Cyclic-AMP dependent protein kinase (PKA) is a key intracellular signal transduction kinase that is modulated by Gs- and Gi-coupled GPCRs. Under normal physiological conditions, PKA exists as an inactive holoenzyme made up of two catalytic subunits and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits (PKACa) are released to perform various downstream phosphorylation events. However, aberrant PKA activation can cause various diseases including Cushing’s Syndrome, which is an endocrine disorder caused by the overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system. This disorder can be caused by pituitary adenomas that release unregulated amounts of ACTH, adrenal adenomas that release unregulated amounts of cortisol without ACTH stimulation, and ectopic tumors outside the hypothalamus-pituitary-adrenal axis that produce ACTH. In recent genomic studies of patients with ACTH-independent Cushing’s Syndrome, the L205R-PKACamutant has been discovered. Through various studies on the mutant enzyme multiple research groups learned that the single point mutation causes a loss in sensitivity to cAMP signaling, a loss in binding to PKA regulatory subunits, and unregulated phosphorylation of PKACasubstrates, which ultimately leads to the increased cortisol biosynthesis in these patients. The first part of this work describes the enzymology and inhibition studies of known inhibitors against both wt- and L205R-PKACa. Early in the enzymology studies we developed at medium throughput endpoint assay that used Rhodamine-kemptide as the substrate and as a chromophore separating substrate and phosphorylated product using a reverse-phase HPLC method. The analysis of the substrate peptide against both wild-type and mutant enzyme showed a 6-fold decrease in the KMand a 2-fold decrease in kcat, and a similar but lower order of magnitude effect was observed for the studies with ATP. The inhibition studies were performed using the substrate competitive inhibitor PKI(5-24), which showed a 253-fold higher potency towards the wild-type enzyme over the mutant while the ATP-competitive inhibitor was determined to be equipotent. Using this information we used modeling studies to aid in the development of mutant selective functional inhibitors for the substrate-binding pocket. Additionally, we begun to explore the use of Proteolysis Targeting Chimeras, or PROTACs, as another means for targeting the L205R mutant enzyme.

Das Ceramid Transfer Protein (CERT) ist einer der geschwindigkeitsbestimmenden Proteine in der de novo Biosynthese von Sphingomyelin. Es ist verantwortlich für den nicht-vesikulären Transfer von Ceramid vom Endoplasmatischen Retikulum zum Golgi-Apparat. Die Inhibition von CERT wird als potenzielle Behandlung für Krankheiten wie Infektionen, Krebs oder Gain-of-Function-Mutationen des CERT Gens diskutiert. Kürzlich, wurde Lomitapide als potenter Inhibitor des CERT-vermittelten intermembran-Transfers identifiziert. Im ersten Teil dieser Arbeit wird die Synthese von Lomitapide Derivaten mit verbesserter Wirksamkeit und Selektivität präsentiert. Die synthetisierten Analoga wurden in vitro mithilfe eines liposomalen Transferassays auf ihre Inhibition der CERT-Transferaktivität getestet. Zusätzlich konnte durch die Messung des Ceramid-Sphingomyelin-Verhältnisses nach Inhibitor Behandlung die Aktivität in cellulo bestätigt werden. Die Selektivität gegenüber dem Mikrosomalen Triglycerid Transfer Protein (MTP) wurde durch Messung der MTP-vermittelten Sekretion von apoB ermittelt. Unter den synthetisierten Analoga zeigten einige verbesserte CERT-Transfer Inhibition und niedrigere Inhibition der apoB Sekretion, sogar bei fünffacher Konzentration verglichen mit Lomitapide. Obwohl die Bewertung der biologischen Aktivität noch im Gange ist, wurde eine vorläufige Struktur-Aktivitäts-Beziehung etabliert. Es wurden strukturelle Bestandteile identifiziert, die wichtig für die CERT-Inhibition sind und andere welche variabel sind, um die Wirksamkeit und Selektivität in Zukunft noch weiter zu steigern. Der zweite Teil dieser Arbeit beschreibt die Synthese von Lomitapide-basierten proteolysis targeting chimeras (PROTACs) für CERT. PROTACs haben sich im letzten Jahrzehnt zu einem vielversprechenden therapeutischen Ansatz entwickelt und mehrere potenzielle Wirkstoffe hervorgebracht. PROTACs sind heterobifunktionale Moleküle, die sich den zellulären Weg der Proteinzersetzung zunutze machen, indem sie das gewünschte Protein zur Zersetzung markieren. Es wurde eine erste Serie von CERT PROTACs mit vielversprechender Abbauwirkung synthetisiert, welche eine bevorzugte Zersetzung von CERT aber nicht CERTL andeuten. CERTL ist eine längere Spleiß-Variante, welche vornehmlich im Herz, Gehirn und den Skelettmuskeln exprimiert wird. Eine zweite Serie von PROTACs mit variierter Linker Kettenlänge wurde synthetisiert. Untersuchung des Einflusses auf die apoB Sekretion aus HepG2 Zellen zeigte sogar bei 50-facher Konzentration einen niedrigeren Einfluss auf diese als Lomitapide. / The ceramide transfer protein (CERT) is one of the rate-limiting proteins in the de novo biosynthesis of sphingomyelin, facilitating the non-vesicular transfer of ceramide from the Endoplasmic Reticulum to the Golgi-apparatus. Inhibition of CERT has been proposed as a potential treatment for pathogenesis like infectious diseases, cancer, or disease-causing gain-of-function mutations within the CERT gene. Recently Lomitapide has been identified as a potent inhibitor of CERT-mediated intermembrane transfer. In the first part of this thesis, the synthesis of Lomitapide derivatives with improved potency and selectivity is presented. The synthesized analogs were tested in vitro for their inhibition of CERT-transfer using a liposomal transfer assay. Additionally, the activity could be confirmed in cellulo by monitoring the ceramide-sphingomyelin-ratio after inhibitor treatment. Selectivity against the microsomal triglyceride transfer protein (MTP) has been determined by monitoring the MTP-mediated cellular secretion of apoB. Among the synthesized analogs, several showed improved CERT-transfer inhibition and lower inhibition of apoB secretion even at five-fold higher concentrations compared to Lomitapide. Although the biological evaluation is still underway, a preliminary structure-activity-relationship has been established and identified structural motifs important for CERT inhibition and modifiable moieties to increase potency and selectivity even further in the future. The second part of the thesis describes the synthesis of Lomitapide-based proteolysis targeting chimeras (PROTACs) for CERT. PROTACs have evolved in the last decade as a promising therapeutic technique and resulted in the development of several drugs which are currently in clinical trials. PROTACs are heterobifunctional small molecules that mediate the degradation of the target protein by hijacking the cellular proteasomal pathway. A first series of synthesized CERT PROTACs showed promising preliminary results for CERT degrader activity and indicated a preferred degradation of CERT over CERTL, a longer splicing variant expressed in the heart, brain, and skeletal muscles. Motivated by this a second generation of PROTACs with varying linker chain lengths was synthesized. Investigation of their inhibition of apoB secretion from HepG2 cells revealed lower activity on secretion than Lomitapide even at 50-fold concentrations for a set of CERT PROTACs.

The use of small molecules to probe the function of proteins is referred to as chemical genetics. The Proteolysis Targeting Chimera (PROTAC) is a chemical genetic tool that contains the ligand for a target protein of interest and the recognition motif for an E3 ubiquitin ligase attached by a linker. The PROTAC is capable of binding to and recruiting specific target proteins to the intracellular degradation system, the ubiquitin proteasome system (UPS). While the approach has had success it has not been optimized to be used on a broader scale. Optimization efforts focused on elucidating the ideal linker length between the ligand and the E3-ligase recognition motif, the preferred location for attachment of the linker to the two moieties, and the possibility for a dimeric PROTAC comprised of two ligands. An estrogen receptor (ER)-targeting PROTAC was chosen as a model for optimization attempts as the ER is known to have pathological significance in breast cancer. Optimization of the PROTAC technology will not only provide a novel tool to probe ER biology, but may also offer a novel approach to breast cancer therapies. The ER targeting PROTAC constitute the 17β-estradiol (E2), as the ligand for ER and a pentapeptide derived from HIF-1α as the E3-ligase recognition motif, joined by a linker. Following the successful synthesis and evaluation of a number of PROTACs, it was revealed that an optimum ER-targeting monomeric PROTAC (KC-3) has a spacer of 16 atoms between the E2 and HIF-1α pentapeptide. The spacer is attached at the C-7α position on E2 and at the N-terminus of the HIF-1α pentapeptide. It was also established that the PROTAC is capable of targeting the ER for degradation in a proteasome and E3- ligase dependent manner, which translated to a decrease in the proliferation of MCF-7 cells with an IC50 similar to that of tamoxifen. KC-3, in comparison with E2, displayed lower agonistic activity on an ER-regulated downstream target, the progesterone receptor (PR). A dimeric PROTAC more effectively binds and degrades the ER in a proteasome dependent manner, suggesting that the dimeric ligand approach may be applied to the design of other PROTACs.

The increasing knowledge of molecular drivers of tumorigenesis has fueled targeted cancer therapies based on specific inhibitors. Beyond “classic” oncogene inhibitors, epigenetic therapy is an emerging field. Epigenetic alterations can occur at any time during cancer progression, altering the structure of the chromatin, the accessibility for transcription factors and thus the transcription of genes. They rely on post-translational histone modifications, particularly the acetylation of histone lysine residues, and are determined by the inverse action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Importantly, HDACs are often aberrantly overexpressed, predominantly leading to the transcriptional repression of tumor suppressor genes. Thus, histone deacetylase inhibitors (HDACis) are powerful drugs, with some already approved for certain hematological cancers. Albeit HDACis show activity in solid tumors as well, further refinement and the development of novel drugs are needed. This review describes the capability of HDACis to influence various pathways and, based on this knowledge, gives a comprehensive overview of various preclinical and clinical studies on solid tumors. A particular focus is placed on strategies for achieving higher efficacy by combination therapies, including phosphoinositide 3-kinase (PI3K)-EGFR inhibitors and hormone- or immunotherapy. This also includes new bifunctional inhibitors as well as novel approaches for HDAC degradation via PROteolysis-TArgeting Chimeras (PROTACs).

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

Most patients diagnosed with cancer will receive systematic chemotherapy at some point during their illness, which almost always cause severe side effects for the patients such as, anemia, nausea and vomiting. The problems with today’s chemotherapy is not only that it cause severe side effects, but also that the cancer may develop resistance to the therapy, which is why the development of a new type of therapeutic agent is in dire need. The ubiquitin proteasome system (UPS) is a vital machinery for the cancer cells to maintain protein homeostasis, which also make them vulnerable to any disruption of this system. In recent years, a new technology has been developed that utilize the UPS by chemically bringing an E3 ubiquitin ligase into close proximity of a protein of choice and tagging the protein with ubiquitin for degradation. This technology is called proteolysis targeting chimera (PROTAC). In this project, we managed to theoretically develop a new type of cancer therapeutic agent, that utilize the PROTAC system together with the first-in-class proteasome regulatory subunit, POH1, inhibitor Capzimin as a warhead. By using Capzimin as a warhead it should be possible to polyubiquitinate POH1, and thus induce proteotoxic stress in the cancer cells to trigger apoptosis. This theoretically developed drug is therefore called Capzimin-PROTAC, which should be able to trigger apoptosis in cancer cells, and at the same time being relatively safe to normal healthy cells.

Le développement d’agents anticancéreux s’étend de la découverte d’une cible thérapeutique à la commercialisation du médicament. La chimie médicinale intervient dans la conception et la synthèse de molécules. Les essais réalisés in vitro puis in vivo sont ensuite déterminants pour évaluer le mode d’action, l’efficacité et la sélectivité du principe actif. À cette étape, si une molécule n’est pas sélectionnée, son étude s’arrête. Afin de tirer profit du temps et de l’argent investis dans son développement, des stratégies s’inspirant de ces composés peuvent être utilisées, en modifiant leur structure afin d’améliorer leur efficacité. La première méthode exploitée ici consiste à synthétiser des molécules bifonctionnelles, incorporant le pharmacophore du principe actif d’intérêt, ainsi qu’un ligand capable d’induire la dégradation de sa cible. Ces molécules sont appelées AUTAC ou PROTAC, selon le mode de dégradation induit, soit l’autophagie ou la protéolyse. La deuxième méthode repose sur le développement de molécules hybrides, capables d’interagir avec différentes cibles. Deux principes actifs sont alors liés de manière covalente afin de synthétiser une nouvelle molécule dont les propriétés pharmacocinétiques diffèrent. L’hybride pourrait être ainsi plus efficace, cumulant les activités de ses deux ligands de base. Pour viser les cellules cancéreuses, une stratégie attrayante est le ciblage de certaines voies métaboliques. L’ATP étant une source d’énergie essentielle pour l’intense prolifération des cellules cancéreuses, réduire sa production en ciblant la phosphorylation oxydative dans la mitochondrie et la glycolyse peut être un traitement efficace. Les dérivés de biguanides, comme la metformine, sont connus pour inhiber la phosphorylation oxydative. Par ailleurs, le NAD+ étant un cofacteur nécessaire à de nombreux processus biologiques essentiels, notamment la glycolyse, diminuer sa production peut alors induire des effets anticancéreux. De nouveaux dérivés de biguanides et d’inhibiteurs de la NAMPT sont donc synthétisés afin de développer des molécules bifonctionnelles ciblant le métabolisme des cellules cancéreuses. / The development of new anticancer agents covers all the steps from the discovery of a target to the commercialisation of a drug. Medicinal chemists are responsible for the design and synthesis of molecules but the future of the drug relies on their activity in vitro and in vivo. However, compounds that failed because of their lack of efficiency or selectivity can be reused to take advantage of the time and money invested on their development. To develop more potent drugs, these compounds can be modified using different strategies. One of them consists in synthesising bifunctional molecules, also called chimeras, incorporating a pharmacophoric moiety from the drug and a ligand inducing its target’s degradation. These molecular degraders are named according to the mode of degradation involved: AUTAC for Autophagy Targeting Chimera or PROTAC for Proteolysis Targeting Chimera. Another strategy is to design a dual targeting agent based on two different drugs bound covalently. The resulting hybrid is then able to interact with each ligand’s target but exhibits a new pharmacokinetic profile that might increase its efficiency. Targeting cancer cell metabolism is an attractive strategy that can be applied to many different types of cancer. Since ATP is the main energy source of the cells, its production is necessary to insure the intense proliferation rate of cancer cells. Targeting the oxidative phosphorylation in mitochondria and the glycolysis, the two main ways to produce ATP, can be effective to inhibit the proliferation of cancer cells. Biguanides derivatives such as metformin are known to inhibit OXPHOS. Furthermore, NAD+ being an essential cofactor involved in many biological processes including glycolysis, targeting its production by the NAD salvage pathway allows the discovery of effective anticancer agents. New biguanides and NAMPT inhibitors derivatives are thus synthesized for the development of bifunctional molecules targeting the energy metabolism of cancer cells.

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