Targeting the splice factor kinases SRPK1 and CLK1 in leukaemia

This study was aimed at investigating the effect of inhibiting SRPK1 in leukaemic cells. It was also aimed at exploring the potential utility of combining conventional leukaemia chemotherapy (such as imatinib) with compounds that inhibit SRPK1.SRPK1 is best known for its role in the phosphorylation of serine/argenine rich proteins (SR-proteins) which are responsible for constitutive and alternative mRNA splicing. Studies have associated elevated levels of SRPK1 with tumour growth, proliferation and invasiveness with inhibition resulting in decreased tumour growth and altering the choice of alternative splice site. Imatinib mesylate and azacytidine remain the drugs of choice for the management of chronic myeloid leukaemia (CML) and acute myelogenous leukaemia (AML) respectively. Studies have shown that both imatinib and azacytidine are able to reduce the growth of proliferating Bcr/Abl+ and AML cells principally through the induction of apoptotic cell death.SRPK1 was inhibited using the small molecule inhibitor SPHINX. SPHINX was combined with either imatinib in a CML cell line (K562) or azacytidine in an AML cell line (Kasumi-1) for up to 72hrs. Results suggest that the SPHINX compound affects the ability of SRPK1 to phosphorylate its substrates in all three cell lines (TK6, K562 and Kasumi-1). Inhibition of SRPK1 was found to reduce cell viability in Kasumi-1 cells and at higher concentration, affect K562 cell viability consistent with the work of Sanidas et al.,(2010). There was also an indication that SRPK1 could be regulating its own expression through a feedback loop in a cell line-dependent manner.Studies with imatinib mesylate and azacytidine showed that both imatinib mesylate and azacytidine are able to reduce cell growth and viability in a dose and time-dependent manner. On combining them with SPHINX, a combination of azacytidine and SPHINX had an additive effective on Kasumi-1 cells but not with imatinib mesylate in K562 cells. Results also showed that imatinib affected the alternative splicing of caspase 9 favouring a pro-apoptotic isoform, caspase 9a. Imatinib mesylate alone also caused an apparent reduction in the expression of SRPK1, CLK1 and SRSF1, suggesting that pathways imatinib affects cell signalling pathways that regulate the expression of these oncogenic splice factor kinases and splice factors. In summary, this thesis presents evidence that targeting SRPK1 could potentially provide therapeutic benefit in the treatment of a range of leukaemias; further research is now needed to explore this novel approach

Modulation of human apurinic/apyrimidinic endonuclease 1 (APE1) functions for breast cancer therapy

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2016DNA repair is required for the maintenance of genome stability. In the last years DNA repair pathways have emerged as important targets for cancer therapy. Since standard anticancer agents are mainly DNA-damaging drugs, its combination with DNA repair inhibitors may contribute to improve treatment outcomes. Among the multiple effectors involved in DNA repair, the multifunctional base excision repair (BER) protein apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most attractive druggable targets in this field. APE1 is the major endonuclease in BER participating in the repair of different DNA lesions including toxic abasic sites. In addition to the DNA repair activity, APE1 also acts independently as a reduction/oxidation signalling protein modulating the activation and DNA binding ability of several transcription factors implicated in cell survival and tumour promotion and progression. In this context, this thesis is focused on the combination of APE1 pharmacological inhibitors with conventional anticancer agents in the highly aggressive human breast cancer MDA-MB-231 cell line. Endonuclease activity has been the most studied function of APE1 in cancer therapy. Methoxyamine (MX), a commercially available indirect inhibitor of APE1 DNA repair function, was evaluated in combination with doxorubicin (Dox) in MDA-MB-231 cells. The chemotherapeutic drug Dox is widely used in the treatment of advanced breast cancer and may act, in part, by inducing oxidative DNA damage. MX had little effects in viability and colony formation of MDA-MB-231 cells. However, a significant increase in the frequency of micronucleated cells and an alteration in the pattern of micronuclei distribution were observed suggesting an increase in Dox genotoxicity. The differential results obtained in terms of cytotoxicity and genotoxicity showed that a therapeutic strategy based on APE1 inhibition is likely to have no relevance for the improvement of outcomes of Dox treatment. Although several putative inhibitors of APE1 endonuclease activity have been reported they still lack potential to be translated to the clinical setting. Therefore, in this thesis a structure-based virtual screening (SBVS) study based on molecular docking analysis of National Cancer Institute (NCI) database of compounds was performed to identify novel small-molecule inhibitors of APE1. The evaluation of SBVS study most promising compounds in a fluorescence-based APE1 endonuclease activity assay revealed three APE1 inhibitors. Compound 22 was a potent APE1 inhibitor showing inhibitory effects at nanomolar concentrations, while compounds 37 and 41 inhibited the enzyme in the micromolar range. These novel scaffolds for the design of more potent APE1 inhibitors did not affect the viability of non-tumourigenic human breast epithelial MCF10A cell line highlighting their promising features. The importance of APE1 modulation is beyond its functions in DNA repair. Therefore, E3330, a commercially available inhibitor of APE1 redox function, was also evaluated as single agent and in combination with the taxane drug docetaxel (DTX) in MDA-MB- 231 cells. DTX has anti-migratory and anti-angiogenic effects and is frequently used in advanced breast cancer refractory to anthracycline-based regimens. Consequently, relevant endpoints of cell migration and invasion were studied in addition to cell viability, proliferation and cell cycle profile assessment. Minor effects were observed in cell proliferation. However, E3330 alone significantly reduced the collective cell migration evaluated by the wound-healing assay without affecting chemotaxis and chemoinvasion. The combination of E3330 with DTX significantly decreased invasion of MDA-MB-231 cells suggesting a potential therapeutic role in metastatic breast cancer. The results described in this work emphasise the importance of preclinical studies of APE1 functions in cancer therapy and highlight the potential of novel drug combinations based on APE1 inhibitors reinforcing the role of targeting DNA repair in cancer treatment

Discovery and development of novel inhibitors for the kinase Pim-1 and G-Protein Coupled Receptor Smoothened

Investigation of the cause of disease is no easy business. This is particularly so when one reflects upon the lessons taught us in antiquity. Prior to the beginning of the last century, diagnosis and treatment of diseases such as cancers was so bereft of hope that there was little physicians could offer in the way of comfort, let alone treatment. One of the major insights from investigations into cancers this century has been that those involved in research leading to treatments are not dealing with a singular malady but multiple families of diseases with different mechanisms and modes of action. Therefore, despite the end game being similar in cancers, that of uncontrolled growth and replication leading to cellular dysfunction, different diseases require different approaches in targeting them. This leads us to a particular broad treatment approach, that of drug design. A drug is, in the classical sense, a small molecule that, upon introduction into the body, interacts with biochemical targets to induce a wider biological effect, ideally with both an intended target and intended effect. The conceptual basis underpinning this `lock-and-key' paradigm was elucidated over a century ago and the primary occupation of those involved in biochemical research has been to determine as much information as possible about both of these protein locks and drug keys. And, as inferred from the paradigm, molecular shape is all-important in determining and controlling action against the most important locks with the most potent and specific keys. The two most important target classes in drug discovery for some time have been protein kinases and G Protein-Coupled Receptors (GPCRs). Both classes of proteins are large families that perform very different tasks within the body. Kinases activate and inactive many cellular processes by catalysing the transfer of a phosphate group from Adenosine Tri-Phosphate (ATP) to other targets. GPCRs perform the job of interacting with chemical signals and communicating them into a biological response. Dysfunction in both types of proteins in certain cells can lead to a loss of biological control and, ultimately, a cancer. Both of kinases and GPCRs have entirely different chemical structures so structural knowledge therefore becomes crucial in any approach targeting cells where dysfunction has occurred. Thus, for this thesis, a member from each class was investigated using a combination of structural approaches. From the kinase class, the kinase Proviral Integration site for MuLV (Pim-1) and from the GPCR class, the cell membrane-bound Smoothened receptor (SMO). The kinase \pimone\ was the target of various approaches in \autoref{chap:three}. Although a heavily studied target from the mid-2000's, there is a paucity of inhibitors targeting residues more remote from structural characteristics that define kinases. Further limiting extension possibilities is that \pimone\ is constitutively active so no inhibitors targeting an inactive state are possible. An initial project (\pone) used the known binding properties of small molecules, or, `fragments' to elucidate structural and dynamic information useful for targeting \pimone. This was followed by three projects, all with the goal of inhibitor discovery, all with different foci. In \ptwo, fragment binding modes from \pone\ provided the basis for the extension and development of drug-like inhibitors with a focus on synthetic feasibility. In contrast, inhibitors were found in \pthree\ via a large-scale public dataset of purchasable molecules that possess drug-like properties. Finally, \pfour\ took the truncated form of a particularly attractive fragment from \pone\ that was crystallised with \pimone, verified its binding mode and then generated extensions with, again, a focus on synthetic feasibility. The GPCR \smo\ has fewer molecular studies and much about its structural behaviour remains unknown. As the most `druggable' protein in the Hedgehog pathway, structural studies have primarily focussed on stabilising its inactive state to prevent signal transduction. Allied to this is that there are generally few inhibitors for \smo\ and the drugs for cancers related to its dysfunction are vulnerable to mutations that significantly reduce their effectiveness or abrogate it entirely. The elucidation of structural information in therefore of high priority. An initial study attempting to identify an unknown molecule from prior experiments led to insights regarding binding characteristics of specific moieties. This was particularly important to understand not just where favourable moieties bind but also sections of the \smo\ binding pocket with unfavourable binding. In both subsequent virtual screens performed in Chapter 4, the primary aim was to find new drug-like inhibitors of \smo\ using large public datasets of commercially-available molecules. The initial screen retrieved relatively few inhibitors so the binding pocket was modified to find a structural state more amenable to small molecule binding. These modifications led to a significant number of new, chemically novel inhibitors for \smo, some structural information useful for future inhibitors and the elucidation of structure-activity relationships useful for inhibitor design. This underpins the idea that structural information is of critical importance in the discovery and design of molecular inhibitors

Cyclic Nucleotide Signaling and the Cardiovascular System

The cyclic nucleotides 3',5'-adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play important roles in the control of cardiovascular function under physiological and pathological conditions. In this book, which is a reprint of a Special Issue of the Journal of Cardiovascular Development and Disease entitled "Cyclic Nucleotide Signaling and the Cardiovascular System", internationally recognized experts give an overview of this vibrant scientific field. The first series of articles deal with the localization and function of membrane-bound and soluble adenylate cyclases, followed by articles on the roles of phosphodiesterase isoforms in the heart. Cyclic nucleotide signaling takes place in nanodomains and the A-kinase anchor proteins (AKAPS) are essential for the compartmentalized assembly of signaling proteins into functional complexes. Reviews on the role of AKAP proteins in the physiology and pathophysiology of the heart are also included in this book. Cyclic nucleotides act through effector proteins and articles on EPAC and POPDC proteins inform the reader of recent developments on these topics. A major advancement in our understanding of cyclic nucleotide signaling came through the use of genetically encoded cAMP sensor molecules, and a series of articles review the current insight that these reporter molecules have provided. The final set of articles in this book deals with the association of the cyclic nucleotide pathway and cardiovascular disease as well as the development of novel therapeutic approaches. Thomas Brand and Enno Klussmann Special Issue Editor

Integration of HSP90 inhibition in combinational immunotherapies targeting receptor tyrosine kinase EphA2

Limitations in CD8+ T cell recognition of tumor cells due to defects in their antigen processing machinery or the selection of variants expressing low or absent levels of cognate tumor antigens have been previously identified as impediments to effective cancer immunotherapy. Hence, treatment regimens that coordinately promote enhanced activation of anti-tumor CD8+ T cells, improved delivery of such effector cells into tumor sites, and augmented recognition of tumor or tumor-associated stromal cells by therapeutic CD8+ T cells, would be expected to yield greater clinical benefit. Using an MCA205 sarcoma model, I show that in vitro treatment of tumor cells with the HSP90 inhibitor 17-DMAG results in the transient (proteasome-dependent) degradation of the HSP90 client protein EphA2 and the subsequent increased recognition of tumor cells by Type-1 anti-EphA2 CD8+ T cells. In vivo administration of 17-DMAG to tumor-bearing mice led to: i.) slowed tumor growth; ii.) enhanced/prolonged recognition of tumor cells by anti-EphA2 CD8+ T cells; iii.) reduced levels of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the tumor microenvironment (TME); and iv.) activation of tumor-associated vascular endothelial cells in association with elevated levels of Type-1 tumor infiltrating lymphocytes (TIL). When combined with EphA2-specific active vaccination or the adoptive transfer of EphA2-specific CD8+ T cells, 17-DMAG cotreatment yielded a superior tumor therapeutic regimen that was capable of rendering animals free of disease

Advances and Novel Treatment Options in Metastatic Melanoma

The book presents several studies reporting advances on melanoma pathogenesis, diagnosis and therapy. It represents a milestone on the state of the art, updated at 2021, and also presents the current knowledge on the future developments in melanoma field

Structure, Activity, and Function of Protein Methyltransferases

This collection of review articles describes the structure, function and mechanism of individual protein methyltransferase enzymes including protein lysine methyltransferases, protein arginine methyltransferases, and also the less abundant protein histidine methyltransferases and protein N-terminal end methyltransferases. The topics covered in the individual reviews include structural aspects (domain architecture, homologs and paralogs, and structure), biochemical properties (mechanism, sequence specificity, product specificity, regulation, and histone and non-histone substrates), cellular features (subcellular localization, expression patterns, cellular roles and function, biological effects of substrate protein methylation, connection to cell signaling pathways, and connection to chromatin regulation) and their role in diseases. This review book is a useful resource for scientists working on protein methylation and protein methyltransferases and those interested in joining this emerging research field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Development of WNT inhibitors as novel anti-cancer drugs

La voie de signalisation WNT joue un rôle essentiel dans le développement embryogénique, où elle contrôle la prolifération, la différenciation et la migration des cellules. En physiologie adulte, son activité se limite aux niches de cellules souches responsables de la régénération. Dans certains cas, cependant, la voie WNT est faussement activée, conduisant à une prolifération cellulaire non contrôlée, également connue sous le nom de cancer. L'activation aberrante de la signalisation WNT se produit également dans le cancer du sein triple négatif (TNBC), un sous-type de cancer du sein très agressif dépourvu de thérapies ciblées. Ce travail propose donc de cibler le TNBC par inhibition de la voie WNT suractivée. Le candidat-médicament idéal doit prouver qu'il a des effets anticancéreux contre le TNBC, tout en n'étant pas nocif pour la physiologie cellulaire normale. Notre hypothèse de travail est que le ciblage des niveaux de la membrane plasmique, en particulier les principaux récepteurs de la voie, les Frizzleds (FZD), pourrait obtenir de tels résultats. En ciblant le sous-ensemble spécifique de FZD surexprimé dans le tissu tumoral, mais pas dans le tissu sain, il devrait être possible d'éviter l'inhibition des branches WNT nécessaires au fonctionnement physiologique normal. De plus, étant classés comme GPCR, les FZD ont le potentiel d'être hautement médicamenteux, ce qui en fait des candidats cibles idéaux. Il existe de nombreuses façons d'aborder le développement de nouvelles molécules médicamenteuses : le repositionnement d'entités médicamenteuses approuvées ou le criblage de nouvelles molécules sont deux méthodes prometteuses. Dans ce travail, une bibliothèque de petites molécules a été criblée à l'aide d'une pipeline de test à haut débit spécifique au TNBC pour cibler les GPCR FZD dans le cancer, développé en interne. Cela a conduit à la découverte d'une nouvelle classe de molécules de pyrazole substituées par un diphényle avec des effets inhibiteurs du WNT et des propriétés anticancéreuses in vitro et in vivo. Des recherches détaillées sur la ou les cibles exactes des molécules nouvellement découvertes ont prouvé qu'elles se lient bien au FZD. Les molécules phares ont des profils de toxicité améliorés par rapport aux inhibiteurs du WNT actuellement connus, ce qui en fait une nouvelle génération très prometteuse d'inhibiteurs du WNT à petites molécules. Pris ensemble, ce travail montre une approche robuste pour l'identification et le développement préclinique d'inhibiteurs de WNT avec des propriétés anticancéreuses pour le TNBC avec le potentiel d'être étendu à d'autres indications de cancer. -- The WNT signalling pathway plays an essential role in embryogenic development, where it controls cell proliferation, differentiation and migration. In adult physiology, its activity is limited to stem cell niches responsible for regeneration. In some instances, however, the WNT pathway is falsely activated, leading to uncontrolled cell proliferation, also known as cancer. Aberrant activation of WNT signalling also occurs in triple-negative breast cancer (TNBC), a very aggressive breast cancer subtype lacking targeted therapies. This work, therefore, proposes to target TNBC by inhibition of the over-activated WNT pathway. The ideal drug candidate must prove to have anti-cancer effects against TNBC, while not being deleterious for normal cell physiology. It is our working hypothesis that targeting the plasma membrane levels, especially the main pathway receptors, the Frizzleds (FZDs), could achieve such results. By targeting the specific subset of FZDs overexpressed in the tumour tissue, but not in healthy tissue, it should be possible to avoid inhibition of the WNT branches necessary for normal physiological functions. Additionally, being classed as GPCRs, FZDs have the potential of being highly druggable, making them ideal target candidates. There are many ways to approach the development of new drug molecules: repositioning of approved drug entities or the screening of new molecules are both promising methods. In this work, a library of small molecules was screened using a TNBC-specific high-throughput assay pipeline for targeting FZD GPCRs in cancer, developed in-house. This led to the discovery of a new class of diphenyl-substituted pyrazole molecules with WNT-inhibitory effects and anti-cancer properties in vitro and in vivo. Detailed investigations into the exact target(s) of the newly discovered molecules proved that they are indeed FZD binding. The lead compounds have improved toxicity profiles compared to currently known WNT inhibitors, making them a very promising new generation of small molecule WNT inhibitors. Taken together, this work shows a robust approach to the identification and pre-clinical development of WNT inhibitors with anti-cancer properties for TNBC with the potential to be expanded to other cancer indications