The Toll-like receptor 4 antagonist TAK-242 induces antidepressant-like effects in a rat learned helplessness model of depression through BDNF-TrkB signaling and AMPA receptor activation.

Finding from animal models of depression indicated that Toll-like receptor 4 (TLR4) is associated with the pathophysiology of depression. Herein, the TLR4 antagonists TAK-242 and baicalin induced antidepressant-like effects in a rat learned helplessness model of depression. The antidepressant-like effects of both TLR4 antagonists were blocked by the TrkB inhibitor ANA-12. Also, the antidepressant-like effects of TAK-242 were blocked by the treatment with AMPA receptor antagonist NBQX. The antidepressant-like effects of the TLR4 antagonist TAK-242 involves BDNF-TrkB signaling and AMPA receptor activation.

Structural Optimization and Structure-Activity Relationship Studies of 6,6-Dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one Derivatives as A New Class of Potent Inhibitors of Pan-Trk and Their Drug-Resistant Mutants.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Design, synthesis and biological evaluation of macrocyclic derivatives as TRK inhibitors.

Tropomyosin receptor kinases (TRKA, TRKB, TRKC) are transmembrane receptor tyrosine kinases, which are respectively encoded by NTRK1, NTRK2, and NTRK3 genes. Herein, we reported the design, synthesis and Structure-Activity Relationship (SAR) investigation of a series of macrocyclic derivatives as new TRK inhibitors. Among these compounds, compound 9e exhibited strong kinase inhibitory activity (TRKG595R IC50 = 13.1 nM) and significant antiproliferative activity in the Ba/F3-LMNA-NTRK1 cell line (IC50 = 0.080 µM) and compound 9e has shown a better inhibitory effect (IC50 = 0.646 µM) than control drug LOXO-101 in Ba/F3-LMNA-NTRK1-G595R cell line. These results indicate that compound 9e is a potential TRK inhibitor for further investigation.

Discovery of the Next-Generation Pan-TRK Kinase Inhibitors for the Treatment of Cancer.

The neurotrophic receptor tyrosine kinase (NTRK) genes including NTRK1, NTRK2, and NTRK3 encode the tropomyosin receptor kinase (Trk) proteins TrkA, TrkB, and TrkC, respectively. So far, two TRK inhibitors, larotrectinib sulfate (LOXO-101 sulfate) and entrectinib (NMS-E628, RXDX-101), have been approved for clinical use in 2018 and 2019, respectively. To overcome acquired resistance, next-generation Trk inhibitors such as selitrectinib (LOXO-195) and repotrectinib (TPX-0005) have been developed and exhibit effectiveness to induce remission in patients with larotrectinib treatment failure. Herein, we report the identification and optimization of a series of macrocyclic compounds as potent pan-Trk (WT and MT) inhibitors that exhibited excellent physiochemical properties and good oral pharmacokinetics. Compound 10 was identified via optimization from the aspects of chemistry and pharmacokinetic properties, which showed good activity against wild and mutant TrkA/TrkC in in vitro and in vivo studies.

TrkB/BDNF Signaling Could Be a New Therapeutic Target for Pancreatic Cancer.

BACKGROUND/AIM: Tropomyosin-related kinase B (TrkB)/brain-derived neurotrophic factor (BDNF) signaling plays a role in inducing malignant phenotypes in several aggressive types of cancers. To create a conclusive therapy targeting TrkB/BDNF signaling in solid refractory cancers, the biological significance of TrkB/BDNF signaling was analyzed in pancreatic ductal adenocarcinoma (PDAC) cells. MATERIALS AND METHODS: Three PDAC cell lines were used as target cells to investigate proliferation and invasiveness. Small interfering RNA (siRNA) and the TrkB tyrosine kinase inhibitor k252a were used as TrkB/BDNF signaling inhibitors. RESULTS: All PDAC cell lines expressed TrkB and BDNF. When TrkB and BDNF were inhibited by siRNA or k252a, the invasiveness of PANC-1 and SUIT-2 cells significantly decreased. When TrkB was inhibited by siRNA or k252a, proliferation was significantly inhibited in PDAC cells. CONCLUSION: TrkB/BDNF signaling may be a new therapeutic target for PDAC. Therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy for refractory solid cancer.

Evolving role of entrectinib in treatment of NTRK-positive tumors.

Targeted therapy has shown to be a very effective treatment in tumors with specific genomic drivers. Trk has proven to be one such target. Efforts to target the Trk fusion with specific inhibitors have shown remarkable responses in a tumor agnostic fashion, with responses seen even in patients with intracranial metastasis. Entrectinib is a first-generation Trk inhibitor with impressive activity in early phase trials performed in patients with NTRK fusion positive solid tumors and ROS1 positive non-small-cell lung cancers with subsequent approval for those indications. Entrectinib was also found to be effective in treatment of brain metastasis and generally well tolerated.

Lay abstract Advances in medical science has allowed us to analyze genes within cancer cells and target abnormal genes more precisely. One such target is called NTRK, which carries genetic information and has been targeted using a medication called entrectinib. This medication is also very effective in patients with cancers that has spread to the brain. This medication can be used in any type of cancer if the cancer cells possess the abnormal DNA. Some of the side effects of entrectinib include weight gain, lightheadedness, throwing up, taste changes, swelling of legs, lack of energy and so on. Based on the benefit of entrectinib seen in clinical trials the medication was approved by the US FDA for treatment of any type of cancer with the NTRK problem. We hope that this new approach to cancer treatment will result in patients having greater benefit and live longer.

Inhibiting BDNF/TrkB.T1 receptor improves resiniferatoxin-induced postherpetic neuralgia through decreasing ASIC3 signaling in dorsal root ganglia.

BACKGROUND: Postherpetic neuralgia (PHN) is a devastating complication after varicella-zoster virus infection. Brain-derived neurotrophic factor (BDNF) has been shown to participate in the pathogenesis of PHN. A truncated isoform of the tropomyosin receptor kinase B (TrkB) receptor TrkB.T1, as a high-affinity receptor of BDNF, is upregulated in multiple nervous system injuries, and such upregulation is associated with pain. Acid-sensitive ion channel 3 (ASIC3) is involved in chronic neuropathic pain, but its relation with BDNF/TrkB.T1 in the peripheral nervous system (PNS) during PHN is unclear. This study aimed to investigate whether BDNF/TrkB.T1 contributes to PHN through regulating ASIC3 signaling in dorsal root ganglia (DRGs). METHODS: Resiniferatoxin (RTX) was used to induce rat PHN models. Mechanical allodynia was assessed by measuring the paw withdrawal thresholds (PWTs). Thermal hyperalgesia was determined by detecting the paw withdrawal latencies (PWLs). We evaluated the effects of TrkB.T1-ASIC3 signaling inhibition on the behavior, neuronal excitability, and inflammatory response during RTX-induced PHN. ASIC3 short hairpin RNA (shRNA) transfection was used to investigate the effect of exogenous BDNF on inflammatory response in cultured PC-12 cells. RESULTS: RTX injection induced mechanical allodynia and upregulated the protein expression of BDNF, TrkB.T1, ASIC3, TRAF6, nNOS, and c-Fos, as well as increased neuronal excitability in DRGs. Inhibition of ASIC3 reversed the abovementioned effects of RTX, except for BDNF and TrkB.T1 protein expression. In addition, inhibition of TrkB.T1 blocked RTX-induced mechanical allodynia, activation of ASIC3 signaling, and hyperexcitability of neurons. RTX-induced BDNF upregulation was found in both neurons and satellite glia cells in DRGs. Furthermore, exogenous BDNF activated ASIC3 signaling, increased NO level, and enhanced IL-6, IL-1ß, and TNF-α levels in PC-12 cells, which was blocked by shRNA-ASIC3 transfection. CONCLUSION: These findings demonstrate that inhibiting BDNF/TrkB.T1 reduced inflammation, decreased neuronal hyperexcitability, and improved mechanical allodynia through regulating the ASIC3 signaling pathway in DRGs, which may provide a novel therapeutic target for patients with PHN.

Tissue-Specificity of Antibodies Raised Against TrkB and p75NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases.

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF.

Discovery of pyrazolo-thieno[3,2-d]pyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: Design, synthesis, and biological evaluation.

Tropomyosin receptor kinase (TRK) represents an attractive oncology target for cancer therapy related to its critical role in cancer formation and progression. NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. In this paper, we described the discovery of the type-II pan-TRK inhibitor 4c through the structure-based drug design strategy from the original hits 1b and 2b. Compound 4c exhibited excellent in vitro TRKA, TRKB, and TRKC kinase inhibitory activity and anti-proliferative activity against human colorectal carcinoma derived cell line KM12. In the NCI-60 human cancer cell lines screen, compound 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. Western blot analysis demonstrated that 4c and its urea cousin 4k suppressed the TPM3-TRKA autophosphorylation at the concentrations of 100 nM and 10 nM, respectively. The work presented that 2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamides could serve as a novel scaffold for the discovery and development of type-II pan-TRK inhibitors for the treatment of TRK driven cancers.

Class I HDAC inhibition improves object recognition memory consolidation through BDNF/TrkB pathway in a time-dependent manner.

There is increasing evidence showing that HDACs regulates BDNF (brain-derived neurotrophic factor) expression through its interaction with the Bdnf gene promoter, a key regulator to consolidate memory. Although the nuclear mechanisms regulated by HDACs that control BDNF expression have been partially described recently, the temporal events for memory consolidation remain unknown. Hence, in this work, we studied the temporal pattern for the activation of the BDNF/TrkB pathway through class I HDAC inhibition to enhance object recognition memory (ORM) consolidation. To this end, we inhibited class I HDAC into the insular cortex (IC) and a weak ORM protocol was used to assess temporal expression and function of the BDNF/TrkB pathway in the IC. We found that cortical class I HDAC inhibition enhanced long-term ORM, coincident with a clear peak of BDNF expression at 4 h after acquisition. Furthermore, the tyrosine kinase B (TrkB) receptor blockade at 4 h, but not at 8 h, impaired the consolidation of ORM. These results suggest that histone acetylation regulates the temporal expression of BDNF in cortical circuits potentiating the long-term recognition memory.

Microinjection of the BDNF receptor antagonist ANA-12 into the nucleus accumbens and medial-prefrontal cortex attenuates morphine-induced reward memory, and alterations of BDNF levels and apoptotic cells in rats.

This study was designed to examine the effects of intra- nucleus accumbens (NAc) of BDNF receptor antagonist ANA-12 on the acquisition and expression and intra- medial-prefrontal cortex (mPFC) of ANA-12 on the extinction and reinstatement of morphine-induced conditioned place preference (CPP) and also BDNF levels and apoptotic neurons in the NAc and mPFC of rats. In this study, adult male Wistar rats (200-250 g) were used. Two separate cannulas were inserted bilaterally into the NAc and/or mPFC. ANA-12 (3 µg/0.5 µl/side) was injected into the NAc and/or mPFC to evaluate the rewarding effects of morphine using a CPP paradigm. Then, the levels of BDNF and apoptotic in the NAc and mPFC were assessed at the end of each treatment phase using ELISA and TUNEL methods, respectively. All of vehicle-treated rats following morphine CPP showed the increase of BDNF levels and apoptotic neurons in the NAc and mPFC. ANA-12 significantly attenuated the acquisition and expression of morphine-induced CPP, BDNF levels and apoptotic neurons in the NAc during the acquisition, but not the expression phase. Also, ANA-12 significantly facilitated the extinction, but no effect on reinstatement of morphine CPP, and decreased BDNF levels and apoptotic neurons in the mPFC during the extinction, but not the reinstatement. We conclude that blocking TrkB with ANA-12 showed therapeutic effects on morphine-associated reward memory and neuronal death in the NAc and mPFC induced by morphine CPP. Thus, the BDNF-TrkB signaling may be important in the acquisition, expression, extinction, but not the reinstatement of morphine CPP.

Mulberry fruit improves memory in scopolamine-treated mice: role of cholinergic function, antioxidant system, and TrkB/Akt signaling.

Objectives: Although mulberry fruit possesses some biological activities, it is not known how it protects neuronal cells in neurodegenerative diseases. Here, we examined whether mulberry fruit extract (MFE) protected neuronal cells against oxidative stress-induced neurodegeneration.Methods: In this experiments, glutamate challenged hippocampal neuronal HT-22 cell lines as an in vitro model and scopolamine-induced memoty-impairment mice model were used.Results: MFE improved cell viability and glutathione level as well as reducing reactive oxygen species level in glutamate-treated HT-22 cells. Additionally, MFE suppressed apoptotic bodies and mitochondrial depolarization through regulating expression of apoptosis-related proteins. Furthermore, MFE elevated expression of p-TrkB, p-Akt, p-CREB, BDNF, and antioxidant enzymes as well as nuclear translocation of Nrf2. In contrast, the inclusion of K252a, a TrkB inhibitor, or MK-2206, an Akt selective inhibitor, neutralized the neuroprotective actions of MFE. Separately, MFE attenuated scopolamine-induced amnesia via regulating the activities of enzymes related with cholinergic function and the antioxidant system in mice. Additionally, MFE protected neuronal cells in the hippocampal CA1 and CA3 regions in brain of mice.Conclusions: MFE protects neuronal cells against oxidative stress-induced apoptosis through upregulating the expression of BDNF and antioxidant enzymes by stabilizing the activation of the TrkB/Akt pathway. Such an effect of MFE, which includes rich polyphenols, may provide information for its application as a food supplement for the prevention and treatment of neurodegenerative diseases.

The Secretome of Aged Fibroblasts Promotes EMT-Like Phenotype in Primary Keratinocytes from Elderly Donors through BDNF-TrkB Axis.

Age-related changes in the dermis can play a primary role in tumor initiation promoting the unrestrained proliferation of precancerous keratinocytes (KCs) through cytokines and GF secretion. We found a high percentage of epithelial-to-mesenchymal transition-like colonies raising in primary human KC cultures from old subjects after treatment with aged fibroblast supernatants (SPNs). Continuous extracellular signals were required for maintaining these changes. Conversely, the secretome did not induce epithelial-to-mesenchymal transition-like colonies in KCs from young subjects. SPN-treated aged KCs displayed the activation of pathways involved in the disjunction of cell‒cell adhesion, extracellular matrix remodeling, manifestation of a mesenchymal phenotype, and dedifferentiation programs. Moreover, they recovered proliferation and clonogenic ability and showed enhanced migration. We identified an age-related increase of the BDNF secretion from fibroblasts as well as of the expression of its receptor TrkB in KCs. BDNF treatment of aged KCs induced TrkB phosphorylation and recapitulated the modifications promoted by aged fibroblast SPN. Furthermore, the treatment with a specific antibody against BDNF or a TrkB antagonist inhibited the paracrine signaling preventing SPN-mediated morphological and molecular changes. Finally, BDNF induced signs of matrix invasion in a three-dimensional organotypic model. Therefore, we demonstrate that aged fibroblast SPN promotes phenotypic plasticity in KCs from the elderly through BDNF-TrkB axis.

Inhibiting NTRK2 signaling causes endometriotic lesion regression.

Endometriosis is a common gynecological disease in reproductive-age women. Although the hormone-dependent therapy is the first line treatment for endometriosis, it is not a curative regimen and associated with severe side-effects, which significantly decrease the life quality of affected individuals. To seek a target for treatment of endometriosis, we focused on plasma membrane proteins that are elevated in ectopic cells and exert beneficial effects in cell growth and survival. We performed bioinformatics analysis and identified the neurotrophic receptor tyrosine kinase 2 (NTRK2) as a potential candidate for treatment. The expression levels of NTRK2 were markedly upregulated in the lesions of clinical specimen as well as in the mouse endometriotic-like lesion. Mechanistic investigation demonstrated that upregulation of NTRK2 is induced by hypoxia in a hypoxia-inducible factor 1 alpha-dependent manner. Knockdown of NTRK2 or administration of ANA-12, a selective antagonist of NTRK2, significantly induced endometriotic stromal cells death, suggesting it may be a potential therapeutic agent. In vivo study using surgery-induced endometriosis mice model showed ANA-12 (1.5 mg/kg body weight) treatment induced apoptosis of endometriotic cells and caused the regression of ectopic lesions. Taken together, our findings suggest a possible mechanism responsible for the aberrant expression of NTRK2 in endometriotic lesions and this may be involved in the pathogenesis of endometriosis.

Rapid-acting and long-lasting antidepressant-like action of (R)-ketamine in Nrf2 knock-out mice: a role of TrkB signaling.

The transcription nuclear factor-erythroid factor 2-related factor 2 (Nrf2) plays a key role in inflammation that is involved in depression. We previously reported that Nrf2 knock-out (KO) mice exhibit depression-like phenotypes through systemic inflammation. (R)-ketamine, an enantiomer of ketamine, has rapid-acting and long-lasting antidepressant-like effects in rodents. We investigated whether (R)-ketamine can produce antidepressant-like effects in Nrf2 KO mice. Effects of (R)-ketamine on the depression-like phenotypes in Nrf2 KO mice were examined. Furthermore, the role of TrkB in the antidepressant-like actions of (R)-ketamine was also examined. In the tail-suspension test (TST) and forced swimming test (FST), (R)-ketamine (10 mg/kg) significantly attenuated the increased immobility times of TST and FST in the Nrf2 KO mice. In the sucrose preference test (SPT), (R)-ketamine significantly ameliorated the reduced preference of SPT in Nrf2 KO mice. Decreased expression of synaptic proteins (i.e., GluA1 and PSD-95) in the medial prefrontal cortex (mPFC) of Nrf2 KO mice was significantly ameliorated after a single injection of (R)-ketamine. Furthermore, the pre-treatment with the TrkB antagonist ANA-12 (0.5 mg/kg) significantly blocked the rapid and long-lasting antidepressant-like effects of (R)-ketamine in Nrf2 KO mice. Furthermore, ANA-12 significantly antagonized the beneficial effects of (R)-ketamine on decreased expression of synaptic proteins in the mPFC of Nrf2 KO mice. These findings suggest that (R)-ketamine can produce rapid and long-lasting antidepressant-like actions in Nrf2 KO mice via TrkB signaling.

FDA Approval Summary: Entrectinib for the Treatment of NTRK gene Fusion Solid Tumors.

The FDA-approved entrectinib on August 15, 2019, for the treatment of adult and pediatric patients 12 years of age and older with solid tumors that have a neurotrophic tyrosine receptor kinase (NTRK) gene fusion without a known acquired resistance mutation, are metastatic or where surgical resection is likely to result in severe morbidity, and have progressed following treatment or have no satisfactory alternative therapy. Approval was based on demonstration of a durable overall response rate of 57% (95% confidence interval: 43-71), including a complete response rate of 7%, among 54 entrectinib-treated patients with 10 different tumor types harboring an NTRK fusion enrolled in one of three single-arm clinical trials. The durations of response ranged from 2.8 months to 26.0+ months; 68% of responses lasted ≥ 6 months. The most serious toxicities of entrectinib are congestive heart failure, central nervous system effects, skeletal fractures, hepatotoxicity, hyperuricemia, QT prolongation, and vision disorders. Adverse reactions were manageable through dose interruptions (46%), dose reductions (29%), or discontinuation of entrectinib (9%). This is the third approval of a cancer drug for treatment of a tissue agnostic, biomarker-defined cancer.

Brain-Derived Neurotrophic Factor Regulates Ishikawa Cell Proliferation through the TrkB-ERK1/2 Signaling Pathway.

(1) Background: Endometrial regulation is a necessary condition for maintaining normal uterine physiology, which is driven by many growth factors. Growth factors produced in the endometrium are thought to be related to the proliferation of endometrial cells induced by estradiol-17ß (E2). In this study, we found that E2 can induce the secretion of brain-derived neurotrophic factor (BDNF) in Ishikawa cells (the cells of an endometrial cell line). Furthermore, Ishikawa cells were used in exploring the regulatory role of BDNF in endometrial cells and to clarify the potential mechanism. (2) Methods: Ishikawa cells were treated with different concentrations of BDNF (100, 200, 300, 400, and 500 ng/mL). The mRNA expression levels of various proliferation-related genes were detected through quantitative reverse transcription polymerase chain reaction, and the expression of various proliferation-related genes was detected by knocking out BDNF or inhibiting the binding of BDNF to its receptor TrkB. The expression levels of various proliferation-related genes were detected by performing Western blotting on the TrkB-ERK1/2 signaling pathway. (3) Results: Exogenous BDNF promoted the growth of the Ishikawa cells, but the knocking down of BDNF or the inhibition of TrkB reduced their growth. Meanwhile, BDNF enhanced cell viability and increased the expression of proliferation-related genes, including cyclin D1 and cyclin E2. More importantly, the BDNF-induced proliferation of the Ishikawa cells involved the ERK1/2 signaling pathway. (4) Conclusions: The stimulating effect of exogenous E2 on the expression of BDNF in the uterus and the action of BDNF promoted the proliferation of the Ishikawa cells through the TrkB-ERK1/2 signal pathway.

Cholic Acid Protects In Vitro Neurovascular Units against Oxygen and Glucose Deprivation-Induced Injury through the BDNF-TrkB Signaling Pathway.

Ischemic stroke (IS) can disrupt various types of brain cells in the neurovascular unit (NVU) at both the structural and functional levels. Therefore, NVU is considered to be a more comprehensive target for the treatment of IS. It is necessary to develop drugs which targeted multiple mechanisms and cell types on NVU against IS. As a component of bile acid, cholic acid has been reported to be able to diffuse across phospholipid bilayers and further cross the blood-brain barrier (BBB). However, the effects exerted by cholic acid (CA) on the NVU after stroke remain unclear. Based on our previous research, we established and further supplemented the characteristics of the functional in vitro NVU model and its oxygen-glucose deprivation and reoxygenation (OGD/R) model. Then, we investigated the effect of CA on the maintenance of the in vitro NVU after OGD/R and further discussed the specific molecular targets that CA played a role in. For the first time, we found that CA significantly maintained BBB integrity, downregulated apoptosis, and mitigated oxidative stress and inflammation damage after OGD/R. Meanwhile, CA obviously increased the levels of brain-derived neurotrophic factor (BDNF), which were mainly secreted from astrocytes, in the coculture system after OGD/R. The results demonstrated that CA significantly increased the expression of TrkB, PI3K/Akt, MAPK/Erk, and CREB in neurons. These positive effects on the downstream proteins of BDNF were suppressed by treatment with ANA12 which is an inhibitor of TrkB. In conclusion, the present study demonstrates that CA exerted multiple protective effects on the NVU, mediated by increasing the release of BDNF and further stimulating the BDNF-TrkB-PI3K/Akt and BDNF-TrkB-MAPK/Erk signaling pathways in the context of OGD/R-induced injury. These findings indicate that CA possesses the effect of antagonizing multiple mechanisms of IS and protecting multiple cell types in NVU and may be useful as a treatment for IS.

Discovery of First-In-Class Potent and Selective Tropomyosin Receptor Kinase Degraders.

We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice. Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis. Our study also paves the way for pharmacological degradation of TRK.

2,3,5,4'-tetrahydoxystilbene-2-O-ß-D-glucoside eliminates staurosporine-induced cytotoxicity by restoring BDNF-TrkB/Akt signaling axis.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (THSG) is the major active ingredient in Plygonum multiflorum that displays a great deal of health-benefits including anti-oxidation, anti-hyperlipidemia, anti-cancer, anti-inflammation and neuroprotection. However, it is unclear whether THSG exerts neuroprotective functions by regulating neurotrophic factors and their associated signaling pathways. In this study, hippocampal neurons were challenged with staurosporine (STS) to establish a neural damage model. We found that STS-induced cytotoxicity introduced significant morphological collapse and initiating cell apoptosis, along with the down regulation of BDNF and TrkB/Akt signaling axis. In contrast, neurons pretreated with THSG showed resistance to STS-induced toxicity and maintained cell survival. THSG rescued STS induced dysfunctions of BDNF and its associated TrkB/Akt signaling, and restored the expression of Bcl-2 and Caspase-3. However, inhibition of TrkB activity by K252a or Akt signaling by LY294002 abolished the neuroprotective effects of THSG. Therefore, BDNF and TrkB/Akt signaling axis is a promise target for THSG mediated neuroprotective functions.