A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing

Abstract

2Abbreviations: BCA, bicinchoninic acid; CV, coefficient of variation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMCs, peripheral blood mononuclear cells; RPPA, reverse phase protein array; SSMD, strictly standardised mean difference; TNFR1, tumour necrosis factor receptor-1; TRAPS, TNF receptor associated periodic syndrome; WT, wild type. 3ABSTRACT: TNF Receptor Associated Periodic Syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro-inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti-inflammatories based on their ability to down-regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimised, high through-put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS-associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro-inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four ten-fold concentrations on cell lines expressing both wild-type (WT) TNFR1 and TRAPS-associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reversephase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y-TNFR1 transfectants towards the level seen in the WT-TNFR1 transfectants. Seven high-ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y-TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro-inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose-dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF-kB and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing.

Consistent Biomarkers and Related Pathogenesis Underlying Asthma Revealed by Systems Biology Approach

“…Grassi et al found that antibiotic lomefloxacin can treat acute exacerbations of chronic bronchitis [62]. Using the signalome screening method, Todd et al reported that lomefloxacin showed significant effects on multiple pro-inflammatory signaling pathways that are constitutively activated in the auto-inflammatory disease TNF receptor [63]. However, no study has been performed concerning the usage of lomefloxacin for asthma treatment.…”

TNF receptor signalling in autoinflammatory diseases

“…Using cell line models depicting TRAPS, as well as PBMC lysates from C33Y-TRAPS patients relative to controls, the RPPA approach was combined with the screening of existing pharmacologically active compounds to identify key compounds which modulate the TRAPS signalome. The fluoroquinolone antibiotic, lomefloxacin, as well as others from the same class of compounds was found to have the most significant effects on pro-inflammatory pathways in TRAPS (116). This approach would be useful for other autoinflammatory diseases to identify cellular pathways in the disease state as well as candidates for drug repurposing.…”

“…An interesting investigative approach has been developed for TRAPS, whereby new technologies, -'PPA), can be used to detect and quantify intracellular signalling molecules associated with a particular disease phenotype (116). Using cell line models depicting TRAPS, as well as PBMC lysates from C33Y-TRAPS patients relative to controls, the RPPA approach was combined with the screening of existing pharmacologically active compounds to identify key compounds which modulate the TRAPS signalome.…”

Periodic fever syndromes and the autoinflammatory diseases (AIDs)

“…The study of signaling pathways and its regulation (signaloma) through the application of reverse-phase protein microarray (RPPA) technology has been recently adopted for TRAPS [ 72 ]. So far, the focus of the treatment of these patients has been the blockage of proinflammatory cytokines instead of targeting intracellular proinflammatory signaling pathways.…”

“…The most ranked drug was the antibiotic lomefloxacin that significantly reduced the expression of several molecules across the Jak/Stat, MAPK, NF-kB and PI3K/AKT pathways. Although at a preliminary stage, this would be matter of future research for TRAPS and other AIDs [ 72 ].…”