation aufzeigte. Zudem konnte durch ein weiteres Screening mit Computer-gestützten Verfahren auf Nur77 der Chemotyp von 41 noch weiter optimiert werden und führte zur Identifizierung von Verbindung 68 (EC50 = 2 ± 1 μM). Diese zeichnete sich durch eine hohe Potenz aus, die zu einer beachtenswerten Aktivierung von Nur77 (169 ± 18% maximale Aktivierung) führte. Die Untersuchung der strukturellen Erweiterung von 43 (IC50 = 47 ± 8 μM) führte zur Verbindung 75, die eine 3,5-fache Steigerung des inversen Agonismus auf NOR-1 zeigte. Die Erkenntnisse dieser Entdeckung ermöglichte den Rückschluss, dass das Einführen von voluminösen Resten, wie Brom oder Phenyl eine invers agonistische Potenz im unteren mikromolaren Bereich bewirkte. Die Identifizierung der Verbindungen 41 und 68 für Nur77 sowie 43 und 76 für NOR-1 könnten dazu beitragen, ein tieferes Verständnis der molekularen Mechanismen hinter der Aktivierung von Nur77 und NOR-1 zu erlangen und einen vielversprechenden chemischen Ausgangspunkt für die Entwicklung von noch wirksameren und selektiveren Liganden bieten.
Im anderen Teil dieser Forschungsarbeit stand die Synthese eines selektiven allosterischen PPARγ-Liganden im Fokus, um mit diesem die allosterische Modulation von PPARγ zu charakterisieren. Den Ursprung der Idee lieferte Garcinolsäure, dass in der Lage ist, PPARγ orthosterisch und allosterisch zu binden. Aufgrund der komplexen biologischen Effekte und der geringen synthetischen Zugänglichkeit konnte 37 nicht als Ausgangspunkt für dieses Vorhaben dienen. Auf der Suche nach einer geeigneten Ausgangsverbindung wurde durch ein in vitro Screening mit einer hauseigenen Sammlung von synthetischen PPARγ-Modulatoren, bei dem die orthosterische Bindungsstelle von PPARγ durch den irreversiblen Antagonisten GW9662 blockiert wurde, Verbindung 39 identifiziert. Diese ist wie 37 in der Lage PPARγ ortho- und allosterisch zu binden, weist aber eine bessere synthetische Zugänglichkeit auf. Die Co-Kristallisation von 39 mit der PPARγ-Ligandenbindungsdomäne zeigte, dass die orthosterische Bindungstasche (BT) keinen Platz für eine Verlängerung des Moleküls bietet, die allosterische BT ist dagegen Lösungsmittel exponiert, wodurch eine Verlängerung möglich schien. Daraufhin wurde die Hypothese aufgestellt, dass eine Verlängerung von 39 eine orthosterische Bindung verhinderte und dadurch eine selektive allosterische Bindung ermöglichen könnte. Aus diesem Grund wurde eine modifizierte Struktur von 39 verwendet, um eine einfache Einbringung eines Linkers in das Molekül zu ermöglichen. Durch verschiedenste Modifikationen und Anpassungen wurde 104 als potenzieller selektiver allosterischer Ligand synthetisiert. Die Testung von 104 im Reportergenassay zeigte eine schwache Aktivierung von PPARγ allein, jedoch offenbarte sich bei der Kombination mit dem orthosterischen Agonisten Pioglitazon eine dosisabhängige Steigerung der Aktivität von PPARγ. Diese Ergebnisse deuteten darauf hin, dass trotz der Bindung von 104 eine Bindung von 33 in die orthosterische BT immer noch möglich war. Diese Annahme konnte anschließend auch durch zellfreie Experimente (Isotherme Titrationskalorimetrie, MS-basierte-PPARγ-Ligandenbindungs-Assay) bestätigt werden. Der eindeutige Beweis für die selektive allosterische Bindung von 104 lieferte die Co-Kristallisation von 104 mit der PPARγ-LBD. Zusätzlich offenbarte sich, durch den strukturellen Vergleich der Bindungsmodi von anderen PPARγ-Liganden, der außergewöhnliche Bindungsmodus von 104, da 104 im Vergleich zu anderen Liganden selektiv die allosterische BT, ohne Überlappung in die orthosterische BT, besetzte. Weitere Untersuchungen, wie der Einfluss von 104 auf die Rekrutierung von Co-Regulatoren, die Differenzierung von adipozytären Stammzellen und die Genexpression zeigten eine bisher einmalige Modulation von PPARγ, die auf die selektive allosterische Modulation zurückzuführen war. Mit 104 wurde ein innovatives und vielfältig einsetzbares Werkzeug zur Erforschung der allosterischen Modulation von PPARγ entdeckt, dessen Geschichte an diesem Punkt noch nicht zu Ende ist.
Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death.
In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
Autophagy is an important degradation pathway mediating the engulfment of cellular material (cargo) into autophagosomes followed by degradation in autophagosomes.
Different stress stimuli, e.g. nutrient deprivation, oxidative stress or organelle damage, engage autophagy to maintain cellular homeostasis, recycle nutrients or remove damaged cell organelles. Autophagy not only degrades bulk cytoplasmic material but also selective autophagic cargo, for example lysosomes (lysophagy), mitochondria (mitophagy), ER (ER-phagy), lipid droplets (lipophagy), protein aggregates (aggrephagy) or pathogens (xenophagy). Selective autophagy pathways are regulated by selective autophagy receptors which bind to ubiquitinated cargo proteins and link them to LC3 on the autophagosomal membrane.
Ubiquitination is an essential post-translational modification controlling different cellular processes such as proteasomal and lysosomal degradation or innate immune signaling.
M1-linked (linear) poly-Ubiquitin (poly-Ub) chains are exclusively assembled by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the M1 poly-Ub-specific OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN). In addition to key functions in innate immune signaling and nuclear factor-κB (NF-κB) activation, M1 ubiquitination is also implicated in the regulation of autophagy.
LUBAC and OTULIN control autophagy initiation and maturation and the autophagic clearance of invading bacteria via xenophagy. However, additional functions of LUBAC- and OTULIN-regulated M1 ubiquitination in autophagy are largely unknown and it also remains unexplored if LUBAC and OTULIN control other selective autophagy pathways in addition to xenophagy. This study aimed to unravel the role of LUBAC- and OTULIN-controlled M1 ubiquitination in bulk and selective autophagy in more detail.
In this study, characterization of OTULIN-depleted MZ-54 glioblastoma (GBM) cells revealed that OTULIN deficiency results in enhanced LC3 lipidation in response to autophagy induction and upon blockade of late stage autophagy with Bafilomycin A1 (BafA1). Furthermore, electron microscopy analysis showed that OTULIN-deficient cells have an increased number of degradative compartments (DGCs), confirming enhanced autophagy activity upon loss of OTULIN. APEX2-based autophagosome content profiling identified various OTULIN-dependent autophagy cargo proteins. Among these were the autophagy receptor TAX1BP1 which regulates different forms of selective autophagy (e.g. lysophagy, aggrephagy) and the glycan-binding protein galectin-3 which serves key functions in lysophagy, suggesting a role of OTULIN and M1 poly-Ub in the regulation of aggrephagy and lysophagy.
Abstract 2
To study aggrephagy, protein aggregation was induced with puromycin which causes premature termination of translation and accumulation of defective ribosomal products (DRiPs). Loss of OTULIN increased the number of M1 poly-Ub-positive foci and insoluble proteins and reduced the levels of soluble TAX1BP1 and p62 in response to puromycin-induced proteotoxic stress.
Intriguingly, upon induction of lysosomal membrane permeabilization (LMP) with the lysosomotropic drug L-Leucyl-L-Leucine methyl ester (LLOMe), M1 poly-Ub strongly accumulated at damaged lysosomes and colocalized with TAX1BP1- and galectin-3-positive puncta. M1 poly-Ub-modified lysosomes formed a platform for NF-κB essential modulator (NEMO) and inhibitor of κB (IκB) kinase (IKK) complex recruitment and local NF-κB activation in a K63 poly-Ub- and OTULIN-dependent manner. Furthermore, inhibition of lysosomal degradation enhanced LLOMe-induced cell death, suggesting pro-survival functions of lysophagy following LMP. Enrichment of M1 poly-Ub at damaged lysosomes was also observed in human dopaminergic neurons and in primary mouse embryonic cortical neurons, confirming the importance of M1 poly-Ub in the response to lysosomal damage.
Together, these results identify OTULIN as a negative regulator of autophagy induction and the autophagic flux and reveal OTULIN-dependent autophagy cargo proteins.
Furthermore, this study uncovers novel and important roles of M1 poly-Ub in the response to lysosomal damage and local NF-κB activation at damaged lysosomes.
The simultaneous inhibition of HDACs and BET proteins has shown promising anti-proliferative effects against different cancer types, including the difficult to treat pancreatic cancer. In this work, the strategy of concurrently targeting HDACs and BET proteins was pursued by developing different types of dual inhibitors.
By developing a novel scaffold that selectively inhibits HDAC1/2 together with BET proteins in cells, an effective tool for the investigation of pancreatic cancer, and other diseases which are sensitive to epigenetic processes, was created. The compound’s small size further gives the opportunity to further develop the inhibitor towards optimized pharmacokinetic properties, potentially resulting in a drug for cancer treatment.
A second novel approach that was pursued, was the development of a small-molecule degrader, targeting HDACs and BET proteins. Through synthesizing a variety of different molecules, a compound that was capable of lowering BRD4 levels and, at the same time, increasing histone acetylation was developed. While additional mechanistic investigations are needed to verify the degradation, the potent antiproliferative effects in pancreatic cancer cells encourage further studies following this alternative new strategy.