# Discovery of small molecules inhibiting Toll-like receptor-mediated inflammation

> **NIH NIH R01** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2021 · $515,751

## Abstract

ABSTRACT
Members of the Toll-like receptor (TLR) family recognize various pathogen- and host tissue-derived
molecules, and initiate immune responses physiologically via inflammatory processes. Exaggerated or
prolonged TLR activation, however, leads to pathological inflammation as observed in a large number of
etiologically diverse diseases, such as bacterial sepsis, autoimmune diseases and cancer. Despite the
apparent need, no effective small molecule inhibitors of the canonical TLR signaling pathway are available,
neither as probes to explore TLR biology nor as drugs to treat patients. The goal of this project is to identify
small molecule inhibitors of TLR-specific signaling pathways. One factor impeding drug development is the
signaling mechanism involved, i.e. a series of protein interactions that are inherently difficult for targeted drug
development. Specifically, TLRs signal via the hierarchically acting proteins TIRAP, MyD88, IRAKs and
TRAF6, the latter defining the border towards common, TLR-non-specific pathways. Thus, signaling events up-
stream of TRAF6 represent the desirable ‘drug target window’. We have developed a unique, cellular high
throughput screening (HTS) platform that retains the advantages of phenotypic screening, i.e. the testing of
complex responses in the natural environment of cells but, at the same time, mitigates the major disadvantage
by eliminating non-specific compounds. Key feature of this system are drug-inducible (GyrB-fused) forms of
TIRAP, MyD88 and TRAF6, whose responses reflect the ‘signaling level’ of compound activity. We have
validated this approach using the St. Jude bioactive compound library, which allowed in consecutive screening
steps facile elimination of non-specific hits (>99% of hits blocking TRAF6), and identification of one compound
as selective TLR inhibitor. Using this compound as tool molecule, we established a hit advancement algorithm
including SAR analysis, quantitative proteomics, protein interaction- and cellular thermal shift assays (CETSA),
collectively validating this approach as discovery tool for TLR antagonists. Here we propose to perform a HTS
of the St. Jude collection (~650,000 compounds) to identify a set of TLR inhibitory compounds with defined
chemotype. The primary screen (single concentration) based on TIRAP-GyrB identifies compounds that act at
any possible level of the TLR pathway. The secondary screen (dose-response) based on TIRAP-, MyD88-
and TRAF6-GyrB cells defines the signaling level. Compounds inhibiting TRAF6 will be discarded as TLR-non-
specific. Inhibition of TIRAP or MyD88 will define TLR-specific compounds. TLR-specific compounds will be
validated by physiological TLR stimulation and prioritized based on chemoinformatics analysis and
chemistry inspection. SAR analysis will be conducted to validate chemotype tractability based on
available analogs and exploratory chemistry, and mentioned biochemical algorithm will be used to define
mechanism of action and d...

## Key facts

- **NIH application ID:** 10201505
- **Project number:** 5R01AI139014-05
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** HANS HAECKER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $515,751
- **Award type:** 5
- **Project period:** 2018-06-14 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10201505

## Citation

> US National Institutes of Health, RePORTER application 10201505, Discovery of small molecules inhibiting Toll-like receptor-mediated inflammation (5R01AI139014-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10201505. Licensed CC0.

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