Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemistry

Major Professor

Haitao Mark Ji, Ph.D.

Committee Member

Derek Duckett, Ph.D.

Committee Member

Ernst Schonbrunn, Ph.D.

Committee Member

James Leahy, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Keywords

Wnt/β-catenin Signaling, Structure-Based Drug Design, Pharmacophore-Based Drug Design, Transcription Factor, Undruggable Target

Abstract

The Wnt/β-catenin signaling pathway is associated with activating genes that support cell proliferation, differentiation, and survival. Excessive activation of Wnt/β‐catenin signaling has been associated with development of several types of cancer and activation of stem-like cancer cells. Direct targeting of β‐catenin, the central hub in this signaling pathway, is a promising strategy to suppress the hyperactive β‐catenin signaling but has proven to be highlychallenging. Substantial efforts have been made to discover compounds that bind with β‐catenin, block β‐catenin mediated protein–protein interactions (PPIs) and suppress β‐catenin signaling. β‐catenin/BCL9 PPI has a smaller contacting area (1450 Å2) and a moderate binding affinity (KD = 470 nM), representing a promising binding site for inhibitor design to suppress Wnt/β‐catenin signaling. In this dissertation, two projects to design and synthesize new inhibitors for β-catenin/BCL9 protein-protein interaction are presented.

Structure-based design and optimization were performed to develop small-molecule β-catenin/B-cell lymphoma 9 (BCL9) PPI inhibitors and improve their inhibitory activities. Compound ZL3138 with a novel 1-benzoyl 4-phenoxypiperidine scaffold was discovered to disrupt the β-catenin/BCL9 PPI with a Ki of 0.96 μM in AlphaScreen competitive inhibition assays and displayed good selectivity for β-catenin/BCL9 over β-catenin/E-cadherin PPIs. The binding mode of new inhibitors was characterized by structure−activity relationship and site-directed mutagenesis studies. Protein pull-down assays indicate that this series of compounds directly binds with β-catenin. Cellular target engagement and co-immunoprecipitation experiments demonstrate that ZL3138 binds with β-catenin and disrupts the β-catenin/BCL9 interaction without affecting the β-catenin/E-cadherin PPI in living cells. Further cell-based studies show that ZL3138 selectively suppresses transactivation of Wnt/β-catenin signaling, regulates transcription and expression of Wnt target genes, and inhibits growth of Wnt/β-catenin dependent cancer cells.

Although ZL3138 displays potent activity in cell-based study, its less desirable chemical structure and physicochemical properties post some challenges for further optimization. In the absence of a co-crystal structure of a small-molecule β-catenin/BCL9 PPI inhibitor with β-catenin, we conducted pharmacophore elucidation studies of existing β-catenin/BCL9 PPI inhibitors and performed pharmacophore-driven optimization to discover a new pharmacophore for the disruption of β-catenin/BCL9 interaction. The simple incorporation of this new pharmacophore resulted in new inhibitors that show a dramatic 15-fold increase in β-catenin/BCL9 PPI disruption. Utilizing our structure-activity relationship (SAR) findings, we have effectively pinpointed three essential pharmacophores and synthesized multiple compounds exhibiting nanomolar activity. These compounds not only serve as valuable tools for chemical biology research but also open up a promising avenue for future inhibitor optimization.

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