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. 2019 May 24;10:1176.
doi: 10.3389/fimmu.2019.01176. eCollection 2019.

Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease

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Free PMC article

Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease

Aleksandra Antonczyk et al. Front Immunol. .
Free PMC article

Abstract

Interferon regulatory factors (IRFs) are a family of homologous proteins that regulate the transcription of interferons (IFNs) and IFN-induced gene expression. As such they are important modulating proteins in the Toll-like receptor (TLR) and IFN signaling pathways, which are vital elements of the innate immune system. IRFs have a multi-domain structure, with the N-terminal part acting as a DNA binding domain (DBD) that recognizes a DNA-binding motif similar to the IFN-stimulated response element (ISRE). The C-terminal part contains the IRF-association domain (IAD), with which they can self-associate, bind to IRF family members or interact with other transcription factors. This complex formation is crucial for DNA binding and the commencing of target-gene expression. IRFs bind DNA and exert their activating potential as homo or heterodimers with other IRFs. Moreover, they can form complexes (e.g., with Signal transducers and activators of transcription, STATs) and collaborate with other co-acting transcription factors such as Nuclear factor-κB (NF-κB) and PU.1. In time, more of these IRF co-activating mechanisms have been discovered, which may play a key role in the pathogenesis of many diseases, such as acute and chronic inflammation, autoimmune diseases, and cancer. Detailed knowledge of IRFs structure and activating mechanisms predisposes IRFs as potential targets for inhibition in therapeutic strategies connected to numerous immune system-originated diseases. Until now only indirect IRF modulation has been studied in terms of antiviral response regulation and cancer treatment, using mainly antisense oligonucleotides and siRNA knockdown strategies. However, none of these approaches so far entered clinical trials. Moreover, no direct IRF-inhibitory strategies have been reported. In this review, we summarize current knowledge of the different IRF-mediated transcriptional regulatory mechanisms and how they reflect the diverse functions of IRFs in homeostasis and in TLR and IFN signaling. Moreover, we present IRFs as promising inhibitory targets and propose a novel direct IRF-modulating strategy employing a pipeline approach that combines comparative in silico docking to the IRF-DBD with in vitro validation of IRF inhibition. We hypothesize that our methodology will enable the efficient identification of IRF-specific and pan-IRF inhibitors that can be used for the treatment of IRF-dependent disorders and malignancies.

Keywords: IRF; TLR; inflammation; inhibition; interferon; transcriptional regulation.

Figures

Figure 1
Figure 1
(A) Phylogenetic tree of the DNA-binding domain of IRF family proteins in vertebrates. Homologous protein sequences were searched using the NCBI BLAST server and aligned using ClustalW. Phylogenetic analyses were performed using the Neighbor-Joining method within the Mega 7.0 program. Data were analyzed using Poisson correction, and gaps were removed by pairwise deletion. The bootstrap values of the branches were obtained by testing the tree 10,000 times. Bootstrap values larger than 70% are shown next to the branches based on 10,000 replications. (B) Functional domains of human IRF proteins. DBD, DNA-binding domain; AD, activation domain; LK, linker region; IAD, IRF association domain type 1 (IAD1) or type 2 (IAD2); AR, auto-inhibitory region; P, phosphorylation site; 5W, five tryptophan repeats—“tryptophan cluster”; STAT-BD, STAT-binding domain. (C) DNA binding modes of IRFs. LINE—nucleotides involved in interaction with IRF—DBD; N, any nucleotide; R, purine; Y, pyrimidine. IRF3 homodimer, IRF3/IRF7 heterodimer and ISGF3 are bound to the consensus ISRE sequence with two ISRE half-sited “GAAA.” IRF4/PU.1 complex bind to the composite binding site, while NF-κB binds κB DNA element.
Figure 2
Figure 2
Schematic representation of IRFs in the TLR and IFN pathways. The three subfamilies of IFN signal through distinct receptors: IFN-II signals via a receptor which consist of two interferon gamma receptor (IFNγR)-1 and two IFNγR-2 chains (first left). IFN-I signal via the IFNAR receptor expressed in nearly all cell types and tissues (second left). IFN-III subfamily uses the interferon lambda receptor consisting of IL10R2 and IFNLR1 (third from left). While the signaling of IFNAR and IFN-lambda relies on phosphorylation of JAK1 and tyrosine kinase 2 TYK2, the interferon gamma receptor triggers STAT signaling via phosphorylation of JAK1 and JAK2. IFN-II specifically triggers STAT1 homodimer formation (most left), while IFN-I and IFN-III trigger ISGF3, (second from left), or STAT2/IRF9 in absence of ISGF3 (third of left). These complexes translocate to the nucleus to bind DNA on recognition sequences (GAS or ISRE, see bottom-left). The initial IFN stimulation leads to the early expression of ISGs and the transcription of IRF1/5/7/8/9 and STAT1, STAT2. The accumulation of newly synthesized transcription factors leads to a secondary, prolonged wave of ISG expression (bottom-left), contributing to antiviral activity and host defense. TLR4 signaling occurs through a MyD88-dependent (middle-right) and MyD88 independent (right) signaling cascade. In the MyD88 dependent signaling MyD88 recruits IRAK4 and IRAK1 leading to their phosphorylation, which in turn associates IRAK with TRAF6. TRAF6 activates TAK1, which in turn leads to phosphorylation of IKKα/β. The phosphorylation of these proteins results in their degradation and enables the translocation of NF-κB to the nucleus where it binds NF-κB binding sites (middle-right). The MyD88 independent signaling activates TRIF, which in turn via IKKε and TBK1 signaling phosphorylate IRF3 and IRF7 at their C-terminal serine/threonine cluster (right). Upon phosphorylation these IRFs translocate to the nucleus and bind ISRE or PRD sites on the DNA. TLR3 (most-right) also signals through TRAF and IRF3, or via the PI3K-Akt pathway. TLR7 and 9 signaling (right, down) goes via MyD88, TRAF, and IRF7, or via phosphorylation of IRF5. Down below in the figure the subsequent DNA recognition sites are listed, together with the general biological effects of gene activation, such as interferon production, prolonged ISG production and host defense. TLR3 and 4 signaling leads to upregulation of IFN beta, triggering the IFN-I pathway. This, together with the IRFs whose expression is upregulated by pathway activation (listed down below in the figure) provide cross-talk between the TLR and IFN pathways (pink arrow).
Figure 3
Figure 3
Schematic representation of the wide variety of IRF-mediated transcriptional regulatory mechanisms and their function. Independent of TLR and IFN stimulation, PU.1/IRF binding regulates leukocyte development and differentiation, while IRF1 homodimers, u-ISGF3 and u-STAT2/IRF9 maintain ISG expression in homeostasis (upper panel). After stimulation of the TLR and IFN pathways, DNA binding of IRF homo- and heterodimers (second panel from above), IRF/STAT complexes (middle panel), IRF/PU.1 complexes (second panel from bottom), or NF-κB/IRF (bottom panel) dependent mechanisms initiate or enhance ISG transcription. Potential IRF inhibition strategies. Red sticks indicate several points at which IRFs activity might be blocked by targeting: (1) ligand binding to the receptor e.g., TLR; (2) active components of the receptors, such as Jak2; (3) important IRF regulators and activators, such as NF-κB; (4) events such as phosphorylation, homo- and hetero- dimers formation; (5) critical mediators downstream of IRFs; (6) complex formation with other TF such as STATs; (7) IRFs ability to translocate to the nucleus. IRFs activity might be also modulated by preventing DNA binding, either directly (8) or by blocking interaction with binding partners (9) such as PU.1 or NF-κB. IRFs transcription (10) can be disrupted by RNAi and ncRNA mechanisms.
Figure 4
Figure 4
Binding conformations of pre-selected compounds from Natural Products ZINC database subset in different IRF DNA-binding domains. (A) Top-scored IRF1-specific binding compounds in apo-DBD of IRF1 and IRF8; (B) Top-scored IRF8-specific binding compounds in apo-DBD of IRF1 and IRF8; (C) Top-scored pan-IRF1/8 binding compounds in apo-DBD of IRF1 and IRF8. Graphical representation, that has been used, describes in detail binding mode of top-scored conformation of the inhibitor in the active pocket of apoIRF-DBD. dsDNA fragment of the respective holoIRF-DBD/IRE complexes superimposed on the apo-form implicates the position of selected target cavity for inhibitory compound. The best binding conformation of each potential inhibitor is shown in stick representation (carbon—gray; oxygen—red; nitrogen—blue; phosphorus—orange and hydrogen—white). IRF1 and IRF8 apo-DBDs are in the cartoon representation with visible secondary structure, multi-colored with amino acid side chains that interact with compounds shown as lines. dsDNA fragment of the respective IRF-holo-DBD/IRE complexes is shown in 60%-transparent cartoon representation and colored in pale-cyan with nucleobases colored in light-pink. Ligand docking results were obtained using Surflex-Dock 2.6 software.
Figure 5
Figure 5
Therapeutic, diagnostic, and prognostic space occupied by IRFs and IRF-mediated transcription regulatory mechanisms. IRF-containing complexes involved in transcriptional regulation, including IRF homodimers, STAT/IRF, PU.1/IRF, and NF-κB/IRF (central), can serve as diagnostic and therapeutic targets in different ways. The analysis of IRFs transcriptional mechanisms together with GWAS and characterization of gene expression signatures might provide disease diagnostic and prognostic markers (left side). Novel IRF-specific and pan-IRF inhibitors combined with an appropriate delivery system have the potential to be effective therapeutic agents for numerous immune related diseases (right side). We propose nanoparticles as promising tool for IRF inhibitory compound administration.

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