Inhibitory effect on the proteasome regulatory subunit, RPN11/POH1, with the use of Capzimin-PROTAC to trigger apoptosis in cancer cells

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Linköping University | Department of Physics, Chemistry and Biology Type of thesis, bachelor 16 | Educational Program: Chemistry – Molecular design Spring term 2020 | LITH-IFM-G-EX—20/3845--SE

Inhibitory effect on the proteasome regulatory subunit,

RPN11/POH1, with the use of Capzimin-PROTAC to

trigger apoptosis in cancer cells.

Andreas Holmqvist

Supervisor, Pádraig D’Arcy Co-supervisor, Hamid Shirani Examiner, Peter Nilsson

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URL för elektronisk version

ISBN

ISRN: LITH-IFM-G-EX--20/3845--SE

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Serietitel och serienummer ISSN

Title of series, numbering ______________________________

Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title

Inhibitory effect on the proteasome regulatory subunit, POH1, with the use of

Capzimin-PROTAC to trigger apoptosis in cancer cells.

Författare

Andreas Holmqvist

Nyckelord

Keyword

Proteolysis targeting chimera (PROTAC), Cancer therapeutics, Ubiquitin proteasome system (UPS), Cancer, Chemotherapy, Proteasome inhibitor, Capzimin

Sammanfattning

Abstract

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, thereby tagging the protein 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 a 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 newly developed drug is therefore called Capzimin-PROTAC, which could trigger apoptosis in cancer cells, while not being relatively harmful to normal healthy cells.

Datum

Date

Avdelning, institution

Division, Department

Department of Physics, Chemistry and Biology Linköping University

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Abstract

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.

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Keywords: Proteolysis targeting chimera, PROTAC, Cancer therapeutics, Ubiquitin proteasome system, UPS, Cancer, Chemotherapy, Proteasome inhibitor, Capzimin

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Abbreviations

BET Bromodomain extraterminal protein

BETi Bromodomain extraterminal protein inhibitor

CRBN Cereblon

Cs2CO3 Cesium carbonate

DMF Dimethylformamide

EtOAc Ethyl acetate

GC Gas chromatography

HPLC High-performance liquid chromatography

HSPs Heat shock protein

IC50 The half maximal inhibitory concentration

LC-MS Liquid chromatography-mass spectrometry

Leu56 Leucine number 56

MelJuSo Human melanoma cell-line

MeOH Methanol

PEG Polyethylene glycol

Phe133 Phenylalanine number 133

PROTAC Proteolysis targeting chimera

Pro89 Proline number 89

Thr129 Threonine number 129

Ub-YFP Ubiquitinated yellow fluorescence protein

UPS Ubiquitin proteasome system

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Inhibitory effect on the proteasome regulatory subunit, POH1, with

the use of Capzimin-PROTAC to trigger apoptosis in cancer cells.

1. Introduction 1.1 Background

1.1.1 Cancer

Cancer is one of the most common diseases and one of the leading causes of deaths in

Sweden. According to the National Board of Health and Welfare in Sweden (1), cancer ranks as second in the most common underlying cause of death in 2018, accounting for 24 and 27 percent of all deaths among women and men respectively. One of the problems with treating cancer is the heterogeneous nature of the cancer cell population, meaning that most of the cancer cells consist of different variants of mutations, and thereby it becomes difficult to target a particular cancer type. Instead, the most common approach in cancer therapy is chemotherapy using drugs such as alkylating agents (Figure 1a) and anti-metabolites (Figure

1b). These treatments are non-selective and can target both cancer cells and normal cells,

within the patient triggering apoptosis and cell death. These approaches will often cause severe side effects for the patient. However, some cancers may already be resistant to

chemotherapy or develop resistance to the drug, and thus will not have an effect on the cancer cells. Another frequent strategy in treating cancer is by surgically removing the malignant tumor tissue. This procedure required the surgeon to completely remove all of the cancer tissue, together with some healthy tissue to minimize the risk of recurring tumors, and that there has been no metastatic spread of the tumor. Additionally, surgical procedures always come with risks, such as loss of organ function, infections and bleedings. This is why a new approach in cancer therapy is needed, one that is both effective and able to target all the different types of cancer variants (2).

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Figure 1. a) A concise description of the mechanism of action for alkylating agents, where diamond represents

the alkylating agents reacting to the DNA-sequence, which leads to replication error and cell death.

b) A concise description of the mechanism of action for anti-metabolites, where the purple and grey/yellow sticks represent anti-metabolites replacing the purine or pyrimidine which leads to replication error and thus cell death.

1.1.2 Chemotherapy

Most of the people diagnosed with cancer will receive systematic chemotherapy at some point during their illness. Chemotherapeutical agents strive to cause lethal cytotoxic effects to the cells, which either inhibits cell replication or induces apoptosis. Their general mechanism of action is directed to DNA or metabolic sites that are essential for cell replication. One type of chemotherapeutical drugs are alkylating agents. These drugs are extremely reactive and will form covalent bonds with all macromolecules having a nucleophilic center, for example DNA bases. The main cytotoxic effect comes from when the alkylating agent covalently bonds with DNA bases and inhibits DNA replication. When the DNA-strands no longer can be replicated, the cell will either trigger apoptosis or will not be able to proliferate and the tumor will stop growing. However, since alkylating agents are reactive and bind to DNA bases, all the alkylating agents are mutagenic and carcinogenic. Therefore, the drug treatment itself can cause a secondary malignance by themselves (3). Resistance to alkylating agents may occur in some type of cancers, in the form of, an increase in DNA-repair systems or decreased drug permeability (4).

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1.1.3 The Ubiquitin-proteasome system

All cells in the body contain proteasomes, which is a large protein complex responsible for the intracellular degradation of proteins. For the proteasome to start degrading a protein, the protein first has to be marked for degradation with ubiquitin by a E3 ubiquitin ligase. The E3 ubiquitin ligase is a highly selective targeting enzyme that recognizes unwanted or misfolded protein and tags them with a polyubiquitin-chain. This is done by covalently attach the polyubiquitin-chain to a target protein acceptor site for instance a Lysine (5). When a protein is marked with ubiquitin, first then can a proteasome recognize the protein and one of the subunits of the proteasome located at the “lid”, called POH1/RPN11, will remove and recycle the ubiquitin from the protein and degradation of the protein will begin (Figure 2) (6). The ubiquitin-proteasome system (UPS) is especially important in cancer cells. Most tumor cells have shown to have an increased requirement for protein synthesis and metabolism, which in turn make them vulnerable for proteasome inhibition. Studies have demonstrated that malignant cells are more sensitive to the cytotoxic effects induced by proteasome

inhibitors than normal healthy cells. The reason behind the increase of sensitivity in malignant cells compared to normal cells remain unknown (7).

Figure 2. A concise explanation of the ubiquitin proteasome system and the required steps for degradation of

protein. Step 1, the E3 ubiquitin ligase ubiquitinates the target protein at lysine residue. Step 2, the ubiquitinated protein is recognized by the proteasome. Step 3, RPN11/POH1 subunit deubiquitinate and recycle the ubiquitin thereby enabling the proteasome to start degrading the protein. Step 4, the protein is degraded into free amino acids, and the proteasome is now available to degrade another tagged protein.

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1.1.5 Proteasome inhibitors

Proteasome inhibitors are considered as new potential chemotherapeutical agents. By inhibiting the proteasome, the cells will no longer be able to degrade polyubiquitinated proteins. This will result in polyubiquitinated proteins forming toxic aggregates within the cell, triggering apoptosis (Figure 3) (8). Studies have shown that resistance may occur towards proteasome inhibitor as well as for other drugs (9, 10). The molecular mechanism of this type of resistance is still not entirely determined. However, previous studies on the proteasome inhibitor bortezomib, have shown that point mutations in the binding pocket of the proteasome is an important mechanism for the acquired resistance (9). There are some strategies to overcome resistance to proteasome inhibitors. For example, develop a method to increase the drugs potency, increase the bioavailability of the drug or target specific subunits of the protein, which are sensitive to mutations (10).

Figure 3. A concise explanation the mechanism of action for proteasomes regulatory subunit POH1 inhibitor

Capzimin and which of the required steps for protein degradation that gets interrupted. Step 1, the E3 ubiquitin ligase ubiquitinates the target protein at lysine residue. Step 2, the ubiquitinated protein is recognized by the proteasome. Step 3, the proteasome is inhibited and can no longer deubiquitinate and recycle the ubiquitin, nor degrade the protein, thus the polyubiquitinated protein will start to aggregate and trigger apoptosis.

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1.1.6 The proteasome inhibitor Capzimin

There are several proteasome inhibitors (Figure 4) that has been used for clinical studies, such as the Bortezomib and Salinosporamide A. Bortezomib works by reversible binding to the catalytic site of the 26S proteasome, thereby inhibiting its function (11), while

Salinosporamide A works by irreversibly inhibiting the 20S proteasome (12). Another

proteasome inhibitor that has shown to be selective to one of the proteasome’s subunit, POH1, is Capzimin (13). Capzimin has shown to trigger apoptosis in leukemia, lung cancer and breast cancer, with an IC50 concentration of ~0.6 µM. Capzimin has also been shown to interact with POH1’s active site in a bidentate manner as well as the residues in the distal ubiquitin binding site. It has been suggested that Capzimin acts by chelating the Zn2+ ion, which inhibits POH1 (14). By inactivating POH1, studies have shown that, the proteasome loses its function to deubiquitinate proteins, which then leads to an increase of misfolded proteins and protein-aggregation within the cell triggering apoptosis.

Figure 4. Molecular structure of the proteasome inhibitors Bortezomib, Salinosporamide A and Capzimin.

1.1.7 PROTAC-technology

In recent year, a new technology has emerged that exploit UPS for targeted protein degradation, called Proteolysis-targeting chimera (PROTAC). PROTAC consists of a “double-headed” molecule which has affinity for a E3 ubiquitin ligase and is connected via a flexible linker to another molecule called a warhead (Figure 5). The warhead consists of a molecule that has affinity for a target protein of choice. By utilizing this system, it is

theoretically possible to target any protein within the cell for degradation, as long as it has a viable ubiquitin acceptor site. It has also been suggested that PROTAC requires a lower concentration then those required for inhibition to attain an equal pharmaceutical effect (15). By using a substance with a high selectivity and low IC50-concentration, together with the PROTAC-technology, it should be theoretically possible to reduce the IC50-value of the compound, resulting in a more effective inhibitor.

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Figure 5. A concise explanation of PROTAC’s mechanism of action where the yellow box represents PROTAC’s

warhead, and the green box represents the E3 ubiquitin ligase recruit unit. Step 1, PROTAC’s E3 recruit unit binds to the E3 ubiquitin ligase. Step 2, the warhead of the PROTAC molecule binds to the target protein bringing the connected E3 ubiquitin ligase and the target protein of choice into close proximity and the protein becomes ubiquitinated at lysine residue. Step 3, PROTAC now dissociate from the E3 ubiquitin ligase and the target protein. Step 4, the polyubiquitinated protein is recognized by the proteasome and the subunit

RPN11/POH1 starts to deubiquitinate and recycle the ubiquitin. The proteasome can thereafter start degrading the protein in to free amino acids. Step 5, the proteasome degrades the protein. Step 6, the PROTAC-molecule react with a new E3 ubiquitin ligase and cycle starts over.

1.1.8 PROTAC for cancer therapeutics

Researchers and pharmaceutical companies have been struggling with drug resistance in cancer therapeutics for years. As a result of PROTAC’s unique properties, compared to those of traditional inhibitors, researchers and pharmaceutical companies has started to investigate the potential of PROTAC in cancer therapeutics. Since PROTAC works by degrading the target protein, instead of inhibiting them, the cell has to develop a different kind of resistance (16). The BET-PROTAC developed by Arvinas have shown to be more potent and selective to bromodomain extraterminal protein (BETs) than bromodomain extraterminal protein inhibitors (BETi). In their studies they found that the developed BET-PROTACs had an IC50 value, for induction of apoptosis in Mino cells, at 16±3 nM, whereas the BETi had an IC50 value at 398±15 nM (17). Also, according to previous studies, the difference between the two compounds, when it came to drug resistance, cells that were resistant to the BET-PROTAC did not contain any secondary mutations that affected the substrates binding to the target. Rather, resistance was primarily due to genomic changes that affected the expression of the corresponding E3 ligase complexes. In a report where Arvinas BET-PROTAC was studied, they found that the acquired resistance came from a CRBN deletion, which codes for the E3 ubiquitin complex cereblon (18). This genomic alteration has been observed in many other cancer types. Their study suggests that there is a lot of potentials for PROTAC in cancer therapeutics and that it requires more studies involving PROTAC’s and cancer therapeutics.

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1.1.9 Capzimin-PROTAC

By using Capzimin as a PROTAC warhead, it should theoretically be possible for the

proteasome’s subunit POH1 to be tagged with ubiquitin. This would in turn lead to inhibition of the proteasome and as well as proteasome aggregation, which would trigger apoptosis in the cell (Figure 6). While only one Capzimin molecule will be able to inhibit one proteasome, one Capzimin-PROTAC molecule should be able to inhibit numerous proteasomes and

thereby, lower the IC50 concentration and become a more potent drug. This could be a solution to the acquired proteasome inhibitor resistance due to the increase in the drugs potency.

Figure 6. A concise explanation of how the different steps of Capzimin-PROTAC’s mechanism of action could

look like. Step 1, PROTAC’s E3 recruit unit binds to the E3 ubiquitin ligase. Step 2, the Capzimin warhead binds into RPN11/POH1’s active site and brings the VHL E3 ubiquitin ligase into close proximity and thus becomes ubiquitinated at available lysine residue. Step 3, Capzimin-PROTAC now dissociate from the E3 ubiquitin ligase and RPN11/POH1. Step 4, RPN11/POH1 is now ubiquitinated and inhibited and thus the proteasome can no longer degrade protein. Step 5, the polyubiquitinated proteasome starts to aggregate with other

polyubiquitinated protein and become toxic for the cell. The cycle continues until cell death.

1.2 Purpose and goal

The purpose of this study is to both investigate and validate Capzimin-PROTAC’s ability to target the proteasomes subunit POH1, and to validate the effect of applying the PROTAC technology on an already known proteasome inhibitor. The aim of this project is to develop a new potential drug in cancer therapeutics, that can both target several cancer variants, and reduce adverse effects. This project consists of both cellular and in vitro assays, together with the synthesis of the Capzimin-PROTAC compound.

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1.3 Limitations

The time of this project was limited to 10 weeks, which is not enough time for

troubleshooting the synthesis if the synthesis could not be. Also, due to the circumstances of the COVID-19 pandemic, this project will not be able to be carried out practically. Instead, this project had to be limited to a theoretical project based on previous studies and expected results. And further, how this project could be carried out in practice.

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2. Planning of the process and design of the molecule

2.1 Planning of the process

The time of the project was aimed to be 10 weeks, with start in the end of March. The project started with establishing a project plan in the shape of a GANTT-schedule (Appendix 1), in which the time for different assignments of the project were planned. This was followed by a detailed planning report reviewed by the examiner. The planning report consisted of some background information of the project, purpose, laboratory work, goal and the GANTT-schedule. Even though the project was aimed to be 10 weeks, the designing of the Capzimin-PROTAC molecule took place as early as January. This was done to save some time for when the project started.

The two introductory weeks for the project were used for preparation of the GANTT-schedule, chemical inventory, literature research, literature analysis and establishing the planning report. This was followed by three days of in vitro studies in order to validate the cytotoxicity of the proteasome inhibitor Capzimin. The following four weeks were used for synthesis, purification and confirmation of the compound. As soon as the compound was confirmed, three consecutive days were used for an in vitro study to validate Capzimin-PROTAC’s cytotoxicity. Thereafter, two weeks were used to compile all the data and write the final report for the project. The last two weeks were used to prepare the presentation and opposition as well as do the presentation and opposition, followed by the final hand-in of the report. However, due to the circumstances of the COVID-19 pandemic, which lead to that this project could not be performed in practice, the four weeks that was supposed to be used for the synthesis was instead used for literature study and analysis, analyzing theoretical data and writing of the report.

2.2 Designing the compound

When the designing of the Capzimin-PROTAC molecule took place, certain questions regarding the design began to emerge. Some of the questions that needed to be answered was which E3 ubiquitin ligase should be targeted, what length of the linker should be used and where the linker should be connected to the warhead. To answer this, a lot of literature studies and analysis were performed during January and February.

2.2.1 Designing the linker

The distance between the ligand and recruited protein is essential when designing the PROTAC compound. If the distance is too short, only one of the two recruiting units will be able to bind in and the substance will lose its primarily function. If the distance is too long, both of the recruiting units will be able to bind in but the distance between the two recruited proteins will be too far and the compound will lose some of its activity. In one previous study, several linker lengths were tested together with a VHL-E3 ubiquitin ligase recruiting unit (19). The results indicated that the optimal distance between the E3 ubiquitin ligase recruit unit and the warhead, for PROTAC activity, is a 16-atom chain length. However, the linker that would be used for this project was a linker with an 18-atom chain length, since this was the closest linker length to 16 that could be ordered for this project. The results from the previous study also indicated that the length of the linker did not affect the binding affinity, only the activity of the compound (19).

2.2.2 Designing the E3 ubiquitin ligase recruit unit

The two most common E3 ubiquitin ligase recruit units that has been used with PROTAC is

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protein in clear cell renal cell carcinoma, located primarily in the cytoplasm. The VHL-protein itself do not exhibit any enzymatic activities but forms a complex with other VHL-proteins which has E3 ubiquitin ligase activity (20). The CRBN-protein is a thalidomide binding protein that has shown to be important in several different mechanisms for the cells survival, in which one is to form a complex that has E3 ubiquitin ligase activity (21).

In a previous study carried out by Miriam et al. (21) VHL and CRBN was tested against each other. This was performed to find out which of the two E3 ubiquitin ligases were the most potent of the two when it came to protein. This was done by connecting two molecules, one with affinity for CRBN and one with affinity for VHL with a linker, and then try to hijack the E3 ubiquitin ligases against each other. The results showed that the VHL-ligase were able to degrade CRBN more efficient and quicker than CRBN could degrade VHL and thus indicates that the VHL-ligase is most potent of the two (21). This results lead to the E3 ubiquitin ligase recruit unit chosen for this project, together with an 18-atom chain, where the one with affinity for the VHL-ligase and not CRBN (Figure 7).

Figure 7. The molecular structure of the 18-atom chain and VHL E3 ubiquitin ligase recruit unit for the

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2.2.3 Designing the Capzimin-PROTAC

When designing the Capzimin-PROTAC molecule, it was important to know how the different atoms and fragments of the molecule interacted with the targeted protein POH1 (Figure 8a). According to a computational study done by Kumar et al. (14) the 8QT fragment of Capzimin interacts with the catalytic Zn2+ and the azole moiety provides hydrophobic interactions with Phe133, Leu56 and Pro89. The oxygen on the amide provides hydrogen bonding with Thr129. The hydrogen on the secondary amide does not provide any

interactions in stabilizing the binding of the compound. This information led to the conclusion that conversion of the secondary amide into a tertiary amide would be the best option to keep the distortion of the compound’s affinity to a minimum (Figure 8b).

Figure 8. a) Description of the different molecular fragments of Capzimin and how they interact with the

proteasome’s subunit POH1. b) The molecular structure of the Capzimin-PROTAC molecule.

a)

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3. Theory

3.1 Validation of Capzimin and Capzimin-PROTAC

To validate the effect of Capzimin and Capzimin-PROTAC on cells, the substances will be tested to determine both the IC50 concentration and the concentration, at which the UPS is disrupted, that will be indicated by Ub-YFP on a human melanoma cell-line (MelJuSo). The MelJuSo cells contain a Ub-YFP that is constantly degraded under normal proteasome conditions. If proteasome inhibition works, the Ub-YFP reporter will start to accumulate, producing a yellow fluorescence in the cells that can be detected by an Essen incucyte live scanning microscope and thereby indicating the mechanism of action.

MelJuSo parental cells, lacking Ub-YFP, will be used as a control to determine any auto fluorescence of the compounds. To determine cell proliferation, a WST-1 assay kit will be used. The WST-1 reagent is reduced in the presence of active mitochondria, producing a purple color change in the media which act as a surrogate marker for cell survival. When the IC50 and UPS inhibition has been determined, the cells will be analyzed for HSPs, caspase and ubiquitin expression using Western Blotting. However, if no cytotoxicity is observed with the Capzimin-PROTAC, then it could indicate that there is some defect on the PROTAC synthesis or that the N-alkylation disrupts the compounds POH1 affinity.

By analyzing HSPs, Caspase and Ubiquitin expression, it will be possible to see how the drug interacts within the cell and if the compound has the desired effect. By inhibiting the

proteasome, the cell is prone to induce proteotoxic stress, caused by an accumulation of miss-folded proteins, which can be detected by an increase in HSPs, Ubiquitin and Caspase. Due to PROTAC’s mechanism of action in bringing VHL and POH1 into a close proximity, it should be possible to observe a molecular weight shift in POH1 due to ubiquitination. By performing these analyzes, it should be possible to confirm if the Capzimin-PROTAC system works. In summary these three experiments will indicate the concentrations necessary to inhibit cell proliferation and determine the mechanism of action.

3.2 Synthesis

Capzimin consists of a thiol group that is prone to alkylation. To inhibit thiols reactivity, the thiol has to be deactivated, which will be done via a thiol acetylation (Figure 9).

Figure 9. Deactivation of thiol via thiol-acetylation

By converting the thiol into a thioester, the 8QT fragment of Capzimin becomes protected from further alkylations, and thus the secondary amide becomes more accessible for alkylation (Figure 10). However, previous syntheses performed on secondary amides has used iodide as a leaving group instead of chloride. This is due to iodides relatively low charge density in comparison with chloride. Nonetheless, this does not make the synthesis

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also be two different possible synthesis methods presented for this reaction if method one would not work.

Figure 10. Reaction scheme for acetylation of thiol and N-alkylation of the secondary amide.

After the N-alkylation is performed, the thiol has to become activated again. This will be carried out by de-acetylating the thioester (Figure 11).

Figure 11. Deacetylation of thioester.

As soon as the deacetylation of the thiol has succeeded, the synthesis is complete. Hereafter, the compound has to be purified and verified before validation of the Capzimin-PROTAC can be executed.

3.3 Purification

To purify the synthesized compound, a High-performance liquid chromatography (HPLC) purification will be performed. This is due to difficulties using conventional separation methods such as, silica gel column chromatography together with a synthesis yield of maximum 15.5 mg product. What will be used instead is an HPLC purification. Normal HPLC uses high pressure to force the nonpolar solvent, such as acetonitrile and water, through a close C18 column. The separation is based on how fast the analyte equilibrates between the stationary and mobile phase. The more polar the analyte is, the more time will the analyte spend in the stationary phase, and thus the analyte elutes slower from the column. By using HPLC purification, the synthesized compound should be able to get separated from the residual products and become pure enough for the in vitro studies (22).

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3.4 Verification

To verify if the synthesis had been a successful, the compound needs to be analyzed via Liquid chromatography mass-spectrometry (LC-MS). MS is a technique used to analyze the masses of atoms, molecules or fragments of molecules. To obtain a mass spectrum of a molecule, the molecule first evaporates into gas phase and become ionized. Then, the ionized molecule accelerates through an electric field and is expelled into the analyzer tube. In the analyzer tube, the molecule becomes exposed to a magnetic field perpendicular to their direction of flight. The magnetic field then rapidly changes so that only atoms, molecules or fragments with a specific mass reaches the detector. The spectrum is then obtained by varying the magnetic fields strength so different masses always reaches the detector. By using LC-MS as verification, it will be possible to see how pure the compound is and if the synthesis has been successful. However, if the synthesis was unsuccessful it will also be possible to see in which step the synthesis failed (22).

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4. Material and method 4.1 In vitro study

4.1.1 Determine the IC50 value of Capzimin and Capzimin-PROTAC

To establish the IC50 value of both Capzimin and Capzimin-PROTAC, MelJuSo Ub-YFP proteasome reporter cell lines were plated at a density 10,000 cells per well of a 96 well plate. Cells were plated in normal cell culture medium (DMEM+ 10% FBS) at a final volume of 100 µL per well. Two plates were seeded for viability and live cell imaging experiments.

4.1.2 Viability

Cells were exposed to log scale concentrations of Capzimin and Capzimin-PROTAC at concentrations intervals of 100 µM, 10 µM and 0.1 µM. The log scale was for initial estimation of IC50 values and the more refined concentrations would be used for later

experiments. Cells were then incubated for 48 h in a humidified incubator at 37°C. After the incubation period, 10 µL of the cell viability reagent WST1 was added, according to the WST1 assay protocol, to the wells. Cells were then incubated for 2 h with WST-1 and

absorbance was determined at 420 nm. The IC50 values for Capzimin and Capzimin-PROTAC were then obtained by plotting the absorbance values vs. drug concentration. The obtained IC50 values were then compared to determine the cytotoxicity profile and the bioavailability of the two compounds.

4.1.3 Proteasome inhibition

In this step, the experiments were performed in the same way as in the viability experiment, but with one change. The plates were scanned every hour for detection of Ub-YFP

accumulation at early time points for indication of proteasome inhibition. The Ub-YFP fluorescence signal was plotted against time to determine the compound’s mechanism of action. To verify the Ub-YFP accumulation, a western-blot analysis was performed to confirm HSPs, caspase and ubiquitin expression.

4.2 Synthesis

4.2.1 Thiol acetylation of Capzimin

To a 25 mL round-bottom flask, 15.0 µmol Capzimin (~4.7 mg, ≥95% Sigma-Aldrich) were added, followed by addition of 22.5 µmol acetic anhydride. The acetic anhydride was used as both solvent and reagent for this reaction. The solution was then mixed with a magnetic stirrer. When the Capzimin had dissolved, the mixture was placed in a preheated oil bath, with a constant temperature of 60°C for 12 hours. After the reaction was completed, and monitored by LC-MS, the resulting mixture was added to a separation funnel and quenched with NaCO3. Ether was then added, and organic phase was separated and washed with water. Thereafter, the mixture was added to a separation funnel and diluted with 1 ml diethyl ether and then washed and quenched twice with sodium bicarbonate. After the ether layer had been quenched, the solution was dried with sodium sulfate (23).

4.2.2 N-alkylation of Capzimin-PROTAC

4.2.2.1 Synthesis of Capzimin-PROTAC 1

To synthesize Capzimin-PROTAC, 15.0 µmol (~5.4 mg) of dried thiol acetylated Capzimin were placed in a 15 ml beaker. Thereafter, tetrahydrofuran was added until all of the dried thiol acetylated Capzimin were completely dissolved. When the solution was completely homogeneous, the solution was added to 60 µmol sodium hydride (1.44 mg, 55% in mineral oil) portion wise at 0°C and stirred at room temperature for 19 minutes under a constant flow of nitrogen gas. After the stirring, the solution was cooled to 0°C. Thereafter, 30 µmol of

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(S,R,S)-AHPC-C6-PEG1-C3-PEG1-butyl chloride (~22.5 mg, ≥95% Sigma-Aldrich) was added to the mixture and stirred at room temperature for 6 hours. After the stirring, the mixture was slowly quenched with water at 0°C and extracted with EtOAc. The solution was poured into a separation funnel and the water layer was removed. Thereafter, the organic layer was washed with brine twice and dried over Na2SO4 and filtered through a Celite pad. The combined filtrate and washings were concentrated through a rotary evaporator. The residue was lastly purified by HPLC purification and analyzed with LC-MS (24).

4.2.2.2 Synthesis of Capzimin-PROTAC 2

To synthesize Capzimin-PROTAC, 15.0 µmol (~5.4 mg) of dried thiol acetylated Capzimin was added to a 50 ml round-bottom flask containing a solution of 30 mmol Cs2CO3 (~4.88 mg) together with 30 µmol of (S,R,S)-AHPC-C6-PEG1-C3-PEG1-butyl chloride (~22.5 mg, ≥95% Sigma-Aldrich) in 15 ml of DMF in a 80°C PEG oil bath with rapid stirring in closed reactor for 50 hours. Thereafter, the mixture was cooled to room temperature and filtered through a Celite pad. The solid compound on the filter was washed with 10 ml of ether and then the combined liquids was washed once more to remove the precipitated salts. The solution was concentrated in vacuum at first 10 torr and at 1 torr with a cold trap at

-78°C. Once the solution had been concentrated, the solution was warmed in a water bath to 40°C. Afterwards, the solution was diluted in 25 ml ether and poured into a separation funnel. The solution was then washed three times in 3 ml of water and in 3 ml brine. After the wash, the organic solution was dried and analyzed in LC-MS to verify that the reaction was

successful (25).

4.2.3 De-thiol acetylation of Capzimin-PROTAC

After the synthesis and purification, the solution was purged in degassed MeOH in ammonia for 20 minutes. Thereafter, the solution was concentrated in vacuum and then analyzed by LC-MS analysis to verify that the deacetylation had been successful (26).

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5. Expected results 5.1 Synthesis

Since previous syntheses performed have had iodine as a leaving group (24, 25), which is a much better leaving group than chlorine, problems could have arisen from the synthesis. What would have been expected is a synthesis with a lower yield as well as well as longer synthesis steps to achieve a desired yield. However, there is no guarantee that the N-alkylation would have worked at all due to the chlorine atom. Additionally, since this would have been a micro synthesis, the purification process could become problematic, given the low yield from the synthesis. Nonetheless, the amount needed for the in vitro study is approximately 5 mg, which is the amount that would have been an expected yield from the synthesis.

5.2 In vitro study

To validate the cytotoxicity of the Capzimin-PROTAC compound, a determination whether the cytotoxicity came from the expected mechanism of action, or just regular inhibition from the warhead had to be performed, and also if there was any proteasome disruption at all. As mention in section, 3. Theory – 3.1 Validation of Capzimin and Capzimin-PROTAC, the MelJuSo cells contain a Ub-YFP reporter that is constantly degraded under normal

proteasome conditions. This reporter would be an indicator for if there was any proteasome disruption within the cells or not. If there was any proteasome disruption within the cell, could this be detected from the Ub-YPF accumulation, producing a yellow fluorescence, which would have been the expected results in this study. However, if there was no Ub-YFP accumulation could this indicate that the N-alkylation of Capzimin disrupted the molecules affinity to the proteasome subunit, POH1, and thus no Ub-YFP accumulation would occur. Nonetheless, according to the computational study done by Kumar et al. (14), the

N-alkylation of Capzimin should not affect the binding to the protein, since the hydrogen of the amide does not provide any stabilization. Rather, what would have been expected is that the affinity of the Capzimin warhead would decrease. This is due to the fact that there would probably be a small shift in the electron density surrounding the amides oxygen atom, which stabilizes the binding of Capzimin to POH1, but at the same time decrease the IC50 value. The expected decrease of IC50 value comes from the fact that one single Capzimin-PROTAC could inhibit several proteasomes via ubiquitination, which would lead to an increase of cytotoxicity.

To determine that the Capzimin-PROTAC works as expected, an analysis on HSP’s, Caspase and Ubiquitin expression via western-blotting could be performed. By doing this it would be possible to determine whether the cytotoxicity came from proteasome ubiquitination rather than proteasome inhibition. If the Capzimin-PROTAC compound works, a molecular weight shift in POH1 could be detected, due to ubiquitination, and thus prove that the PROTAC works, which is what would be expected in this study.

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6. Discussion

Several proteasome inhibitors have been developed, such as Capzimin, Bortezomib and Salinosporamide A (11-13). However, the cancer cells often develop resistance to these types of inhibitors as a result of point mutations in the proteasome. This is seldom the case with PROTAC (18). Rather, resistance linked to PROTAC is due to genomic changes which impact the expression of the corresponding E3 ligase complexes. To overcome this type of resistance, one could combine a Capzimin-PROTAC molecule with affinity to a VHL E3 ligase, with a Capzimin-PROTAC that has affinity to another E3 ligase, such as cereblon. If the cancer cell starts to knock out a gene that encodes for one of the E3 ligases, the compound would still work since the compound is utilizing another E3 ligase as well. However, if the cell would knock out the expression of all the E3 ligases, the cell would die by itself due to disruption of protein homeostasis.

Nevertheless, if the compound had not shown any disruption of the proteasome, it could indicate that the compound had lost its activity. This could have been an outcome of either problems related to the linker or a loss of molecular affinity to the proteasome’s subunit POH1. If any of this would have been the case, the first thing to test would be to try a longer linker, since the linker could have been too short. Thereafter change the alkylation of the warhead to either the oxygen of the amide, or one of the carbons between the azole moiety and the secondary amide if possible. If no desired effect occurred despite longer linker-length and change of alkylation, a compete change of warhead would probably be needed in order to obtain the desired effect.

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7. Conclusion

The concept of this study, which was to theoretically develop a new potential candidate in cancer therapeutic, has been successful. The developed Capzimin-PROTAC substance should be able to efficiently trigger apoptosis by inducing proteotoxic stress, caused by an

accumulation of miss-folded proteins in cancer cells. What now remains is to practically perform this project and confirm the molecule works as theorized.

According to the literature analysis of Capzimin-PROTAC, the molecule should be able to efficiently induce proteotoxic stress in the cancer cells, triggering apoptosis, while being relatively harmless to healthy cells. This could be because the cancer cell genomes are

scattered with genomic alterations that result genes coding for defected proteins which cannot obtain native form, that could form toxic aggregates within the cancer cells if not degraded (13).

If the Capzimin-PROTAC works, the next step would be to start testing the compound in vivo to see how it will affect living organisms, and to find out if Capzimin-PROTAC could be used as a potential candidate in cancer therapeutics.

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Acknowledgement

First and foremost, I would like to thank my supervisor RA. Pádraig D’Arcy for helping and guiding me throughout this project, without him this project would never have happened. I would also like to thank my co-supervisor Sr. PE Hamid Shirani for helping and guiding me through everything involving the synthesis and purification process. Thanks to my examiner Prof. Peter Nilsson for all the support and feedback throughout this project. Also, a special thanks to Prof. Stig Linder for introducing me to Pádraig, which made this project possible.

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References

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of solid malignancies. Cancer Chemotherapy and Pharmacology. 2018;81(2):227-43.

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Cancer, Resistance

to Targeted Anti-Cancer Therapeutics. Springer. 2014;Resistance of Targeted Anti-Cancer Therapeutics.

11. Accardi F, Toscani D, Bolzoni M, Dalla Palma B, Aversa F, Giuliani N. Mechanism of Action of Bortezomib and the New Proteasome Inhibitors on Myeloma Cells and the Bone Microenvironment: Impact on Myeloma-Induced Alterations of Bone Remodeling. 2015. 12. Gerardo Della S, Francesca A, Carmela M, Tiziana T, Valeria C, Claudia P. Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update. 2018.

13. Li J, Yakushi T, Parlati F, Mackinnon AL, Perez C, Ma Y, et al. Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11. Nature Chemical Biology. 2017;13(5):486-93.

14. Kumar V, Naumann M, Stein M. Computational Studies on the Inhibitor

Selectivity of Human JAMM Deubiquitinylases Rpn11 and CSN5. Frontiers in Chemistry. 2018;6:480.

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Degradation. Angewandte Chemie International Edition. 2016;55(6):1966-73.

16. Sun X, Rao Y. PROTACs as Potential Therapeutic Agents for Cancer Drug

Resistance. Biochemistry. 2020;59(3):240-9.

17. Sun B, Fiskus W, Qian Y, Rajapakshe K, Raina K, Coleman KG, et al. BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells. Leukemia. 2018;32(2):343-52.

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18. Zhang L, Shen Y, Riley-Gillis B, Vijay P. Acquired resistance to BET-ProTACS (proteolysis-targeting chimeras) caused by genomic alterations in core components of E3 ligase complexes. Molecular Cancer Therapeutics. 2019;18(7):1302-11.

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21. Miriam G, Chiara M, Scott JH, Andrea T, Alessio C. Cereblon versus VHL: Hijacking E3 Ligases Against Each Other Using PROTACs. Web server without geographic relation, Web server without geographic relation (org)2019.

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23. Anbu, Nagarjun, Jacob, Kalaiarasi, Dhakshinamoorthy. Acetylation of Alcohols, Amines, Phenols, Thiols under Catalyst and Solvent-Free Conditions. 2019.

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26. Zhang Y, Mantravadi PK, Jobbagy S, Bao W, Koh JT. Antagonizing the Androgen

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Appendix

Figure 12. GANTT-schedule which was established during the first week of the project, in which the time for different assignments of the project were planned.

Capzimin-PROTAC

Project leader Start date Total Left

Start date 2020-03-30 50 0

400 0 ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ######

Start date End date Days Hours Done

Delivery jobs Task Mon-Fri Days Hours MoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat SuMoTue We Thu FriSat Su

Literature study and project planning Prepare GANT schedule 2020-03-30 2020-04-01 3 GulU U U

Search for litterature 2020-04-01 2020-04-10 8 Grön G G G G G G G G G G

Read literature 2020-04-01 2020-04-10 8 Blå B B B B B B B B B B

Analyse literature 2020-04-02 2020-04-10 7 Röd R R R R R R R R R

Lila Chemical inventory Order Capzimin 2020-03-30 2020-03-31 2 SvartS S

Order PROTAC scaffolds 2020-03-30 2020-03-31 2 GulU U

Inventory of chemicals needed for synthesis 2020-03-30 2020-03-31 2 GrönG G Blå

Capzimin validation In vitro study of Capzimin on MelJuSo cells 2020-04-13 2020-04-15 3 Röd R R R

Lila

Synthesis Thiol acetylation of Capzimin 2020-04-16 2020-04-16 1 Svart S

N-alkylation of Capzimin-PROTAC 2020-04-17 2020-04-21 3 Gul U U U U U

Thiol deacetylation of Capzimin-PROTAC 2020-04-22 2020-04-22 1 Grön G

Blå

Purification HPLC purification 2020-04-23 2020-04-30 6 Röd R R R R R R R R

Lila

Confirm the compound Masspectrometry analysis 2020-04-30 2020-04-30 1 Svart S

NMR analysis 2020-04-30 2020-05-01 2 Gul U U

Grön

Capzimin-PROTAC validation In vitro study of Capzimin-PROTAC on MelJuSo cells 2020-05-02 2020-05-06 3 Blå B B B B B Röd

Report Write introduction Lila

Write materials and methods 2020-04-10 2020-05-13 24 Svart S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S

Write results and discussion 2020-04-10 2020-05-18 27 Gul U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U

Write abstract and conclusion 2020-04-10 2020-05-22 31 Grön G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G

Deliver report Blå

Röd

Presentation Make a powerpoint 2020-05-22 2020-05-26 3 Lila L L L L L

Prepare presentation 2020-05-22 2020-05-26 3 Svart S S S S S

Prepare opposition 2020-05-27 2020-05-30 3 Gul U U U U

Presentation 2020-06-02 2020-06-02 1 Grön G GANTT-schedule Project week 10 Project week 9 Project week 8 Project week 7

Left Start 2020-03-30 Start 2020-04-06 Start 2020-04-13 Start 2020-04-20 Start 2020-04-27 Start 2020-05-04 Start 2020-05-11 Start 2020-05-18 Start 2020-05-25 Start 2020-06-01

Project week 2

Project week 1 Project week 5 Project week 6

Work days Work hours

Inhibitory effect on the proteasome regulatory subunit, RPN11/POH1, with the use of Capzimin-PROTAC to trigger apoptosis in cancer cells