Vito Ruggiero

Pivotal Advance: Inhibition of MyD88 dimerization and recruitment of IRAK1 and IRAK4 by a novel peptidomimetic compound.

MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL‐1R and several TLRs. Central to its function is the ability of its Toll/IL‐1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL‐1R‐associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido‐mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB‐loop of the MyD88‐tIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL‐1β‐mediated activation of NF‐κB transcriptional activity. After oral administration, ST2825 dose‐dependently inhibited IL‐1β‐induced production of IL‐6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG‐induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL‐1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.

Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88E52A) and 58 (MyD88Y58A) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88K95A) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27–72)), comprising the Glu52 and Tyr58 residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27–72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-κB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

Background: MyD88 is an adaptor protein that plays a crucial role in the immune response. Results: We identified residues within the TIR domain of MyD88 required for protein self-association. Conclusion: Interference with the surface of homodimerization identified by these residues inhibits MyD88 function. Significance: The inhibition of MyD88 activity could be a good therapeutic strategy for inflammatory and autoimmune diseases. Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu183, Ser244, and Arg288 impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151–189 and GFP-MyD88168–189), comprising the Glu183 residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.

LPS-induced serum TNF production and lethality in mice: effect of L-carnitine and some acyl-derivatives

The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.

Targeting the Toll-like Receptor/Interleukin 1 Receptor Pathway in Human Diseases: Rational Design of MyD88 Inhibitors

Toll-like receptor (TLR)/interleukin (IL) 1 receptor (IL-1R) play a fundamental role in the immune response. These receptors are distributed in various cellular compartments and recognize different components of pathogens. All TLR/IL-1Rs, with the exception of TLR3, interact with MyD88, an intracellular adapter protein that triggers a signaling cascade that culminates in the expression of inflammatory genes. Because aberrant activation of TLR/IL-1Rs can promote the onset of inflammatory or autoimmune diseases and malignancies, this pathway has attracted considerable interest as a potential therapeutic target. Given the central role of MyD88 in TLR/IL-1R signaling, we set out different strategies to develop drugs that can block its function. Structural and functional analysis of the MyD88 domains allowed us to identify crucial residues required for MyD88 homodimerization. Moreover, we developed small cell-permeable peptides and peptidomimetic agents that inhibit MyD88 homodimerization and function. Our results pave the way for the development of new therapeutic drugs for the inhibition of MyD88-dependent signaling.

Design, synthesis, and in vitro activity of peptidomimetic inhibitors of myeloid differentiation factor 88.

We describe the design and synthesis of a peptidomimetic library derived from the heptapeptide Ac-RDVLPGT-NH 2, belonging to the Toll/IL-1 receptor (TIR) domain of the adaptor protein MyD88 and effective in inhibiting its homodimerization. The ability of the peptidomimetics to inhibit protein-protein interaction was assessed by yeast 2-hybrid assay and further validated in a mammalian cell system by evaluating the inhibition of NF-kappaB activation, a transcription factor downstream of MyD88 signaling pathway that allows production of essential effector molecules for immune and inflammatory responses.

Action of lysosomotropic amines on spontaneous and interferon enhanced NK and CTL cytolysis

Cell-mediated cytotoxicities, such as natural killer (NK) and T-dependent cytotoxic (CTL) activities, are inhibited to the same extent by lysosomotropic amines, even when interferon is added to cultures to enhance lysis. It is postulated that the amines block steps common to spontaneous and IFN-enhanced NK and CTL lytic mechanisms.

In vivo and in vitro cytokine modulatory activity of newly synthesised 2-aminotetraline derivatives.

In the present study, the protective effect of newly synthesised 2-aminotetralines was investigated in murine models of toxic shock. A few derivatives protected mice against lethality induced by lipopolysaccharide from different bacterial strains and shock induced by staphylococcal enterotoxin B in mice sensitized by D-Galactosamine (D-Galn). Notably, one derivative, S(−)-2-amino-6-fluoro-7-methoxy-1,2,3,4 tetrahydronaphthalene hydrochloride (ST1214), was also effective when administered orally (30 mg kg−1) in a therapeutic regimen. ST1214 markedly inhibited the production of the proinflammatory cytokines, such as tumor necrosis factor-&agr; (TNF-&agr;), Interleukin-1&bgr; (IL-1&bgr;), Interleukin-12 (IL-12), interferon-&ggr; (IFN-&ggr;), as well as the inflammatory mediator nitric oxide (NO), and concurrently enhanced the production of the anti-inflammatory cytokine IL-10. Moreover, ST1214 dose-dependently reduced TNF-&agr; production by human peripheral blood mononuclear cells and promonocytic THP-1 cells in vitro. In the latter, ST1214 was found to inhibit lipopolysaccharide-induced TNF-&agr; secretion but not cytokine mRNA accumulation. These results suggest that the mechanism of action of ST1214 involves blockade of posttranscriptional events of TNF-&agr; production, apparently independent of p38 and ERK kinase activity. These results show beneficial effects of 2-aminotetralines in murine shock models and indicate a distinct counter-regulatory activity in down-regulating proinflammatory cytokine response, and upregulating IL-10. One derivative, i.e., ST1214, can be regarded as a lead compound in the development of novel drugs effective in anti-inflammatory strategies.

Efficacy of a Nanocochleate-Encapsulated 3,5-Diaryl-s-Triazole Derivative in a Murine Model of Graft-Versus-Host Disease

We have previously demonstrated that the compound 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole exerts immunosuppressive effects in several experimental models of autoimmunity. These results were achieved by subcutaneously administering ST1959 after dissolution in an oily vehicle, because of its poor water solubility. To circumvent this problem, we sought to determine whether nanocochleate technology could be successfully exploited to deliver ST1959 and protect mice undergoing lethal acute graft-versus-host disease (GVHD). Orally-administered encochleated ST1959 significantly protected animals from lethality, resulting in survival rates of 57% and 100% at doses of 2 and 10 mg/kg, respectively, whereas oral administration of 2 mg/kg ST1959, mixed with empty nanocochleates, was completely inactive. Increased survival was associated with diminished serum chemokine levels and donor CD8+ T cells in the spleen of ST1959-treated mice. Moreover, ST1959 treatment significantly counteracted GVHD-induced normocitic anemia by increasing hemoglobin, hematocrit, platelet, and red and white blood cell counts. Overall, these data show that orally-administered encochleated ST1959 significantly protects mice from GVHD.

In vitro activation of murine peritoneal exudate cells (PEC) and peritoneal macrophages by ST 789

ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and tumour growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (M phi) vs the L-M tumour cell line. We also found that ST 789 enhanced the rIFN-gamma-induced NO2- release from cultured PE M phi. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.