Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins

Abstract

Toll-like receptors (TLRs) recognize various pathogen- and host tissue-derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

Triaryl Pyrazole Toll‐Like Receptor Signaling Inhibitors: Structure–Activity Relationships Governing Pan‐ and Selective Signaling Inhibitors

“…1,3,5-Triaryl pyrazoles: inhibition of TLR signaling vs. binding to the estrogen receptor From our initial screen of 4364 unique compounds, we identified the lead compound, methyl-piperidino-pyrazole (MPP),a basic sidec hain-containing pyrazole (BSC-pyrazole) that had been previously optimized as ah igh affinity estrogen receptora (ERa)s elective antagonist. [7][8] Therefore, our initial goal was to decouple ER binding affinity from activity against TLR signaling. In total, we investigated 90 pyrazoles.…”

“…In our initial published study,w ep robedt he requirement for and placementoft he BSCaroundt he pyrazole core,r evealing the necessity of the BSC. [7] Although there was some activity when the BSCw as appended to the N1 and C4 positions, we focused most of our early efforts on the derivatives with a BSC on the C5 ring, as found in our initial hit molecule MPP (compound 1), and eventually movingt od erivatives with a BSC on the C3 ring, as they exhibited less affinity for the ERs (Figure 2). [7] All of the pyrazoles we studied containedt hree aromatic rings at positionsN 1, C3, and C5, because early in our studies we found that when the phenylg roup appended to the N1 positionw as removed, potency decreased significantly (Table S1 in the Supporting Information).…”

“…As reported elsewhere, we have examined thisc ompound further in biological studies. [7] To avoid anyp romiscuous chemotypes that might faili n future studies, we subjected all of our compounds to three publicly availablei ns ilico filters to identify Pan-Assay INterference compoundS (PAINS). [10] All of the compounds passedt wo screens (http://www.cbligand.org/PAINS/ and http://zinc15.docking.org/patterns/home).…”

“…Recently,w eh avee mployed ad rug screening methodt hat relies on the inducible dimerization of the hierarchically acting proteins TIRAP,M yD88 and TRAF6 to identify TSIs. [7] Specific activation of these proteins allows assignment of compound activity to the level of signalt ransduction. Using this approach, we identified aclass of compounds, that is, triaryl pyrazoles appended with ab asic side chain, which interfere with TLR signaling at the level of TIR interaction.…”

“…Interestingly,i nitial structure-activity relationships (SAR) showedt hat alterations to the structure could lead to compounds with selective activity against certain TIR-domain interactions, and thus selectiveT LR inhibition. [7] Herein, we describe the expansion of the SAR for the development of TSI with general TLR inhibitory activity (Pan-TLR), and TSI with more selectiveTLR inhibitory profile.…”