Anti-cancer drug discovery targeting the signal transducer and activator of transcription 3 (STAT3), has been active for more than a decade following a finding that aberrant activity of this transcription factor is involved in more than 70% of cancers. There are two main approaches to directly abating STAT3 activity, one is to inhibit STAT3 homo-dimersation by targeting the SH2 or transcriptional activation domains, and the other is to prevent STAT3 from interacting with DNA targeting its DNA-binding domain (DBD). The aims of this project are to discover novel STAT3 DBD inhibitors for further development and to develop a biophysical assay for the evaluation of STAT3 DBD inhibitors. The foundation to this project involved an observation that a small molecule fragment derived from the STAT3 dimerisation inhibitor BP-1 -102 occupied a site adjacent to the DBD when co-crystallised with a STAT3 (127-688) construct. Consequently, a series of small-molecule fragments were designed based upon the BP-1 -102 fragment. The compounds were synthesised, characterised and biologically evaluated in an attempt to understand the structure-activity relationships (SARs) associated with BP-1 -102. It was anticipated that by modifying the BP-1 -102 structure, the mode of action would be shifted from inhibition of STAT3 dimerisation to blocking STAT3 DNA-binding. However, the small-molecule fragments that were synthesised did not show significantly improved binding affinity compared to the BP-1 -102 fragment. Details of a STAT3 DBD inhibitor, inS3-54, were disclosed in 2014 and 2016, so the focus of the project shifted towards modifying this new inhibitor scaffold. A series of novel STAT3 DBD inhibitors were designed and docked in silico, then synthesised and biologically tested in vitro. Due to the lack of cell-free assays for evaluating STAT3 DBD inhibitors, a new orthogonal fluorescence polarisation (FP) assay was developed and validated to assess ligand binding. The preliminary results suggest some of the new structures are able to inhibit STAT3 DNA-binding in this FP assay after 24 hr incubation. Such compounds are potential lead structures for further development as STAT3 inhibitors.

The signal transducer and activator of transcription (STAT) family of proteins is a group of transcription factors that regulate gene expression related to cell cycle, cell survival, and immune response. Constitutively active STAT3 has been shown to promote the induction and survival of cancer. Given its critical role in both tumor onset and progression, STAT3 has emerged as an attractive target for small molecule therapeutics. The disparity between assays and assay conditions reported in publications for assessing STAT3 inhibitors makes it difficult to compare the potency of one compound to another and to determine a ranking of effective inhibitors. The purpose of this thesis was to identify small molecule inhibitors that target STAT3 directly, and prevent the binding of the DNA-binding domain (DBD) of STAT3 to DNA. This project approached this identification and evaluation of inhibitors through in silico library screening, development of recombinant assays to quantitatively evaluate and rank direct STAT3 inhibitors, and chemical synthesis of STAT3 inhibitors that target the DBD of STAT3. We successfully established a recombinant protein STAT3/DNA-binding ELISA to use in tandem with a recombinant fluorescence polarization assay to identify STAT3 small molecule inhibitors that target either the DBD or the SH2 dimerization domain. The use of the recombinant ELISA is an essential addition to the field of targeting STAT3 activity as it allows for rapid identification of small molecule inhibitors that target the DBD. While other methods have been proposed using recombinant STAT3, the recombinant ELISA is a modified commercially available colorimetric assay that can then be used to standardize the reporting of IC50s against the STAT3 DBD. Using our tandem screening method, we identified a novel STAT3 DBD inhibitor with an EC50 of 59 nM. This is significant in that other inhibitors in this class have reported high micromolar activity for STAT3 inhibition. The design and synthesis of a novel small molecule with nanomolar activity demonstrates the feasibility of using computational screening, in silico inhibitor design, and a tandem evaluation technique to successfully identify small molecules that target the DBD of STAT3.

Authored by the world's leading kinase experts, this is a comprehensive introduction to current knowledge and practice within this emerging field. Following an overview of the major players and pathways that define the kinome, the major part of this work is devoted to current strategies of kinome investigation and manipulation. As such, kinase engineering, peptide substrate engineering, co-substrate design and kinase inhibitor design are discussed in detail, and their potential applications in kinome analysis and kinome-based pharmacotherapy are shown. The result is a toolbox for every kinase researcher: By addressing and comparing current approaches to the study of kinase action, both novice and established researchers will benefit from the practical knowledge contained in this invaluable reference.

This volume, which includes contributions from leading scientists and clinicians in the field, provides definitive, state-of-the-art information on STAT inhibitors in a biological and clinical context. It gives an overview of the biology of the STAT family of transcription factors and their role in cancer etiology. Additionally, it describes the raft of therapeutic approaches being used to inhibit STATs in the context of various cancers, covering the full spectrum of therapeutic approaches to inhibiting STATs, and presenting emerging data from clinical trials.

ABSTRACT: The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided. Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency. We focused on improving the synthetic procedure using microwave-assisted heating, thereby reducing the reaction time and facilitating a combinatorial approach for the synthesis of these isoquinolinols. Isoquinolinols with different aryl groups (R) were synthesized to study the structure activity relationship. A series of isoquinolinols were synthesized by varying the position of the hydroxyl- and methoxy- groups in the isoquinoline core. Chapter Six focuses on the development of Shp2 inhibitors. Based on the hits obtained from HTS screening, compounds were re-synthesized and were evaluated for biological activity for Shp2. A series of compounds were synthesized, containing the isooxindole scaffold, based on the hematopoetic protein tyrosine phosphatase (HePTP) hits reported earlier in literature. Modifications were performed on the isooxindole core to study the SAR. Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.

This approach first entails the docking of fragments into multiple sites on a target engaging in protein-protein interactions. The fragment docking results are analyzed for enriched molecular architectures and are then used for the basis of combinatorial in silico libraries. A library is designed in one site and then the top scoring compounds are selected and used to extend into adjacent sites in an iterative docking and design process. This work describes the synthesis, biochemical, cell-based, and in vivo evaluations of inhibitors designed using this approach.