Development of Small Molecules Towards Novel Protein Targets: From Selective Androgen Receptor Modulators to Affinity Probes for a Novel Immunological Target

• Author / Creator

  Lin, Chih-Hsuan

• Cancer is a group of diseases that result from the uncontrollable growth of cells, which eventually can metastasize to other parts of the body, resulting in spread of the cancer. Chemotherapy, radiotherapy, surgery, and immunotherapy are the main pillars of cancer therapeutics. In recent years, immunotherapy achieved a revolutionary breakthrough with the identification of immune checkpoint proteins and their inhibitors, which has led to the development of several antibody treatments.
  Target deconvolution remains an important task in studying pathologically and clinically significant processes, which pave the way to the development of a drug candidate. Protein identification requires the involvement of medicinal, biological, and organic chemistry. Synthesis of novel small molecules plays an essential role in the process. Small molecules with diverse structures and functionalities are used to probe an active site, such as a ligand binding pocket or protein–protein interaction. Over the years, scientists have developed various techniques and cutting-edge chemistry towards protein identification.
  Barakat and coworkers discovered a novel hit compound with promising immunological activities by being able to promote the production of T cells and cytokine interleukin 2 (IL-2). The target deconvolution revealed that the mechanism of the compound triggering the downstream immune response involves neither of the two common pathways, the programmed cell death protein 1 (PD-1/PD-L1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4/B7-1). Instead, the compound binds to an unknown protein and triggers the downstream immune response. In this project, we executed the structural modifications of the lead compound with different functionalities, which were applied in different strategies in order to identify the unknown protein and probe its mode of action (MOA).
  Chapter 2 outlines the molecular design and preparation of the first- and second- generation probe molecules. The first-generation probe molecule was synthesized with a biotin functionality, which was used towards the streptavidin–biotin affinity purification. The second-generation probe molecules were proposed and synthesized for a proof-of-concept photoaffinity labelling experiment. Benzophenone was incorporated as the photo-crosslinker; the synthesized probe molecules were treated to bovine serum albumin (BSA) for photoaffinity labelling.
  Chapter 3 addresses the utilization of third-generation trifunctional probe molecules and a fourth-generation fluorescent probe molecule. Two distinct trifunctional molecules were prepared by incorporating a photo-crosslinker and a bio-orthogonal handle, affording the benzophenone (BP) and the trifluoromethyl phenyl diazirine (TPD) probe. Later, the TPD probe found applications in a successful BSA photoaffinity labelling experiment by increasing the molecular weight of the protein under UV irradiation. Also, it was used in the synthesis of the fifth-generation probe molecule, in which a biotin handle was installed prior to the cell-based assay. On the other hand, a fluorescent molecule containing a BODIPY fluorophore was prepared and used in multiple microscopic experiments, including real-time high content imaging and confocal microscopy.
  In Chapter 4, a different project aiming at the preparation of a selective androgen receptor modulator (SARMs)-based compounds will be addressed. The compound library consists of SARMs with distinct chemical structures, including aryl propionamide, quinolinone, N-substituted tropine/tropinone, and hydantoin. These compound classes have previously shown to possess performance-enhancing properties, such as increasing body mass and muscle strength; consequently, they have been reported to be abused in the athlete community. These properties also give them the potential to treat muscle wasting conditions. The synthesized analogs were proposed to be analyzed by two-dimensional gas chromatography (GC × GC) by studying their physicochemical properties and retention behaviour, which would be used toward the development of a machine-learning algorithm in order to flag suspicious performance-enhancing drugs (PEDs) in clinical samples. The compounds await for the opportunity to be tested in a cachexia model when an appropriate collaborator is secured.
  Chapter 5 provides a set of general conclusions for the projects that were mentioned in this thesis. Also, it illustrates possible future directions for the immunological chemistry project and reiterates objectives for the SARMs project.

• Subjects / Keywords

  • Small molecules--Immunology

• Graduation date

  Spring 2023

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-z78c-q997

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.