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.

Therapeutic Intervention of Neuroinflammatory Alzheimer Disease Model by Inhibition of Classical Complement Pathway with the Use of Anti-C1r Loaded Exosomes.

Alzheimer's disease (AD) is a complex neurodegenerative disease associated with memory decline, cognitive impairment, amyloid plaque formation and tau tangles. Neuroinflammation has been shown to be a precursor to apparent amyloid plaque accumulation and subsequent synaptic loss and cognitive decline. In this study, the ability of a novel, small molecule, T-ALZ01, to inhibit neuroinflammatory processes was analyzed. T-ALZ01, an inhibitor of complement component C1r, demonstrated a significant reduction in the levels of the inflammatory cytokines, IL-6 and TNF-α in vitro. An LPS-induced animal model, whereby animals were injected intraperitoneally with 0.5 mg/kg LPS, was used to analyze the effect of T-ALZ01 on neuroinflammation in vivo. Moreover, exosomes (nanosized, endogenous extracellular vehicles) were used as drug delivery vehicles to facilitate intranasal administration of T-ALZ01 across the blood-brain barrier. T-ALZ01 demonstrated significant reduction in degenerating neurons and the activation of resident microglia and astrocytes, as well as inflammatory markers in vivo. This study demonstrates a significant use of small molecule complement inhibitors via exosome drug delivery as a possible therapeutic in disorders characterized by neuroinflammation, such AD.

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.

Assessment of BMP responses in an in vitro model of acute ethanol toxicity.

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling. This short-term assay: • Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation • Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels.

Non-Smad, BMP-dependent signaling protects against the effects of acute ethanol toxicity.

This study explores the effect of acute Ethanol (EtOH) exposure on Bone Morphogenetic Protein (BMP)-evoked intracellular signaling, and the concomitant morphological changes induced by EtOH in C2C12 cells and DRG (Dorsal root ganglion) neurons in an in vitro model related to Fetal Alcohol Syndrome Disorder (FASD). All assays were performed within 30 min of BMP stimulation to specifically investigate the earliest events occurring in BMP-evoked intracellular signaling pathways. We show that Smad phosphorylation and nuclear translocation stimulated by BMPs was not altered following acute exposure to EtOH. In contrast, acute EtOH exposure alone caused a striking concentration-dependent decrease in Akt phosphorylation, as well as a loss of adhesion in C2C12 cells. The addition of BMPs before exposure to EtOH was associated with maintenance of Akt phosphorylation, greater cell adhesion in C2C12 cells, and preservation of growth cone complexity in DRG neurons. Thus, for both C2C12 cells and DRG neurons, BMPs, acting through non-canonical BMP signaling pathways, appear to impart some protection against the profound effects of acute EtOH exposure on cellular adhesion and structure.

The Positive Allosteric Modulation of alpha7-Nicotinic Cholinergic Receptors by GAT107 Increases Bacterial Lung Clearance in Hyperoxic Mice by Decreasing Oxidative Stress in Macrophages.

Supplemental oxygen therapy with supraphysiological concentrations of oxygen (hyperoxia; >21% O2) is a life-saving intervention for patients experiencing respiratory distress. However, prolonged exposure to hyperoxia can compromise bacterial clearance processes, due to oxidative stress-mediated impairment of macrophages, contributing to the increased susceptibility to pulmonary infections. This study reports that the activation of the α7 nicotinic acetylcholine receptor (α7nAChR) with the delete allosteric agonistic-positive allosteric modulator, GAT107, decreases the bacterial burden in mouse lungs by improving hyperoxia-induced lung redox imbalance. The incubation of RAW 264.7 cells with GAT107 (3.3 µM) rescues hyperoxia-compromised phagocytic functions in cultured macrophages, RAW 264.7 cells, and primary bone marrow-derived macrophages. Similarly, GAT107 (3.3 µM) also attenuated oxidative stress in hyperoxia-exposed macrophages, which prevents oxidation and hyper-polymerization of phagosome filamentous actin (F-actin) from oxidation. Furthermore, GAT107 (3.3 µM) increases the (1) activity of superoxide dismutase 1; (2) activation of Nrf2 and (3) the expression of heme oxygenase-1 (HO-1) in macrophages exposed to hyperoxia. Overall, these data suggest that the novel α7nAChR compound, GAT107, could be used to improve host defense functions in patients, such as those with COVID-19, who are exposed to prolonged periods of hyperoxia.

The α7 nicotinic acetylcholine receptor agonist, GTS-21, attenuates hyperoxia-induced acute inflammatory lung injury by alleviating the accumulation of HMGB1 in the airways and the circulation.

BACKGROUND: Oxygen therapy, using supraphysiological concentrations of oxygen (hyperoxia), is routinely administered to patients who require respiratory support including mechanical ventilation (MV). However, prolonged exposure to hyperoxia results in acute lung injury (ALI) and accumulation of high mobility group box 1 (HMGB1) in the airways. We previously showed that airway HMGB1 mediates hyperoxia-induced lung injury in a mouse model of ALI. Cholinergic signaling through the α7 nicotinic acetylcholine receptor (α7nAChR) attenuates several inflammatory conditions. The aim of this study was to determine whether 3-(2,4 dimethoxy-benzylidene)-anabaseine dihydrochloride, GTS-21, an α7nAChR partial agonist, inhibits hyperoxia-induced HMGB1 accumulation in the airways and circulation, and consequently attenuates inflammatory lung injury. METHODS: Mice were exposed to hyperoxia (≥99% O2) for 3 days and treated concurrently with GTS-21 (0.04, 0.4 and 4 mg/kg, i.p.) or the control vehicle, saline. RESULTS: The systemic administration of GTS-21 (4 mg/kg) significantly decreased levels of HMGB1 in the airways and the serum. Moreover, GTS-21 (4 mg/kg) significantly reduced hyperoxia-induced acute inflammatory lung injury, as indicated by the decreased total protein content in the airways, reduced infiltration of inflammatory monocytes/macrophages and neutrophils into the lung tissue and airways, and improved lung injury histopathology. CONCLUSIONS: Our results indicate that GTS-21 can attenuate hyperoxia-induced ALI by inhibiting extracellular HMGB1-mediated inflammatory responses. This suggests that the α7nAChR represents a potential pharmacological target for the treatment regimen of oxidative inflammatory lung injury in patients receiving oxygen therapy.

Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA.

BMP7 evokes acute chemotropic PI3K-dependent responses, such as growth cone collapse and monocyte chemotaxis, as well as classical Smad-dependent gene transcription. That these divergent responses can be activated in the same cell raises the question of how the BMP-dependent signaling apparatus is manipulated to produce chemotropic and transcriptional signals. RNA interference and site-directed mutagenesis were used to explore functional and structural BMP receptor requirements for BMP7-evoked chemotropic activity. We show that specific type II BMP receptor subunits, ActRIIA and BMPR2, are required for BMP7-induced growth cone collapse in developing spinal neurons and for chemotaxis of monocytes. Reintroduction of wild-type ActRIIA into monocytic cells lacking endogenous ActRIIA restores BMP7-evoked chemotaxis, whereas expression of an ActRIIA K76A receptor variant fails to rescue. BMP7-evoked Smad-dependent signaling is unaffected by either ActRIIA knockdown or expression of the ActRIIA K76A variant. In contrast, BMP7-evoked PI3K-dependent signaling is significantly disturbed in the presence of ActRIIA K76A. These results support a model for selective engagement of chemotropic BMPs with type II BMP receptors, through specific residues, that results in strict regulation of PI3K-dependent signal transduction.This article has an associated First Person interview with the first author of the paper.

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.

Advances in treatment of pulmonary arterial hypertension: patent review.

INTRODUCTION: Current therapeutic approaches for pulmonary arterial hypertension (PAH) commonly include use of prostacyclins, endothelin pathway antagonists or NO (nitric oxide) pathway modulators. These agents are non-specific and suffer from several important shortcomings including short half-lives, invasive routes of administration, higher dose and frequency requirements, and several dose-related systemic side effects. Hence, discovery of novel agents with improved therapeutic efficacy with respect to survival benefits and the development of non-invasive routes of administration are in critical need. Current research aimed at developing more selective therapies for PAH are focused both on agents acting on novel molecular targets, as well as, novel compounds acting on conventional pathways with improved characteristics. Area covered: The present review covers recently filed (issued/application) patents (2010-2016) describing novel agents acting on investigational targets as well as novel compounds with improved characteristics acting on established targets. Patents describing combinations of conventional and investigational compounds are also discussed. Expert opinion: PAH has recently been considered as cancer-like disease with over-proliferation of pulmonary arterial smooth muscle cells and endothelial cells. New cellular and molecular biological advances have revealed novel target/pathways involved in the pathogenesis and progression of PAH. Thus, discovery of agents that act on these novel pathways provides a promising avenue of research for improving therapeutic approaches for PAH.

Structural distinctions in BMPs underlie divergent signaling in spinal neurons.

BACKGROUND: In dorsal spinal neurons and monocytes, bone morphogenetic protein (BMP)7 activates distinct transduction pathways, one leading to inductive specification and the other to axon orientation and chemotaxis. BMP7-evoked induction, also stimulated by the closely related BMP6, acts through a Smad cascade, leading to nuclear signaling, and is not BMPR subunit selective. Orientation is evoked by BMP7, but not by BMP6, through PI3K-dependent cytoskeletal activation mediated by the type II BMPRs, ActRIIA and BMPRII and is independent of the Smad cascade. The responses can be stimulated concurrently and suggest that BMP7, but not BMP6, can selectively activate BMPR subunits that engage the divergent paths. Although structural and biochemical analyses of selected BMP/BMPR interfaces have identified key regions of interaction, how these translate into function by related BMPs is poorly understood. To determine the mechanisms underlying the distinct activities of BMP7 and the disparate properties of BMP7 and BMP6 in spinal cord development, we have performed a family-wide structure/function analysis of BMPs and used the information to predict and test sites within BMPs that may control agonist properties, in particular the ability of a BMP to orient axons, through interactions with BMPRs. RESULTS: We demonstrate that whereas all BMPs can induce dorsal neurons, there is selectivity in the ability also to orient axons or evoke growth cone collapse. The degree to which a BMP orients is not predictable by overall protein similarity with other BMPs but comparison of sequences of potent and weakly orienting BMPs with that of the non-orienting BMP6 revealed three candidate positions within the BMPs at which the amino acid residues may confer or obstruct orienting ability. Residue swapping analysis has identified one residue, Gln48 in BMP6, that blocks axon orienting ability. Replacing Gln48 with any of the amino acids present at the equivalent residue position in the orienting subset of BMPs confers orienting activity on BMP6. Conversely, swapping Gln48 into BMP7 reduces orienting ability. The inductive capacity of the BMPs was unchanged by these residue swaps. CONCLUSIONS: The results suggest that the presence of the Gln48 residue in BMP6 is structurally inhibitory for BMP/BMPR interactions that result in the activation of intracellular signaling, leading to axon orientation. Moreover, since residue 48 in BMP7 and the corresponding residue in BMP2 are important for type II BMPR binding, our results provide a basis for a mechanistic understanding of the diverse activities of BMPs in spinal cord development.

Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways.

BACKGROUND: Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. RESULTS: We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. CONCLUSIONS: Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway.

ActRIIA and BMPRII Type II BMP receptor subunits selectively required for Smad4-independent BMP7-evoked chemotaxis.

Bone morphogenetic protein (BMP)-evoked reorientation and chemotaxis of cells occurs with rapid onset and involves events local to the cell membrane. The signaling pathways underlying these rapid processes likely diverge from those mediating classical transcriptional responses to BMPs but it remains unclear how BMP receptors are utilized to generate distinct intracellular mechanisms. We show that BMP7-evoked chemotaxis of monocytic cells depends on the activity of canonical type II BMP receptors. Although the three canonical type II BMP receptors are expressed in monocytic cells, inhibition of receptor subunit expression by RNAi reveals that ActRIIA and BMPRII, but not ActRIIB, are each essential for BMP7-evoked chemotaxis but not required individually for BMP-mediated induction. Furthermore, the chemotactic response to BMP7 does not involve canonical Smad4-dependent signaling but acts through PI3K-dependent signaling, illustrating selective activation of distinct intracellular events through differential engagement of receptors. We suggest a model of a BMP receptor complex in which the coordinated activity of ActRIIA and BMPRII receptor subunits selectively mediates the chemotactic response to BMP7.