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COVALENT FRAGMENT SCREENING AND OPTIMIZATION IDENTIFIES NOVEL SCAFFOLDS FOR THE DEVELOPMENT OF INHIBITORS FOR DEUBIQUITINATING ENZYMES

thesis
posted on 2024-04-25, 23:48 authored by Ryan Dean ImhoffRyan Dean Imhoff

Humans encode approximately 100 deubiquitinating enzymes (DUBs) which are categorized into seven distinct subfamilies. Each family and representative has a unique expression, function and binding topology to ubiquitin. In addition to human DUBs, parasites, bacteria, and viruses contain DUBs with unique structures and functions. One subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCH), has four structurally similar human members and two known members within the Plasmodium falciparum genome. Human UCHL1 and UCHL3 are genetically validated targets in oncology and Plasmodium falciparum UCHL3 (PfUCHL3) is a prospective target for antimalarial drug development. Though these three UCH enzymes have potential as therapeutic targets, there is a significant lack of quality small molecule chemical probes to understand the underlying biology and function of the enzymes, pharmacologically validate the targets, and serve as leads for drug development in oncology and malaria.

The UCH enzymes are cysteine proteases, which our lab has leveraged to identify novel covalent small molecule inhibitors of each enzyme. The workflow for each hit identification and optimization campaign is similar. Covalent fragment screening of electrophilic small molecule libraries against the respective recombinant enzyme was performed to identify chemical space around each enzyme. Subsequent medicinal chemistry hit-to-lead optimization was undertaken to improve upon the moderately potent hit molecules to provide improved small molecule inhibitors for each enzyme. Inhibitor identification and optimization for UCHL1 is described in Chapter 2, revealing a novel scaffold and a cocrystal structure reveals a unique binding pose for UCHL1 inhibitors. These molecules were also characterized in breast cancer cells to validate UCHL1 as a therapeutic target in breast cancer. First-in-class covalent inhibitors of UCHL3 are described in Chapter 3. Medicinal chemistry optimization along with a cocrystal structure of the initial hit has revealed the molecular interactions of this novel inhibitory scaffold. PfUCHL3 inhibitor identification is described in Chapter 4. Characterization of these molecules against Plasmodium falciparum is described along with a comparison to a recently identified reversible PfUCHL3 inhibitor. Finally, conclusions and future directions toward the development of potent, drug-like inhibitors of each UCH enzyme is presented in Chapter 5.

Funding

NIH T32 GM125620

PIDD Programatic Grant

History

Degree Type

  • Doctor of Philosophy

Department

  • Medicinal Chemistry and Molecular Pharmacology

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Daniel Flaherty

Additional Committee Member 2

Vincent Jo Davisson

Additional Committee Member 3

Chittaranjan Das

Additional Committee Member 4

Michael Wendt

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