# Discovery of small molecule mutant SMAD4-PPI inducers

> **NIH NIH R37** · EMORY UNIVERSITY · 2022 · $357,994

## Abstract

SUMMARY
Tumor suppressor genes represent a major class of oncogenic “drivers” and offer robust window for therapeutic
intervention. However, direct targeting loss-of-function tumor suppressor genes remains challenging, because
that majority of tumor suppressors do not have enzymatic activity and exert their normal function through protein-
protein interaction (PPI). Noteworthy, a unique class of tumor suppressor mutations are missense mutations
encoding single amino acid substitutions that impair the normal PPI. These tumor suppressor mutations are
defined as “loss-of-interaction” mutation. We aim to directly target the “loss-of-interaction” tumor suppressor
mutations through discovery of small molecule PPI inducers to restore their anticancer functions. SMAD4 is such
a tumor suppressor with “loss-of-interaction” mutations in cancer that disrupt its normal PPI with SMAD3. Using
SMAD4 as a proof-of-concept study, we propose to utilize our newly developed TR-FRET SMAD4-SMAD3 PPI
screening platform to reveal novel small molecule mutant SMAD4-PPI inducer (MuSMADid) that can induce
the mutant SMAD4 PPI with SMAD3 and restore the pathway and cellular response to the tumor suppressive
TGF-b signaling. Preliminary studies showed that the SMAD4-SMAD3 TR-FRET assay is robust and scalable in
1536-well uHTS format and is sensitive to monitor the SMAD4-SMAD3 PPI dynamic at single amino acid
resolution. From a bioactive chemical library, Ro-31-8220, a bisindolylmaleimide derivative, was identified as
potential MuSMADid that induced the mutant SMAD4 PPI with SMAD3 and restored the responsiveness of
SMAD4 mutant colon cancer cells to the TGF-b anti-proliferation signaling. Identification of Ro-31-8220 as a
potential MuSMADid provides strong evidence for direct targeting “loss-of-interaction” SMAD4 mutations.
Together, this preliminary data supports our central premise that novel chemical probes can be discovered as
potential MuSMADid by leveraging the established uHTS TR-FRET assay to screen structurally diverse chemical
libraries. Based on the stages of discovery research, our proposal will focus on Aim 1 “Primary Screen
Implementation” to identify MuSMADid hits with new chemical scaffolds, followed by verification with orthogonal
PPI assays, and on Aim 2 “Functional Validation” to prioritize a list of validated novel small molecule anti-tumor
MuSMADid for future hit-to-lead optimization phase. We will use the uHTS TR-FRET platform to rapidly identify
primary hits and validated hits followed by characterization of their PPI induction and cellular activities in restoring
the TGF-b tumor suppressive signaling. Accomplishing the goals of the proposed study is anticipated to generate
a list of prioritized and confirmed small molecule MuSMADid compounds that show potent biochemical and
biological activities in inducing the mutant SMAD4-SMAD3 PPI and restoring the TGF-b anti-proliferation
signaling pathways. Top ranked anti-tumor MuSMADid with the strongest structura...

## Key facts

- **NIH application ID:** 10458066
- **Project number:** 5R37CA255459-02
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** Xiulei Mo
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $357,994
- **Award type:** 5
- **Project period:** 2021-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458066

## Citation

> US National Institutes of Health, RePORTER application 10458066, Discovery of small molecule mutant SMAD4-PPI inducers (5R37CA255459-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10458066. Licensed CC0.

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