# Deciphering the architecture of TLR signaling complexes

> **NIH NIH R01** · UNIVERSITY OF MARYLAND BALTIMORE · 2020 · $386,250

## Abstract

PROJECT SUMMARY
The Toll-like receptors (TLRs) activated by conserved microbial or endogenous “danger” molecules trigger
intracellular signaling cascades, leading to inflammatory response that is essential for recovery from infection
or sterile tissue damage. The excessive TLR response, however, is injurious to the host and can be fatal. The
intracellular TLR signaling is initiated by recruitment of TLR adapters to activated TLRs through multiple,
homotypic and heterotypic interactions of Toll-Interleukin-1 Receptor homology (TIR) domains present in all
TLRs and TLR adapters. Protein interactions mediated by TIR domains, although critical for triggering of
signaling, are not well understood due to several intrinsic properties that complicate the analysis of TIR
interactions. These complicating factors are (i) the absence of a common TIR-TIR binding motif, (ii) the
topological diversity of TIR interface positions, (iii) the ability of each TIR to establish multiple TIR-TIR
interactions, (iv) the weakness of any binary TIR-TIR interaction, and (v) the highly cooperative nature of
functional TIR-TIR interactions that lead to assembly of TLR signaling complexes.
The longstanding goal of this proposal is to advance the molecular understanding of TIR-TIR recognition
mechanisms and develop TLR targeted candidate therapeutics that abrogate signaling by blocking the
intracellular protein interactions that are consequent to TLR activation. New study will focus on systemic
analysis of TIR-TIR interactions that underlie TLR signaling and will decipher the atomic details for several TIR-
TIR interactions critical for signal transduction. Specifically, in Aim 1, we will develop novel inhibitors of TLR5,
TLR7, TLR8, and TLR9 and evaluate their potency in vivo. Aim 2 is to define the molecular mechanisms of
TIR antagonisms by decoy peptides. Studies of Sub-Aim 2.1 will employ the innovative fluorescence imaging
approach, which we developed recently, to determine the TIR domains specifically targeted by particular
inhibitors in cellular milieu. Sub-Aim 2.2 will employ fluorescence polarization assay (FP) and surface plasmon
resonance analysis (SPR) to determine affinity and kinetics of peptide-TIR interactions in controlled
environment using recombinant TIR domains. These cellular and in vitro studies will provide complementary
and quantitative benchmarks for rapid evaluation of future optimized antagonists. In Sub-Aim 2.3 we will
employ X-ray crystallography to decipher the atomic details of TIR-antagonist interactions. Aim 3 is to optimize
TIR-derived TLR antagonists using several rational and empirical approaches to increase their target-binding
affinity and biological efficacy. The translational aspect of proposed research is the preclinical development of
the in vivo potent TLR inhibitors, each with a defined mechanism of action, in several new specificity categories.
Importantly, antagonists developed in this project target the intracellular signal transduc...

## Key facts

- **NIH application ID:** 9933767
- **Project number:** 5R01AI082299-10
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Vladimir Y. Toshchakov
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $386,250
- **Award type:** 5
- **Project period:** 2010-05-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9933767

## Citation

> US National Institutes of Health, RePORTER application 9933767, Deciphering the architecture of TLR signaling complexes (5R01AI082299-10). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9933767. Licensed CC0.

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