Targeting STING oligomerization with small-molecule inhibitors.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Citrullination profile analysis reveals peptidylarginine deaminase 3 as an HSV-1 target to dampen the activity of candidate antiviral restriction factors.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual's life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer's disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

SARM1, an Enzyme Involved in Axon Degeneration, Catalyzes Multiple Activities through a Ternary Complex Mechanism.

Sterile alpha and toll/interleukin receptor (TIR) motif containing protein 1 (SARM1) is an NAD+ hydrolase and cyclase involved in axonal degeneration. In addition to NAD+ hydrolysis and cyclization, SARM1 catalyzes a base exchange reaction between nicotinic acid (NA) and NADP+ to generate NAADP, which is a potent calcium signaling molecule. Herein, we describe efforts to characterize the hydrolysis, cyclization, and base exchange activities of TIR-1, the Caenorhabditis elegans ortholog of SARM1; TIR-1 also catalyzes NAD(P)+ hydrolysis and/or cyclization and regulates axonal degeneration in worms. We show that the catalytic domain of TIR-1 undergoes a liquid-to-solid phase transition that regulates not only the hydrolysis and cyclization reactions but also the base exchange reaction. We define the substrate specificities of the reactions, demonstrate that cyclization and base exchange reactions occur within the same pH range, and establish that TIR-1 uses a ternary complex mechanism. Overall, our findings will aid drug discovery efforts and provide insight into the mechanism of recently described inhibitors.

Development of LB244, an Irreversible STING Antagonist.

The cGMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway plays a critical role in sensing dsDNA localized to the cytosol, resulting in the activation of a robust inflammatory response. While cGAS-STING signaling is essential for antiviral immunity, aberrant STING activation is observed in amyotrophic lateral sclerosis (ALS), lupus, and autoinflammatory diseases such as Aicardi-Goutières syndrome (AGS) and STING associated vasculopathy with onset in infancy (SAVI). Significant efforts have therefore focused on the development of STING inhibitors. In a concurrent submission, we reported that BB-Cl-amidine inhibits STING-dependent signaling in the nanomolar range, both in vitro and in vivo. Considering this discovery, we sought to generate analogs with higher potency and proteome-wide selectivity. Herein, we report the development of LB244, which displays nanomolar potency and inhibits STING signaling with markedly enhanced proteome-wide selectivity. Moreover, LB244 mirrored the efficacy of BB-Cl-amidine in vivo. In summary, our data identify novel chemical entities that inhibit STING signaling and provide a scaffold for the development of therapeutics for treating STING-dependent inflammatory diseases.

Chemical Derivatization Leads to the Discovery Of Novel Analogs of Azotochelin, a Natural Siderophore, as Promising Anticancer Agents.

Siderophores are structurally unique medicinal natural products and exhibit considerable therapeutic potential. Herein, we report the design and synthesis of azotochelin, a natural siderophore, and an extensive library of azotochelin analogs and their anticancer properties. We modified the carboxylic acid and the aromatic ring of azotochelin using various chemical motifs. We evaluated the cytotoxicity of the compounds against six different cancer cell lines (KB-3-1, SNB-19, MCF-7, K-562, SW-620, and NCI-H460) and a non-cancerous cell line (HEK-293). Among the twenty compounds tested, the IC50 values of nine compounds (14, 32, 35-40, and 54) were between 0.7 and 2.0 µM against a lung cancer cell line (NCI-H460). Moreover, several compounds showed good cytotoxicity profile (IC50 <10 µM) against the tested cancer cell lines. The flow cytometry analysis showed that compounds 36 and 38 induced apoptosis in NCI-H460 in a dose-dependent manner. The cell cycle analysis indicated that compounds 36 and 38 significantly arrested the cell cycle at the S phase to block cancer cell proliferation in the NCI-H460 cell line. The study has produced various novel azotochelin analogs that are potentially effective anticancer agents and lead compounds for further synthetic and medicinal chemistry exploration.

Protein citrullination: inhibition, identification and insertion.

Protein citrullination is a post-translational modification (PTM) that is catalysed by the protein arginine deiminase (PAD) family of enzymes. This PTM involves the transformation of an arginine residue into citrulline. Protein citrullination is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation and DNA damage-induced apoptosis. Aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis (RA), lupus, atherosclerosis and ulcerative colitis. In RA, anti-citrullinated protein antibodies can be detected prior to disease onset and are thus a valuable diagnostic tool for RA. Notably, citrullinated proteins may serve more generally as biomarkers of specific disease states; however, the identification of citrullinated protein residues remains challenging owing to the small 1 Da mass change that occurs upon citrullination. Herein, we highlight the progress made so far in the development of pan-PAD and isozyme selective inhibitors as well as the identification of citrullinated proteins and the site-specific incorporation of citrulline into proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Novel Indolyl-Benzimidazole Compounds Promote in vitro Wound Healing and Osteogenic Differentiation of Pluripotent Cells.

BACKGROUND: Increasing or restoring Bone Morphogenetic Protein- (BMP-) signaling through administration of recombinant BMPs (rBMPs) has demonstrated therapeutic efficacy for treating bone fractures or to enhance repair following spinal surgeries. However, direct use of rBMPs has come up against significant obstacles like high cost and incidence of adverse effects. Recently, we reported our findings on the novel indolyl-benzimidazoles, SY-LB-35 and SY-LB-57, that fully activated BMP receptor signaling demonstrating activity profiles that mirrored rBMPs. Here, we explored the potential of these compounds to substitute for rBMPs in processes like wound healing and osteogenesis. METHODS: Cell-based assays including cell viability, short- and long-term phosphorylation, protein expression, wound healing and bone differentiation assays were carried out in the pluripotent myoblast C2C12 cell line with select assays performed in multiple cell lines. Several assays included conditions in the presence of a selective inhibitor of type I BMP receptor, Activin-like kinase 2 (ALK2), or inhibitors of BMP-stimulated downstream signaling. All assays were repeated at least 3 times with replicates per condition where indicated. Statistical tests were carried out using Student's two-tailed, t-test. RESULTS: Sustained activation of non-canonical BMP signaling pathways was observed after 24-hour exposure to SY-LB-35 and SY-LB-57. Moreover, this treatment increased the expression of targets of BMP-mediated transcription such as the Id1 transcription factor. SY-LB-35 and SY-LB-57 promoted substantial increases in cell viability in three distinct cell types and increased the rate of wound closure in scrape-wounded C2C12 cell cultures. Cell viability and wound closure induced by SY-LB compounds required ALK2-, PI3K- and p38-dependent pathways. In contrast, responses to SY-LB compounds were not affected by ERK inhibition. Expression of bone differentiation markers beginning at 4 hours and evidence of calcium deposition detected after 21 days in C2C12 cell cultures exposed to SY-LB-35 and SY-LB-57 demonstrated the osteogenic potential of these compounds. CONCLUSIONS: The functional similarities between these novel compounds and rBMPs indicates that SY-LB-35 or SY-LB-57, acting as potent activators of BMP receptor signaling and inducers of osteogenic processes, could potentially replace rBMPs for treating BMP-related pathologies such as bone fracture repair or other wound healing processes.

Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA.

Introduction: MZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells show that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidylcitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated. Method: An unbiased analysis of human lung citrullinome was conducted to identify citrullinated proteins that are enriched in several chronic lung diseases, including rheumatoid arthritis-associated interstitial lung disease (RA-ILD), chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, compared to healthy controls. Mass spectrometry, site-specific mutagenesis, and western blotting were used to confirm the citrullination of candidate proteins. Their citrullination was suppressed by pharmacological inhibition or genetic ablation of PAD2 and the impact of their citrullination on the function and differentiation of human B cells was examined with enzyme-linked immunosorbent assay, flow cytometry, and co-immunoprecipitation. Results: Citrullinated MZB1 was preferentially enriched in RA-ILD but not in other chronic lung diseases. MZB1 was a substrate of PAD2 and was citrullinated during the differentiation of human plasmablasts. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. Furthermore, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by pharmacological inhibition of PAD2. Discussion: Our data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD.

Discovery of a novel class of benzimidazoles as highly effective agonists of bone morphogenetic protein (BMP) receptor signaling.

Increasing or restoring Bone Morphogenetic Protein receptor signaling is an effective therapy for conditions such as bone fracture and pulmonary arterial hypertension. However, direct use of recombinant BMPs has encountered significant obstacles. Moreover, synthetic, full agonists of BMP receptor signaling have yet to be identified. Here, we report the discovery of a novel class of indolyl-benzimidazoles, synthesized using a one-pot synthetic methodology, which appear to mimic the biochemical and functional activity of BMPs. The first-in-series compounds, SY-LB-35 and SY-LB-57, stimulated significant increases in cell number and cell viability in the C2C12 myoblast cell line. Cell cycle analysis revealed that these compounds induced a shift toward proliferative phases. SY-LB-35 and SY-LB-57 stimulated canonical Smad and non-canonical PI3K/Akt, ERK, p38 and JNK intracellular signaling pathways, similar to BMP2-stimulated responses. Importantly, increases in Smad phosphorylation and cell viability were dependent on type I BMP receptor activity. Thus, these compounds robustly activate intracellular signaling in a BMP receptor-dependent manner and may signify the first known, full agonists of BMP receptor signaling. Moreover, discovery of small molecule activators of BMP pathways, which can be efficiently formulated and targeted to diseased or damaged areas, could potentially substitute recombinant BMPs for treatment of BMP-related pathologies.

Synthesis and Biological Evaluation of Structurally Diverse Benzimidazole Scaffolds as Potential Chemotherapeutic Agents.

BACKGROUND AND OBJECTIVE: Drug resistance and adverse effects are immense healthcare challenges in cancer therapy. Benzimidazole ring-based small molecules have been effective anticancer agents in drug development. In an effort to develop novel chemotherapeutics, we synthesized and assessed the anticancer and antibacterial activities of a small library of structurally unique benzimidazoles. METHODS: The benzimidazoles were derived from indole, N-alkyl indole, fatty acid, and alpha-amino acid scaffolds providing a panel of diverse structures. The compounds were tested in three different cancer cell lines for cytotoxicity: HepG2 (human hepatocellular carcinoma), HeLa (human cervical carcinoma), and A549 (human lung carcinoma). Mechanism of cell death induced by benzimidazoles was evaluated using fluorescent dye-based apoptosis-necrosis assay, immunoblotting for active caspases, topoisomerase-II activity assay, and cell cycle assay. RESULTS: Cell viability testing revealed that indole- and fatty acid-based benzimidazoles were most potent followed by the amino acid derivatives. Many compounds induced cytotoxicity in a concentration-dependent manner with cellular cytotoxicity (CC50) <20µM in the cell lines tested. Most compounds exhibited cytotoxicity via apoptosis through the intrinsic pathway. Inhibition of topoisomerase activity and cell cycle alterations were not the primary mechanisms of cytotoxicity. In addition, several compounds showed promising activity against S. aureus and S. epidermidis (Minimum Inhibitory Concentration (MIC) of as low as 0.04µmol/mL). CONCLUSION: The reported benzimidazole derivatives possess promising anticancer and antibacterial properties. Additionally, we discovered apoptosis to be the primary mechanism for cancer cell death induced by the tested benzimidazoles. Our findings suggest that further development of these scaffolds could provide drug leads towards new chemotherapeutics.

Tyrosine kinase inhibitor conjugated quantum dots for non-small cell lung cancer (NSCLC) treatment.

Non-small cell lung cancer is a major sub-type of lung cancer that is associated with a poor diagnosis resulting in poor therapy for the disorder. In order to achieve a better prognosis, innovative multi-functional systems need to be developed which will aide in diagnosis as well as therapy for the disorder. One such multi-functional delivery system fabricated is Quantum Dots (QDs). QDs are photo-luminescent inorganic nanoparticles utilized for tumor detection, preclinically. Erlotinib hydrochloride, a tyrosine kinase inhibitor, is a first-generation drug developed to treat NSCLC. Its active metabolite, Desmethyl Erlotinib (OSI-420), exhibits similar anticancer activity as erlotinib. OSI-420 was conjugated to QDs to fabricate a delivery system and was then characterized by FT-IR, H NMR, UV-VIS, particle size, zeta potential, fluorescence spectroscopy and TEM. Drug loading was estimated using UV-VIS spectroscopy (52.2 ±â€¯7.5%). A concentration-dependent release of OSI-420 was achieved using esterase enzymes, which was further confirmed using LC-MS. A cellular uptake study revealed the internalization potential of QDs and QD-OSI 420. A cellular recovery study was performed to confirm the internalization potential. Cell viability studies revealed that QD-OSI 420 conjugates had significantly better efficacy than pure drugs in all tested cell lines. QD conjugated OSI-420 demonstrated an IC60 of 2.5 µM in erlotinib-resistant A549 cell lines, where erlotinib or OSI-420 alone could not exhibit 60% inhibition when evaluated up to 20 µM. Similar cytotoxic enhancement of erlotinib was seen with QD-OSI 420 in other NSCLC cell lines as well. These results were strengthened by 3D-SCC model of A549 which revealed that QD-OSI 420 was significantly better in reducing in-vitro 3D tumor volume, as compared to pure drugs. This study, being one of its kind, explores the feasibility of conjugating OSI-420 with QDs as an alternative to traditional anti-cancer therapy, by improving intracellular drug delivery.

An efficient one-pot conversion of carboxylic acids into benzimidazoles via an HBTU-promoted methodology.

Benzimidazole is a privileged, and routinely used pharmacophore in the drug discovery process. Herein, we report a mild, acid-free and one-pot synthesis of indole, alkyl and alpha-amino benzimidazoles through a novel HBTU-promoted methodology. An extensive library of indole-carboxylic acids, alkyl carboxylic acids and N-protected alpha-amino acids has been converted into the corresponding benzimidazoles in 80-99% yield. Since alpha-aminobenzimidazoles are highly useful synthons as chiral ligands for chemical catalysis, as well as for drug discovery endeavors, our reported method provides direct access to this scaffold in a simple, one-pot operation from commercially available carboxylic acids.