Browsing by Subject "Fragment-Based Screening"

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    Development of targeted degraders and allosteric ligands of NF-κB inducing kinase (NIK): synthesis, biochemical, and biophysical evaluation studies
    (2025-01) Anderson, Jared
    Kinases are commonly targeted in drug discovery campaigns due to their roles in many signaling pathways. Many kinase inhibitors work by occupying the orthosteric binding site and preventing ATP from being used as a substrate. All of the known efforts for targeting the disease relevant kinase NF-κB inducing kinase (NIK) have used a similar approach. This thesis will discuss our efforts in developing novel degraders of NIK by repurposing known orthosteric inhibitors and the development of the first known allosteric NIK ligands and characterization of their binding mode. Dysregulation of NIK has been implicated in a variety of cancers, including multiple myeloma, and immune disorders. Native regulation of NIK involves constant polyubiquitination and subsequent degradation of the protein by the 26s proteasome. Chapter 2 will discuss our efforts towards reinstating this degradation mechanism by using proteolysis targeting chimera (PROTAC) technology. Unfortunately, none of these compounds were able to degrade NIK in multiple myeloma cell lines. These findings serve as a case study into the difficulties of targeted-protein degradation. An allosteric-biased fragment-based screening effort leading to novel allosteric NIK ligands will be discussed in Chapter 3. The primary hit compound 3.1 displayed a Kd of 130 μM and did not show any competition with a non-hydrolysable ATP analogue (AMP-PNP) by NMR or SPR assays. Synthesis of analogues lead to compound 3.4 bearing a cyclopropyl ring and having a binding affinity of 40 μM. Efforts towards tighter binders was difficult due to the lack of structural information. Therefore, Chapter 4 discusses how the covalent probe compound 4.1 was used to adduct a cysteine near the allosteric binding site that was subsequently uncovered using LC-MS-MS peptide mapping experiments. It was uncovered that compound 4.1 was able to selectively adduct to C573 in this allosteric site on the catalytic domain of NIK. Finally, Appendix A focuses on early efforts towards the discovery of allosteric NIK inhibitors from computational screening efforts from our collaborators in the Amaro lab at the University of California – San Diego. While none of these compounds were able to inhibit NIK activity, this served as the starting point that lead to the findings of both Chapter 3 and Chapter 4.
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    Identification and Characterization of Potential BRDT(1) Inhibitors by Fragment-Based Screening Using Differential Screening Fluorimetry and Orthogonal Techniques
    (2019-05) Wisniewski, Andrea
    Almost 60 years after the advent of the female oral contraceptive pill, there are now a plethora of hormonal contraceptive options for women. Despite all of these available methods, 43% of pregnancies in the United states from 2011 to 2015 were unintended. Contraceptive methods for men are limited to condoms and vasectomies, neither of which offer complete protection from unintended pregnancies. Development of effective, reversible, nonhormonal male contraceptives would be a revolution in the area of contraception, complementing currently available methods and offering a new alternative to men. Epigenetics is a rising area of interest, concerned with the differential expression of genes caused by post-translational modification of proteins. Acetylation of lysine residues is regulated by several groups of proteins, including the bromodomains, responsible for recognizing the acetyl modifications and recruiting other effector molecules to impact chromatin remodeling, transcriptional control, and DNA repair. The bromodomain and extra terminal (BET) family of bromodomains has been implicated in many critical processes (such as cell cycle progression, transcriptional elongation) and disease states, including aggressive leukemias, obesity, acute inflammation, and NUT midline carcinoma. One BET protein, BRDT, is expressed exclusively in the testes and is responsible for regulating spermatogenesis. Knockout models of BRDT yield healthy, normal male offspring that are completely infertile. Studies using small molecule inhibitors of BET proteins have shown that BRDT inhibition causes a reversible infertility, making BRDT an attractive target for a non-hormonal male contraceptive. A fragment-based drug design strategy was employed to identify potential BRDT inhibitor leads. Differential scanning fluorimetry (DSF) was optimized for BRDT and used to screen a fragment library. An initial collection of 34 positive- and negative-shifting fragment hits were identified and confirmed from fresh powder by the DSF assay. Protein-observed 19F NMR (PrOF-NMR), fluorescence polarization (FP), amplified luminescent proximity homogenous assay screen (AlphaScreen), and x-ray crystallography were all part of the orthogonal screening cascade used to confirm BRDT binding. In total, 16 fragments were confirmed as ligands of BRDT, including a novel 1,5-benzodiazepinone scaffold. A number of these hits were pursued by our lab as lead molecules. In particular, and indanone scaffold was selected for further investigation. Analogs of the hit fragment were designed to develop structure-activity relationships of the chemotype; the indanone headgroup, linker composition, and substitution of a terminal phenyl group were studied. The analogs were tested by DSF, PrOF-NMR, FP, and AlphaScreen, with the most potent compounds submitted for crystallography experiments. The DSF assay developed for fragment screening was also used to test a number of other compounds for BRDT binding activity, including a pyridone scaffold identified by the DSF screen, several series based on kinase inhibitors shown to bind BET proteins, and a series based on a virtual high throughput screening hit.