Therapeutic Targets For Infectious Diseases Research Articles

Fangchinoline induces antiviral response by suppressing STING degradation

The stimulator of interferon genes (STING), an integral adaptor protein in the DNA-sensing pathway, plays a pivotal role in the innate immune response against infections. Additionally, it presents a valuable therapeutic target for infectious diseases and cancer. We observed that fangchinoline (Fan), a bis-benzylisoquinoline alkaloid, effectively impedes the replication of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), influenza A virus (H1N1 PR8), and herpes simplex virus-1 (HSV-1) in vitro. Fan treatment significantly reduced the viral load, attenuated tissue inflammation, and improved survival in a viral sepsis mouse model. Mechanistically, Fan activates the antiviral response in a STING-dependent manner, leading to increased expression of interferon (IFN) and interferon-stimulated genes (ISGs) for potent antiviral effects in vivo and in vitro. Notably, Fan interacts with STING, preventing its degradation and thereby extending the activation of IFN-based antiviral responses. Collectively, our findings highlight the potential of Fan, which elicits antiviral immunity by suppressing STING degradation, as a promising candidate for antiviral therapy.

Crosstalk between hypoxic cellular micro-environment and the immune system: a potential therapeutic target for infectious diseases.

There are overwhelming reports on the promotional effect of hypoxia on the malignant behavior of various forms of cancer cells. This has been proposed and tested exhaustively in the light of cancer immunotherapy. However, there could be more interesting functions of a hypoxic cellular micro-environment than malignancy. There is a highly intricate crosstalk between hypoxia inducible factor (HIF), a transcriptional factor produced during hypoxia, and nuclear factor kappa B (NF-κB) which has been well characterized in various immune cell types. This important crosstalk shares common activating and inhibitory stimuli, regulators, and molecular targets. Impaired hydroxylase activity contributes to the activation of HIFs. Inflammatory ligands activate NF-κB activity, which leads to the expression of inflammatory and anti-apoptotic genes. The eventual sequelae of the interaction between these two molecular players in immune cells, either bolstering or abrogating functions, is largely cell-type dependent. Importantly, this holds promise for interesting therapeutic interventions against several infectious diseases, as some HIF agonists have helped prevent immune-related diseases. Hypoxia and inflammation are common features of infectious diseases. Here, we highlighted the role of this crosstalk in the light of functional immunity against infection and inflammation, with special focus on various innate and adaptive immune cells. Particularly, we discussed the bidirectional effects of this crosstalk in the regulation of immune responses by monocytes/macrophages, dendritic cells, neutrophils, B cells, and T cells. We believe an advanced understanding of the interplay between HIFs and NF-kB could reveal novel therapeutic targets for various infectious diseases with limited treatment options.

Ferroptotic stress promotes macrophages against intracellular bacteria

Rational: Intracellular bacterial survival is a major factor causing chronic or recurrent infection, leading to the failure of both host defense and/or antibiotic treatment. However, the elimination of intracellular bacteria is challenging as they are protected from antibiotics and host immune attack. Recent studies have indicated that iron helps macrophages against intracellular bacteria, contradictory to traditional “nutritional immunity”, in which iron is considered a key nutrient for bacterial survival in host cells. However, how iron facilitates intracellular bacterial death has not been fully clarified. In this study, we found that ferroptotic stress can help macrophages suppress intracellular bacteria by reversing the importation of ferrous iron into bacterial vacuoles via ferroportin and thereby inducing in situ ferroptosis-like bacterial death.Methods: A macrophage model of bacterial invasion was established to monitor dynamic changes in ferroptotic hallmarks, including ferrous iron and lipid peroxidation. Ferroptosis inducers and inhibitors were added to the model to evaluate the relationship between ferroptotic stress and intracellular bacterial survival. We then determined the spatiotemporal distributions of ferroportin, ferrous iron, and lipid peroxidation in macrophages and intracellular bacteria. A bacterial infection mouse model was established to evaluate the therapeutic effects of drugs that regulate ferroptotic stress.Results: Ferrous iron and lipid peroxidation increased sharply in the early stage of bacterial infection in the macrophages, then decreased to normal levels in the late stage of infection. The addition of ferroptosis inducers (ras-selective lethal small molecule 3, sulfasalazine, and acetaminophen) in macrophages promoted intracellular bacterial suppression. Further studies revealed that ferrous iron could be delivered to the intracellular bacterial compartment via inward ferroportin transportation, where ferrous iron induced ferroptosis-like death of bacteria. In addition, ferroptotic stress declined to normal levels in the late stage of infection by regulating iron-related pathways in the macrophages. Importantly, we found that enhancing ferroptotic stress with a ferroptosis inducer (sulfasalazine) successfully suppressed bacteria in the mouse infection models.Conclusions: Our study suggests that the spatiotemporal response to ferroptosis stress is an efficient pathway for macrophage defense against bacterial invasion, and targeting ferroptosis may achieve therapeutic targets for infectious diseases challenged by intracellular pathogens.

Identification and Structure-Activity Relationships of Dietary Flavonoids as Human Macrophage Migration Inhibitory Factor (MIF) Inhibitors.

Dietary flavonoids are known to have anti-inflammatory and anticancer effects, but their influences on human macrophage migration inhibitory factor (MIF), a vital proinflammatory cytokine recognized as a therapeutic target for infectious diseases and cancers, have been rarely reported. Here, we identified 24 dietary flavonoids that could inhibit the tautomerase activity of MIF, five of which exerted IC50 values lower than the positive control ISO-1 in the micromolar range: morin (IC50 = 11.01 ± 0.45 μM) and amentoflavone (IC50 = 13.32 ± 0.64 μM) exhibited the most potent efficacy followed by apigenin (IC50 = 42.74 ± 4.20 μM), naringin (IC50 = 51.38 ± 2.12 μM), and fisetin (IC50 = 51.99 ± 0.63 μM). X-ray crystallography, molecular docking, and cellular experiments were utilized to illustrate the molecular binding details and structure-activity relationships. Scaffold modifications of flavonoids significantly influenced the potency. What stands out for morin is the unique 2'-OH substitution. In addition, amentoflavone situated at the MIF trimer pore may impact MIF-CD74 signaling. The results also showed that flavonoids could suppress cell chemotaxis and nitric oxide production in RAW264.7 cells. Our results elucidate the molecular mechanism of flavonoids acting on MIF and shed light on developing lead compounds against MIF-involved diseases.

The Functional Deubiquitinating Enzymes in Control of Innate Antiviral Immunity.

Innate antiviral immunity is the first line of host defense against invading viral pathogens. Immunity activation primarily relies on the recognition of pathogen‐associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Viral proteins or nucleic acids mainly engage three classes of PRRs: Toll‐like receptors (TLRs), retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), and DNA sensor cyclic GMP‐AMP (cGAMP) synthase (cGAS). These receptors initiate a series of signaling cascades that lead to the production of proinflammatory cytokines and type I interferon (IFN‐I) in response to viral infection. This system requires precise regulation to avoid aberrant activation. Emerging evidence has unveiled the crucial roles that the ubiquitin system, especially deubiquitinating enzymes (DUBs), play in controlling immune responses. In this review, an overview of the most current findings on the function of DUBs in the innate antiviral immune pathways is provided. Insights into the role of viral DUBs in counteracting host immune responses are also provided. Furthermore, the prospects and challenges of utilizing DUBs as therapeutic targets for infectious diseases are discussed.

Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor and may be a therapeutic target for infectious diseases. Herein, we determined the 3D structure of native SAL, the mutated S116A inactive form, and the inhibitor complex using the anti-obesity drug orlistat to aid in drug development. The determined crystal structures showed a typical α/β hydrolase motif with a dimeric form. Fatty acids bound near the active site in native SAL and inactive S116A mutant structures. We found that orlistat potently inhibits SAL activity, and it covalently bound to the catalytic Ser116 residue. This is the first report detailing orlistat–lipase binding. It provides structure-based information on the production of potent anti-SAL drugs and lipase inhibitors. These results also indicated that orlistat can be repositioned to treat bacterial diseases.

Staphylococcus aureus lipase: purification, kinetic characterization, crystallization and crystallographic study.

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor in S. aureus and may be a therapeutic target for infectious diseases caused by S. aureus. For the purposes of anti-SAL drug development using structure-based drug design, X-ray crystallographic analysis of SAL overexpressed in Escherichia coli was performed. The recombinant protein was purified using a three-step protocol involving immobilized metal-affinity chromatography, cation-exchange chromatography and anion-exchange chromatography flowthrough methods, yielding 40 mg of protein per litre of bacterial culture. Crystals were obtained using the sitting-drop vapor-diffusion technique. Diffraction data to 3.0 Å resolution were collected on the BL44XU beamline at SPring-8 at the zinc peak of 1.2842 Å for SAD phasing. The crystals belonged to space group P4122 or P4322, with unit-cell parameters a = 131.0, b=131.0, c=250.6 Å, and are likely to contain four SAL molecules (408 residues) per asymmetric unit.

The Voltage-Dependent Anion Channels (VDAC) of Mycobacterium avium phagosome are associated with bacterial survival and lipid export in macrophages

Mycobacterium avium subsp. hominissuis is associated with infection of immunocompromised individuals as well as patients with chronic lung disease. M. avium infects macrophages and actively interfere with the host killing machinery such as apoptosis and autophagy. Bacteria alter the normal endosomal trafficking, prevent the maturation of phagosomes and modify many signaling pathways inside of the macrophage by secreting effector molecules into the cytoplasm. To investigate whether M. avium needs to attach to the internal surface of the vacuole membrane before releasing efferent molecules, vacuole membrane proteins were purified and binding to the surface molecules present in intracellular bacteria was evaluated. The voltage-dependent anion channels (VDAC) were identified as components of M. avium vacuoles in macrophages. M. avium mmpL4 proteins were found to bind to VDAC-1 protein. The inactivation of VDAC-1 function either by pharmacological means or siRNA lead to significant decrease of M. avium survival. Although, we could not establish a role of VDAC channels in the transport of known secreted M. avium proteins, we demonstrated that the porin channels are associated with the export of bacterial cell wall lipids outside of vacuole. Suppression of the host phagosomal transport systems and the pathogen transporter may serve as therapeutic targets for infectious diseases.

Proteomic strategies for the discovery of novel diagnostic and therapeutic targets for infectious diseases

Viruses have developed numerous and elegant strategies to manipulate the host cell machinery to establish a productive infectious cycle. The interaction of viral proteins with host proteins plays an important role in infection and pathogenesis, often bypassing traditional host defenses such as the interferon response and apoptosis. Host–viral protein interactions can be studied using a variety of proteomic approaches ranging from genetic and biochemical to large‐scale high‐throughput technologies. Protein interactions between host and viral proteins are greatly influenced by host signal transduction pathways. In this review, we will focus on comparing proteomic information obtained through differing technologies and how their integration can be used to determine the functional aspect of the host response to infection. We will briefly review and evaluate techniques employed to elucidate viral–host interactions with a primary focus on Protein Microarrays (PMA) and Mass Spectrometry (MS) as potential tools in the discovery of novel therapeutic targets. As many potential molecular markers and targets are proteins, proteomic profiling is expected to yield both clearer and more direct answers to functional and pharmacologic questions.

Bacterial proteases: targets for diagnostics and therapy

Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

Genuine Immunomodulation With dSLIM.

Toll-like receptors are sensing modulators of the innate immune system. One member of this protein family, Toll-like receptor (TLR)-9, is increasingly being investigated as therapeutic target for infectious diseases and cancer. Double-Stem Loop ImmunoModulator (dSLIM) is a new TLR-9 agonist in clinical development for patients with metastatic colorectal carcinoma. Compared with other TLR-9 ligands developed as immunomodulators, dSLIM comprises single- and double-stranded DNA, is covalently closed, and consists of natural nucleotide components only. All investigated biologic effects of dSLIM are strongly dependent on CG motifs, and the relevant cellular activation profile of dSLIM is distinct to that of other TLR-9 agonists. Here we describe the structure and biologic profile of dSLIM: in isolated human peripheral blood mononuclear cells (PBMCs), dSLIM induced a unique pattern of cytokine secretion, activated within the PBMC pool particular cell subpopulations, and exhibited specific cytotoxicity on target cells. Using cellular isolation and depletion setups, the mechanism of immunoactivation by dSLIM was deduced to be dependent on, but not restricted to, TLR-9-bearing plasmacytoid dendritic cells. The dSLIM-promoted cellular stimulation directs systemic activation of the immune response as revealed in cancer patients. The observed cellular activation cascades are discussed in the context of cancer therapy.

Application of cutting-edge proteomics technologies for elucidating host-bacteria interactions.

Advanced techniques and high-throughput protein analysis have led proteomics to substantive progress in the understanding of bacterial-host interactions. Mass spectrometry (MS)-based proteomics have been a central methodology in the discovery of new protein involved in the infectious process that leads to thousands of deaths every year. The discovery of novel protein targets, together with de novo drug design, improves the accuracy of early diagnosis, leading to improved new treatments. MS-based proteomics has also been widely applied to structural biology, where proteomic investigation is being used to improve knowledge on the relationship between protein sequence, structure, and function. Thus, the search for therapeutic targets for infectious diseases using these cutting-edge technologies represents the new frontiers for proteomics applications in biomedicine and pharmacology. In this review, the main classical gel-based methods (2-DE, DIGE) are discussed, as well as the advances of gel-free quantitative proteomic techniques, from metabolic and chemical labeling (SILAC, iTRAQ, ICAT, (16)O/(18)O, QconCAT) to nonlabeling (MS spectra counting and peak integration) strategies. Together, these proteomic methods are currently being used in the quest for tailor-made pharmaceutical and biomedical research for bacterial control, where advances in these analytical methods may represent greater improvements in the treatment of a number of infectious diseases.

PHISTO: pathogen–host interaction search tool

Knowledge of pathogen-host protein interactions is required to better understand infection mechanisms. The pathogen-host interaction search tool (PHISTO) is a web-accessible platform that provides relevant information about pathogen-host interactions (PHIs). It enables access to the most up-to-date PHI data for all pathogen types for which experimentally verified protein interactions with human are available. The platform also offers integrated tools for visualization of PHI networks, graph-theoretical analysis of targeted human proteins, BLAST search and text mining for detecting missing experimental methods. PHISTO will facilitate PHI studies that provide potential therapeutic targets for infectious diseases. http://www.phisto.org. saliha.durmus@boun.edu.tr Supplementary data are available at Bioinformatics online.

Dihydrofolate reductase as a therapeutic target for infectious diseases: opportunities and challenges

Infectious diseases caused by parasites continue to take a massive toll on human health in the poor regions of the world. Filling the anti-infective drug-discovery pipeline has never been as challenging as it is now. The organisms responsible for these diseases have interesting biology with many potential biochemical targets. Inhibition of metabolic enzymes has been established as an attractive strategy for anti-infectious drug development. In this field, dihydrofolate reductase (DHFR) is an important enzyme in nucleic and amino acid synthesis and an extensively studied drug target over the past 50 years. The challenges for novel DHFR inhibition-based chemotherapeutics for the treatment of infectious diseases are now focused on overcoming the resistance problem as well as cost-effectiveness. Each year, the large number of literature citations attest the continued popularity of DHFR. It becomes truly the 'enzyme of choice for all seasons and almost all reasons'. Herein, we summarize the opportunities and challenges in developing novel lead based on this target.

Suppression of HIV-1 viral replication and cellular pathogenesis by a novel p38/JNK kinase inhibitor.

To analyze a novel compound, which inhibits serine-threonine protein kinase p38, for its possible bioactivity against HIV-1 infection. Proteins involved in cellular signal transduction pathways represent a novel class of host therapeutic targets for infectious diseases. In this regard the serine/threonine kinase p38 MAPK, a member of the mitogen-activated protein (MAP) kinase superfamily of signal transduction molecules may play an important role in HIV-1 infection. We analyzed the ability of this compound (RWJ67657) to inhibit HIV replication in primary T cells and monocytes. Cellular expression of phospho-p38MAPK was studied by Western blot analysis. Blockade of HIV infection induced apoptosis was measured by Annexin V staining. p38 inhibitor RWJ67657 was effective in inhibiting HIV-1 replication in both T-cell and monocyte cell lines, irrespective of the coreceptor used by the virus for entry into the cell. Importantly, both reverse transcriptase and protease resistant escape mutant viruses were effectively suppressed by RWJ67657. In addition, the tested compounds block HIV-induced T-cell apoptosis, a critical means of T-cell depletion linked to AIDS progression. Several steps in the HIV-1 virus life cycle appear to depend on cellular activation, including activation of the p38 pathway. Without activation virus replication is thought to be blocked due to incomplete reverse transcription and a lack of proviral DNA integration. The data collectively illustrate that inhibition of the p38 pathway can affect HIV-1 replication. Interruption of HIV infection by p38 inhibitors underscores the value of exploring antiviral drugs that target host cellular proteins.