PD-L1 proteinine yönelik görüntüleme ajanı geliştirilmesi ve sentezi
Development and synthesis of moleculer imaging agent for protein PD-L1
Yazar: CEYDA KÖSE
Danışman: DR. ÖĞR. ÜYESİ ONUR ALPTÜRK ; DR. ÖZGÜR YILMAZ
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2020
Konu: Kimya = Chemistry

Özet:
Günümüzde pek çok hastalık gibi olan kanseri saptamak için en basit yaklaşım modülü moleküler görüntülemedir. Bu kapsamda, bir sinyal noktası hedeflenir ve bu nokta hastalığa özgü biyobelirteçlerden biri olmalıdır. Klinik testlerin tanı ve nicel sonuçları, görüntüleme ajanları tarafından sağlanan moleküler görüntü ile ele alınabilir. "Kimyasal Problar" olarak da bilinen bu ajanların biyolojik süreçleri in vivo sistemlerde görselleştirmeyi, karakterize etmeyi ve ölçmeyi sağladığı günümüz koşullarında belirlenmiştir. Bu nedenle ajanlar, seçici bağlanma substratları ile moleküler çekicilik sağlayan moleküller olan reseptör moleküllerine bağlanır.
Bu tez çalışması kansere karşı görüntüleme problarında kullanılabilecek yeni taşıyıcı bileşikler geliştirmeyi hedefler. Buradaki çalışma görüntüleme problarının sinyal kısımlarını, karşılık gelen dokuya taşıyan hedefleme moleküllerinin tasarımı ve hazırlanmasıdır.
Bu problar ile kanser mikro-çevresindeki proteinlerin görüntülenmesi hedeflenmektedir. Kanser dokularının PD-L1 proteninin, hastalık ile birlikte varlığı yoğun olarak artan bileşik, özellikle seçilip buna uygun moleküller geliştirilmesi bu çalışmanın özgün değeridir. Bunu yaparken GVIA peptidinin farmakokoru baz alınarak peptidomimetikler geliştirilmiştir. GVIA proteinin seçilmesinin nedeni, bu proteine uygun peptidomimetiklerin seçilen kanser hücresine özgü biyobelirtece uyum sağladığının tarama çalışmalarında gözlenmesidir. PD-L1 proteini bağışıklık sistemi tarafından hedeflenerek yok edilmeye yarayan bir proteindir. İmmün sistem bunu T hücreleri ile gerçekleştirir. T hücresi (T lenfosit), immün sistemdeki bir beyaz kan hücresi türüdür ve immünolojik süreçlerde önemli rol oynayan yardımcı, katil, düzenleyici hücre tiplerini içerir ve PD-1 bu hücrelerin üzerindedir. Kanser hücreleri PD-1/PD-L1 kompleksi üreterek T-hücrelerinde programlanmış hücre ölümünü mekanizmasını işlevsiz bırakır. PD-1 ve PD-L1 proteinlerinin birleşmesi T hücresinin anti-tümör aktivitesini inhibe eder. Bu kompleks yapının bu tez çalışmasındaki görevi ise artan PD-L1 konsantrasyonundan faydalanarak hastalık tayinine yardımcı moleküller geliştirmektir. Bu proteinine uygun peptidomimetik seçimi ise farmakofor tespiti ile mümkündür.  
PD-L1 proteini ile uyumlu peptidomimetik yapılarının sentezlenmesinin ardından gelecek çalışmalar için istenilen yapı oluşturulmuş olur. Peptidomimetiklerin tasarımında DOTA ligandı ile birleştirilebilir olması her aşamada önceliklerden biri olarak göz önünde bulundurulmuştur. DOTA ligandı ile 68Ga radyonüklid atomunun oluşturduğu kompleks yapı, hedef proteinin görüntüleme ajanı ile birleşiminin ardından PET-BT cihazı tarafından kanser hücrelerinin tanımlanmasını sağlar. Böylece hedeflenen amaç olan kanser görüntülenmesi başarılmış olur. Bu tez kapsamında ise ilk adım olarak peptidomimetik sentezi tamamlanmıştır.

Abstract:
Like many diseases, the most straight forward approach to detect cancer is molecular imaging wherein a signal stems from biomarkers unique to this disease. Of course, diagnosis and quantitative outcome of clinical tests can also be handled with molecular images which are provided by imaging agents. It is worth noting that these agents, also known as "chemical probes", ensure to visualize, characterize, and measure biological process in vivo systems. Hence, agents are bind to receptor molecules that provide molecular attraction with the selective binding substrates. 
The mainstream idea of researches that is applied on cancer, have different aims, such as diagnosis or treatment. Most of researches from them are about to target a receptor molecule or a protein unique to tumour cells. Herein, the overall purpose of this project is to design and synthesize new molecules exhibiting the potential of binding to such receptor molecules. Thus, organic compounds that hold the potential of imaging cancerous cells in a selective manner, are intended to be developed. By achieving this goal, we also believe that the development of novel imaging agents would ultimately eliminate the need for biopsy. To baseline of this thesis, molecules target cancer cells have been developed to be used in the design of imaging agents.
The main role of targeting process belongs to biomarkers, that is used as a chemical probe. These probes have an important role in development progress in drug systems and biomedical researches for many diseases like cancer. Hence, these agents become a bridge between biological processes and clinical outcomes. The vital finding ways for the treatment of targeting disease depend on these outcomes. In recent days, the results and images of biomarkers are accepted as a confidential outcome on the basis of clinical researches and practices. 
Biomarkers provide objective and quantifiable results of biological systems in health care processes. The significant benefit of a biomarkers is that all types of biomarkers ensure the measurable image of the physical state of the body with the time is that undetectable effects are found on the health of patients. As an imaging technique, biomarkers provide the unique ability to interrogate the exact disease and focus such as inflammation or tumour. The word of biomarker is come from the biological marker and it refers to medical sign to analyse synthons of diseases. 
The importance of cancer cell imaging lies within the fact that it eliminates the need for biopsy. A biopsy is the eradication of a certain tissue for analysis. Since it is an invasive procedure, a biopsy could lead to side-effects such as infection or blood collection under the skin and can create further contamination of the diseased area by spreading of the malignant cells. Biopsy also is a procedure that can lead to human error since it is not yet perfected. This could create inaccurate results. 
For the enhancement of the detection of diseases in early stages, in vivo molecular imaging is an important tool. The benefits of molecular imaging are that it aids the physicians to; identify the degree of the disease, select the patient-specific therapeutic treatment, apply a targeted therapy and calculate the molecular-specific effect of treatment. The researches usually aim diagnosis or treatment but the focal point is to target a receptor molecule or a protein that is exclusive to the tumor cells. Imaging Programmed Death Ligand-1 Protein (PD-L1) as a biomarker can be give as an example of this. 
Molecular imaging is a medical imaging model that provides pictures of the inside of the body at a molecular and cellular level. Molecular imaging focuses on locating and monitoring the progression of the disease. With the help of molecular imaging, the chemical and biological processes can be observed. Molecular imaging identifies the disease, locates its exact position at the body, even before the symptoms occur. With the help of biomarkers or organic molecules that have biomarkers, molecular imaging is specific to the destination. 
The diagnosis of many other diseases, including cancer, is determined by imaging. To do so, some peptidomimetic with the consideration of pharmacore of GVIA peptide are developed for targeting for specific biomarker. To define briefly, a peptidomimetic is a small molecule that has a chain designed to substitute a certain portion of peptide. They comprise of modification of an existing peptide, or by designing similar systems that have pharmacore of peptides. This choice stems from the observation that peptide bonds are prone to hydrolysis in the human body.
In order to find the suitable peptidomimetic for the protein specific to the target tissue, the effect map of the protein according to the chemical and physical interaction is first assigned. Relevant ligands are identified, then a three-dimensional protein scheme is made. Radius is for complementarity between ligands and appropriate value, and the angle between atoms is redesigned and placed in the diagram. A few high-impact needs, molecules are sampled in multiple rotameters. All these rearrangements are the main facts of docking systems. The method requires the receptor on the targeted tissue and thus shows tissue selectivity. 
The selectivity is necessary information to be provided for molecular targeting is obtained by proper targeting of the targeting molecule and biomarker. The formation of the image is provided by chemical probes on PD-L1 Protein. 
With this paradigm, we aimed to monitor cancer cells through PD-1. Programmed cell death protein 1, also known as PD-1 is a cell surface receptor that has a significant role in the downregulation of the immune system. The PD-1 provides health-promoting to facilitate self-tolerance of cancer cells by suppressing T cell inflammatory activity. A T cell (T lymphocyte) is a type of white blood cell in the immune system and includes helper, killer, regulatory type of cells that have an important role in immunologic processes. PD-1 is responsible for immune system protection of own cells against autoimmunity through a mechanism of promoting programmed cell death in T-cells by producing PD-1/PD-L1 complex. 
Researches of PD-L1 on tumour cells inhibits the anti-tumour activity of T cells through the engagement of PD-1 and T cells. Through complex protein mechanisms, PD-1 is an inhibitor of the immune system. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. As a basic definition of mechanism, the interaction between PD-L1 on the tumour cells and PD-1 on a T-cell reduces signals which stem from T-cell function, to provide the immune system for killing the tumour cells. The result of this mechanism the disease which is cancer in this project is avoided. Hence, several PD-1 and PD-L1 inhibitors are being trailed within the clinic for use in advanced diseases like cancer.
PD-L1 protein is present in very high rates in the tumor microenvironment and immune-derived cell structures (APCs), including different cancer cells. The mechanism between PD-L1 / T-cells briefly begins when the cancer cell is perceived as a foreign cell and is under attack to be killed by the T-cells. As a result of the interaction of PD-L1 protein on tumor cells with PD-1 on T-cells, regulatory T-cells perceive the cancerous cell as a normal cell and protect some tumor cells from immune attack and decrease anti-cancer function. The PD-L1 / PD-1 protein complex is a structure that is developed by the cancer cell to protect itself and is used to rule out attacks of T-cells.
In this project, we aim to develop some compounds which could be ultimately utilized as imaging biomarker which is PD-L1, against cancer. Therein, our contribution is the design and preparation of the targeting molecules, which carry the signalling moieties to the corresponding tissues. In doing so, we have developed some peptidomimetic with the consideration of pharmacore of GVIA peptide. With this work, we have reasoned that the development of these molecules will pave the way to imaging cancer in a non-invasive way, unlike biopsy. 
The targeting molecules are designed as imaging agent with the DOTA complex molecule and 68Ga radionuclide atom. Via this design, the molecules aim to become imaging agents for PET technique. With this work, we have reasoned that the development of these molecules will pave the way to imaging cancer in a non-invasive way, unlike biopsy. In this way, the targeted cancer cells are aimed imaging complete successfully. As a fundamental aim in this thesis, peptidomimetic synthesis has been completed as a first step of all imaging technique.