Peptide-based allosteric inhibitor targets TNFR1 conformationally active region and disables receptor-ligand signaling complex.

Tumor necrosis factor (TNF) receptor 1 (TNFR1) plays a pivotal role in mediating TNF induced downstream signaling and regulating inflammatory response. Recent studies have suggested that TNFR1 activation involves conformational rearrangements of preligand assembled receptor dimers and targeting receptor conformational dynamics is a viable strategy to modulate TNFR1 signaling. Here, we used a combination of biophysical, biochemical, and cellular assays, as well as molecular dynamics simulation to show that an anti-inflammatory peptide (FKCRRWQWRMKK), which we termed FKC, inhibits TNFR1 activation allosterically by altering the conformational states of the receptor dimer without blocking receptor-ligand interaction or disrupting receptor dimerization. We also demonstrated the efficacy of FKC by showing that the peptide inhibits TNFR1 signaling in HEK293 cells and attenuates inflammation in mice with intraperitoneal TNF injection. Mechanistically, we found that FKC binds to TNFR1 cysteine-rich domains (CRD2/3) and perturbs the conformational dynamics required for receptor activation. Importantly, FKC increases the frequency in the opening of both CRD2/3 and CRD4 in the receptor dimer, as well as induces a conformational opening in the cytosolic regions of the receptor. This results in an inhibitory conformational state that impedes the recruitment of downstream signaling molecules. Together, these data provide evidence on the feasibility of targeting TNFR1 conformationally active region and open new avenues for receptor-specific inhibition of TNFR1 signaling.

Synthesis of bioactive (1→6)-ß-glucose branched poly-amido-saccharides that stimulate and induce M1 polarization in macrophages.

ß-Glucans are of significant interest due to their potent antitumor and immunomodulatory activities. Nevertheless, the difficulty in purification, structural heterogenicity, and limited solubility impede the development of structure-property relationships and translation to therapeutic applications. Here, we report the synthesis of a new class of (1→6)-ß-glucose-branched poly-amido-saccharides (PASs) as ß-glucan mimetics by ring-opening polymerization of a gentiobiose-based disaccharide ß-lactam and its copolymerization with a glucose-based ß-lactam, followed by post-polymerization deprotection. The molecular weight (Mn) and frequency of branching (FB) of PASs is readily tuned by adjusting monomer-to-initiator ratio and mole fraction of gentiobiose-lactam in copolymerization. Branched PASs stimulate mouse macrophages, and enhance production of pro-inflammatory cytokines in a FB-, dose-, and Mn-dependent manner. The stimulation proceeds via the activation of NF-κB/AP-1 pathway in a Dectin-1-dependent manner, similar to natural ß-glucans. The lead PAS significantly polarizes primary human macrophages towards M1 phenotype compared to other ß-glucans such as lentinan, laminarin, and curdlan.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Modulating lysosomal pH: a molecular and nanoscale materials design perspective.

Lysosomes, membrane-bound organelles, play important roles in cellular processes including endocytosis, phagocytosis, and autophagy. Lysosomes maintain cellular homeostasis by generating a highly acidic environment of pH 4.5 - 5.0 and by housing hydrolytic enzymes that degrade engulfed biomolecules. Impairment of lysosomal function, especially in its acidification, is a driving force in the pathogenesis of diseases including neurodegeneration, cancer, metabolic disorders, and infectious diseases. Therefore, lysosomal pH is an attractive and targetable site for therapeutic intervention. Currently, there is a dearth of strategies or materials available to specifically modulate lysosomal acidification. This review focuses on the key aspects of how lysosomal pH is implicated in various diseases and discusses design strategies and molecular or nanoscale agents for lysosomal pH modulation, with the ultimate goal of developing novel therapeutic solutions.

Nanoparticle tumor localization, disruption of autophagosomal trafficking, and prolonged drug delivery improve survival in peritoneal mesothelioma.

The treatment outcomes for malignant peritoneal mesothelioma are poor and associated with high co-morbidities due to suboptimal drug delivery. Thus, there is an unmet need for new approaches that concentrate drug at the tumor for a prolonged period of time yielding enhanced antitumor efficacy and improved metrics of treatment success. A paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) system is described that addresses two unique challenges to improve the outcomes for peritoneal mesothelioma. First, following intraperitoneal administration, eNPs rapidly and specifically localize to tumors. The rate of eNP uptake by tumors is an order of magnitude faster than the rate of uptake in non-malignant cells; and, subsequent accumulation in autophagosomes and disruption of autophagosomal trafficking leads to prolonged intracellular retention of eNPs. The net effect of these combined mechanisms manifests as rapid localization to intraperitoneal tumors within 4 h of injection and persistent intratumoral retention for >14 days. Second, the high tumor-specificity of PTX-eNPs leads to delivery of greater than 100 times higher concentrations of drug in tumors compared to PTX alone and this is maintained for at least seven days following administration. As a result, overall survival of animals with established mesothelioma more than doubled when animals were treated with multiple doses of PTX-eNPs compared to equivalent dosing with PTX or non-responsive PTX-loaded nanoparticles.

Grafting of ZnS:Mn-Doped Nanocrystals and an Anticancer Drug onto Graphene Oxide for Delivery and Cell Labeling.

A facile method for the synthesis of highly fluorescent manganese-doped zinc sulfide (ZnS:Mn) nanocrystals covalently functionalized with polyethylene glycol conjugated graphene oxide (GO-PEG) for drug delivery and cell labeling is reported. First, covalently functionalized GO with PEG-bis(amine) to enhance the solubility and biocompatibility in water and physiological buffers. Second, glutathione (GSH)-coated ZnS:Mn-doped nanocrystals were covalently grafted onto GO-PEG. An acid-amidation process was employed to obtain GO-PEG/ZnS:Mn nanocomposites, which were characterized by UV/Vis, photoluminescence, and Fourier transform infrared spectroscopies, and transmission electron microscopy. Finally, the anticancer drug doxorubicin (DOX) was noncovalently loaded onto these GO-PEG/ZnS:Mn composite particles. High drug entrapment efficiency (100 % due to more GO surface available for binding), slow in vitro release of drug (ca. 40 % at acidic pH), better HeLa cancer cell killing efficiency (ca. 85 %), and cell labeling capability are the important traits of these DOX-loaded nanocomposites. It is believed these novel fluorescent [GO-PEG/ZnS:Mn]-DOX composite particles have great potential as theranostic agents in cancer diagnosis and therapy.