Targeting Myeloid Differentiation Primary Response Protein 88 (MyD88) and Galectin-3 to Develop Broad-Spectrum Host-Mediated Therapeutics against SARS-CoV-2.

Nearly six million people worldwide have died from the coronavirus disease (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although COVID-19 vaccines are largely successful in reducing the severity of the disease and deaths, the decline in vaccine-induced immunity over time and the continuing emergence of new viral variants or mutations underscore the need for an alternative strategy for developing broad-spectrum host-mediated therapeutics against SARS-CoV-2. A key feature of severe COVID-19 is dysregulated innate immune signaling, culminating in a high expression of numerous pro-inflammatory cytokines and chemokines and a lack of antiviral interferons (IFNs), particularly type I (alpha and beta) and type III (lambda). As a natural host defense, the myeloid differentiation primary response protein, MyD88, plays pivotal roles in innate and acquired immune responses via the signal transduction pathways of Toll-like receptors (TLRs), a type of pathogen recognition receptors (PRRs). However, recent studies have highlighted that infection with viruses upregulates MyD88 expression and impairs the host antiviral response by negatively regulating type I IFN. Galectin-3 (Gal3), another key player in viral infections, has been shown to modulate the host immune response by regulating viral entry and activating TLRs, the NLRP3 inflammasome, and NF-κB, resulting in the release of pro-inflammatory cytokines and contributing to the overall inflammatory response, the so-called "cytokine storm". These studies suggest that the specific inhibition of MyD88 and Gal3 could be a promising therapy for COVID-19. This review presents future directions for MyD88- and Gal3-targeted antiviral drug discovery, highlighting the potential to restore host immunity in SARS-CoV-2 infections.

Cells Stimulated with More Than One Toll-Like Receptor-Ligand in the Presence of a MyD88 Inhibitor Augmented Interferon-ß via MyD88-Independent Signaling Pathway.

Host exposure to pathogens engage multiple pathogen recognition receptors (PRRs) including toll-like receptors (TLRs); recruit intracellular signaling adaptor proteins primarily myeloid differentiation primary response protein 88 (MyD88) for activating downstream signaling cascades, which culminate in the production of type I interferons (IFNs), proinflammatory cytokines, and chemokines; and impede pathogen replication and dissemination. However, recent studies highlight that absence of MyD88 increased antiviral type I IFN induction, and MyD88-/- mice showed a higher survival rate compared with the low survival rate of the MyD88+/+ mice, implicating MyD88 limits antiviral type I IFN response. As a single infectious agent may harbor multiple PRR agonists, which trigger different sets of TLR-initiated immune signaling, we examined whether MyD88 inhibition during stimulation of cells with more than one TLR-ligand would augment type I IFN. We stimulated human U87- and TLR3-transfected HEK293-TLR7 cells with TLR-ligands, such as lipopolysaccharides (LPS) (TLR4-ligand) plus poly I:C (TLR3-ligand) or imiquimod (R837, TLR7-ligand) plus poly I:C, in the presence of compound 4210, a previously reported MyD88 inhibitor, and measured IFN-ß response using an enzyme-linked immunosorbent assay. Our results showed that when U87- or TLR3-transfected HEK293-TLR7 cells were stimulated with TLR-ligands, such as poly I:C plus LPS or poly I:C plus R837, IFN-ß production was significantly increased with MyD88 inhibition in a dose-dependent manner. Collectively, these results indicate that during more than one TLR-ligand-induced immune signaling event, impairment of antiviral type I IFN response was restored by inhibition of MyD88 through MyD88-independent pathway of type I IFN signaling, thus, offer a MyD88-targeted approach for type I IFN induction.

MyD88 and beyond: a perspective on MyD88-targeted therapeutic approach for modulation of host immunity.

The continuous emergence of infectious pathogens along with antimicrobial resistance creates a need for an alternative approach to treat infectious diseases. Targeting host factor(s) which are critically involved in immune signaling pathways for modulation of host immunity offers to treat a broad range of infectious diseases. Upon pathogen-associated ligands binding to the Toll-like/ IL-1R family, and other cellular receptors, followed by recruitment of intracellular signaling adaptor proteins, primarily MyD88, trigger the innate immune responses. But activation of host innate immunity strongly depends on the correct function of MyD88 which is tightly regulated. Dysregulation of MyD88 may cause an imbalance that culminates to a wide range of inflammation-associated syndromes and diseases. Furthermore, recent reports also describe that MyD88 upregulation with many viral infections is linked to decreased antiviral type I IFN response, and MyD88-deficient mice showed an increase in survivability. These reports suggest that MyD88 is also negatively involved via MyD88-independent pathways of immune signaling for antiviral type I IFN response. Because of its expanding role in controlling host immune signaling pathways, MyD88 has been recognized as a potential drug target in a broader drug discovery paradigm. Targeting BB-loop of MyD88, small molecule inhibitors were designed by structure-based approach which by blocking TIR-TIR domain homo-dimerization have shown promising therapeutic efficacy in attenuating MyD88-mediated inflammatory impact, and increased antiviral type I IFN response in experimental mouse model of diseases. In this review, we highlight the reports on MyD88-linked immune response and MyD88-targeted therapeutic approach with underlying mechanisms for controlling inflammation and antiviral type I IFN response. HIGHLIGHTS: • Host innate immunity is activated upon PAMPs binding to PRRs followed by immune signaling through TIR domain-containing adaptor proteins mainly MyD88. • Structure-based approach led to develop small-molecule inhibitors which block TIR domain homodimerization of MyD88 and showed therapeutic efficacy in limiting severe inflammation-associated impact in mice. • Therapeutic intervention of MyD88 also showed an increase in antiviral effect with strong type I IFN signaling linked to increased phosphorylation of IRFs via MyD88-independent pathway. • MyD88 inhibitors might be potentially useful as a small-molecule therapeutics for modulation of host immunity against inflammatory diseases and antiviral therapy. • However, prior clinical use of more in-depth efforts should be focused for suitability of the approach in deploying to complex diseases including COPD and COVID-19 in limiting inflammation-associated syndrome to infection.

An increase in p62/NBR1 levels in melioidosis patients of Sri Lanka exhibit a characteristic of potential host biomarker.

Introduction. Melioidosis, caused by Burkholderia pseudomallei, in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei, host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim. We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology. We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results. Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions. ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.

A small molecule inhibitor of MyD88 exhibits broad spectrum antiviral activity by up regulation of type I interferon.

Recent studies highlight that infection with Coxsackievirus B3, Venezuelan equine encephalitis virus (VEEV), Marburg virus, or stimulation using poly I:C (dsRNA), upregulates the signaling adaptor protein MyD88 and impairs the host antiviral type I interferon (IFN) responses. In contrast, MyD88 deficiency (MyD88-/-) increases the type I IFN and survivability of mice implying that MyD88 up regulation limits the type I IFN response. Reasoning that MyD88 inhibition in a virus-like manner may increase type I IFN responses, our studies revealed lipopolysaccharide stimulation of U937 cells or poly I:C stimulation of HEK293-TLR3, THP1 or U87 cells in the presence of a previously reported MyD88 inhibitor (compound 4210) augmented IFN-ß and RANTES production. Consistent with these results, overexpression of MyD88 decreased IFN-ß, whereas MyD88 inhibition rescued IFN-ß production concomitant with increased IRF3 phosphorylation, suggesting IRF-mediated downstream signaling to the IFN-ß response. Further, compound 4210 treatment inhibited MyD88 interaction with IRF3/IRF7 indicating that MyD88 restricts type I IFN signaling through sequestration of IRF3/IRF7. In cell based infection assays, compound 4210 treatment suppressed replication of VEEV, Eastern equine encephalitis virus, Ebola virus (EBOV), Rift Valley Fever virus, Lassa virus, and Dengue virus with IC50 values ranging from 11 to 42 µM. Notably, administration of compound 4210 improved survival, weight change, and clinical disease scores in mice following challenge with VEEV TC-83 and EBOV. Collectively, these results provide evidence that viral infections responsive to MyD88 inhibition lead to activation of IRF3/IRF7 and promoted a type I IFN response, thus, raising the prospect of an approach of host-directed antiviral therapy.

An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency.

INTRODUCTION: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. METHODS: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei. RESULTS: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. CONCLUSION: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.

Author Correction: Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen.

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Small Molecule Analogues of the parasitic worm product ES-62 interact with the TIR domain of MyD88 to inhibit pro-inflammatory signalling.

ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is anti-inflammatory by virtue of covalently attached phosphorylcholine. Previously we have reported that drug-like Small Molecule Analogues (SMAs) of its phosphorylcholine moiety can mimic ES-62 in protecting against disease development in certain mouse models of autoimmune and allergic conditions, due to them causing partial degradation of the TLR/IL-1R adaptor MyD88. We have now taken a molecular modelling approach to investigating the mechanism underlying this effect and this predicts that the SMAs interact directly with the MyD88 TIR domain. Further support for this is provided by assay of LPS-induced MyD88/NF-κB-driven secreted alkaline phosphatase (SEAP) reporter activity in commercially-available stably transfected (TLR4-MD2-NF-κB-SEAP) HEK293 cells, as SMA12b-mediated inhibition of such SEAP activity is blocked by its pre-incubation with recombinant MyD88-TIR domain. Direct binding of SMA12b to the TIR domain is also shown to inhibit homo-dimerization of the adaptor, an event that can explain the observed degradation of the adaptor and inhibition of subsequent downstream signalling. Thus, these new data identify initial events by which drug-like ES-62 SMAs, which we also demonstrate are able to inhibit cytokine production by human cells, homeostatically maintain "safe" levels of MyD88 signalling.

Rational design of peptide derivatives for inhibition of MyD88-mediated toll-like receptor signaling in human peripheral blood mononuclear cells and epithelial cells exposed to Francisella tularensis.

Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of MyD88-mediated toll-like receptor (TLR) signaling by Francisella tularensis. Fifty-three tripeptide derivatives were synthesized to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1ß in human peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to LPS extracts from F. tularensis. From 53 compounds synthesized and tested, ten compounds were identified as effective inhibitors of F. tularensisLPS-induced cytokines. Compound stability testing in the presence of human liver microsomes and human serum resulted in the identification of tripeptide derivative 7 that was a potent, stable, and drug-like small molecule. Target corroboration using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of 1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling represent a promising strategy toward host-directed adjunctive therapeutics for inflammation associated with biothreat agent-induced sepsis.

Innate immune response to Burkholderia mallei.

PURPOSE OF REVIEW: Burkholderia mallei is a facultative intracellular pathogen that causes the highly contagious and often the fatal disease, glanders. With its high rate of infectivity via aerosol and recalcitrance toward antibiotics, this pathogen is considered a potential biological threat agent. This review focuses on the most recent literature highlighting host innate immune response to B. mallei. RECENT FINDINGS: Recent studies focused on elucidating host innate immune responses to the novel mechanisms and virulence factors employed by B. mallei for survival. Studies suggest that pathogen proteins manipulate various cellular processes, including host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement. Immune-signaling molecules such as Toll-like receptors, nucleotode-binding oligomerization domain, myeloid differentiation primary response protein 88, and proinflammatory cytokines such as interferon-gamma and tumor necrosis factor-α, play key roles in the induction of innate immune responses. Modifications in B. mallei lipopolysaccharide, in particular, the lipid A acyl groups, stimulate immune responses via Toll-like receptor4 activation that may contribute to persistent infection. SUMMARY: Mortality is high because of septicemia and immune pathogenesis with B. mallei exposure. An effective innate immune response is critical to controlling the acute phase of the infection. Both vaccination and therapeutic approaches are necessary for complete protection against B. mallei.