Discovery of novel TrkA allosteric inhibitors: Structure-based virtual screening, biological evaluation and preliminary SAR studies.

Tropomyosin receptor kinases A (TrkA) is a potential therapeutic target for the treatment of numerous tumor types and chronic pain. However, most of the reported TrkA inhibitors are ATP competitive pan-Trks inhibitors that lack subtype selectivity. A selective TrkA inhibitor may provide valuable therapeutic benefits. Here, we described the discovery of novel TrkA allosteric inhibitors by structure-based virtual screening. A promising hit (D5261, TrkA cell IC50 = 3.32 µM) was selected for further studies. The binding free energy between TrkA and D5261 was calculated. In addition, the preliminary structure-activity relationship (SAR) studies with D5261 were investigated. The results suggest that D5261 can be used as a starting point for development of TrkA allosteric inhibitors.

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.

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.

Identification and characterization of the Cucurbitacins, a novel class of small-molecule inhibitors of Tropomyosin receptor kinase a.

BACKGROUND: NGF-TrkA is well known to play a key role in propagating and sustaining pruritogenic signals, which form the pathology of chronic pruritus. Inhibition of NGF-TrkA is a known strategy for the treatment of pruritus. In the present paper, we describe the identification, in vitro characterization, structure-activity analysis, and inhibitory evaluation of a novel TrkA inhibitory scaffold exemplified by Cucurbitacins (Cus). METHODS: Cus were identified as TrkA inhibitors in a large-scale kinase library screen. To obtain structural models of Cus as TrkA inhibitors, AutoDock was used to explore their binding to TrkA. Furthermore, PC12 cell culture systems have been used to study the effects of Cus and traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) extracts on the kinase activity of TrkA. RESULTS: Cus block the phosphorylation of TrkA on several tyrosine sites, including Tyr490, Tyr674/675, and Tyr785, and inhibit downstream Akt and MAPK phosphorylation in response to NGF in PC12 cell model systems. Furthermore, traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) containing Cu extracts were shown to inhibit the phosphorylation of TrkA and Akt. These data reveal mechanisms, at least partly, of the anti-pruritus bioactivity of Cus. CONCLUSION: Taken together, with the recent discovery of the important role of TrkA as a therapeutic target, Cus could be the basis for the design of improved TrkA kinase inhibitors, which could someday help treat pruritus.

Emerging Treatments and Novel Pathways in Pruritus.

Itch treatment is a major challenge in the dermatologist's practice. We encounter patients suffering from pruritus on a regular basis, and often lack diverse treatment options to adequately respond to the patients' needs. In the last 20 years, novel pathways have been investigated that were beyond the scope of histamine. Although most did not result in a molecule available on the Canadian market, it is interesting and important as health care providers to stay up to date with new neuronal pathways involved in itch transmission and potential new therapeutic options. In this review, we will discuss pathways targeted in new topical treatments such as antagonist of proteinase-activated receptor-2, the endocannabinoid system, neurotrophins and tropomyosin-related kinase A receptor, the transient receptor potential-vanilloid or transient receptor potential-melastatine ion channels. New systemic therapies are now focusing on antagonizing the neurokinin receptor, modulating the opioidergic system, or targeting itch cytokines such as interleukin-31.

TRK Inhibitors: Clinical Development of Larotrectinib.

PURPOSE OF REVIEW: In this review, we highlight the pre-clinical development, recent clinical studies, and future directions of larotrectinib in patients with NTRK fusion-positive tumors. RECENT FINDINGS: The tropomyosin receptor kinase family, TrkA, TrkB, and TrkC, transmit extracellular signals via a variety of intracellular pathways to promote normal neuronal development. TrkA, B, and C are encoded by NTRK1, 2, and 3, respectively. NTRK chromosomal alterations, most commonly gene fusions, have been identified as driver mutations in a broad range of malignancies. Small molecule tyrosine kinase inhibitors of Trk, including larotrectinib, have shown broad clinical activity across multiple tumor types with NTRK fusion events. Although the prevalence of NTRK alterations is low, the exceptional activity of larotrectinib makes NTRK alterations an important predictive biomarker to screen for in any cancer.

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors.

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.

A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement.

Tropomyosin-related kinase A (TRKA) fusion was originally detected in colorectal carcinoma that had resulted in expression of the oncogenic chimeric protein TPM3-TRKA. Lately, many more rearrangements in TRK family of kinases generating oncogenic fusion proteins have been identified. These genetic rearrangements usually result in fusion of cytoplasmic kinase domain of TRK to another gene of interest resulting in constitutive kinase activity. Estimation of TRK inhibitor potency in a cellular context is required for drug discovery programs and is measured by receptor phosphorylation levels upon compound administration. However, since a large chunk of the TRK protein is lost in this rearrangement, it's difficult to set up sandwich ELISA for detection of receptor phosphorylation in any cell assay harboring these fusion proteins. In order to address this issue, we developed a novel and robust in-cell ELISA method which quantifies the phosphorylation of TRK kinase (Tyr 674/675) within the KM12 cells. This cell based method is more versatile & economical than conventional ELISA using engineered overexpressing cell line and/or western blot methods. Performance reliability & robustness for the validated assay were determined by %CV and Z factor in assays with reference molecule larotrectinib. This in-cell ELISA method can be used with any TRKA rearranged oncogenic fusion cell type and can be extended to other TRK isoforms as well. We have used this assay to screen novel molecules in KM12 cells and to study pharmacodynamic properties of compounds in TRKA signaling.

The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Accumulating evidence indicates that neurotrophic factor-like substances involved in the induction of neurotrophic factor synthesis may aid in the treatment of neurological disorders, such as Alzheimer's disease. Yokukansan (YKS), a traditional Kampo medicine, has been used for the treatment of anxiety and mood disorders. In the present study, we aimed to identify the signaling pathways associated with YKS-mediated enhancement of nerve growth factor (NGF)-induced neurite extension in rat pheochromocytoma (PC12) cells. Akt and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation levels were assessed by western blot analysis, in the presence of YKS and following the treatment with TrkA inhibitor, K252a. YKS treatment (NGF+YKS 0.5 group) enhanced NGF-induced neurite outgrowth and phosphorylation/activation of Akt and ERK1/2 in PC12 cells. Moreover, YKS-induced effects were inhibited by the treatment with the TrkA receptor antagonist K252a (NGF+YKS 0.5+K252a group); no significant difference in neurite outgrowth was observed between K252a-treated (NGF+YKS 0.5+K252a group) and NGF-K252a-treated cells (NGF+K252a group). However, neurite outgrowth in K252a-treated cells (NGF+K252a and NGF+YKS 0.5+K252a group) reached only one-third of the level in NGF-treated cells (NGF group). NGF-mediated Akt phosphorylation increased by YKS was also inhibited by K252a treatment (NGF+YKS 0.5+K252a group), but no significant difference in ERK1/2 phosphorylation was observed between NGF-YKS-K252a- and NGF-treated cells (NGF group). Our results indicate that YKS treatment enhanced NGF-induced neurite outgrowth via induction of Akt and ERK1/2 phosphorylation, following the binding of NGF to the TrkA receptor. These findings may be useful in the development of novel therapeutic strategies for the treatment of Alzheimer's disease.

Role of TrkA signalling and mast cells in the initiation of osteoarthritis pain in the monoiodoacetate model.

OBJECTIVE: Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). METHOD: In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D2 (PGD2) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. RESULTS: In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD2 production. Finally, we observed that a PGD2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. CONCLUSION: Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

New targets bring hope in squamous cell lung cancer: neurotrophic tyrosine kinase gene fusions.

Neurotrophic tyrosine kinase genes encode for the Trk-family proteins TrkA, TrkB, and TrkC, which have an important role in the development of the nervous system; however, they have been identified as oncogenic fusions in solid tumors (NTK-1, NTRK-2, and NTRK-3) and are associated with poor survival in lung cancer. These three new fusions can be detected by fluorescent in situ hybridization or next-generation sequencing in less than 5% of the lung tumors. There are several ongoing clinical trials of NTRK oncogenes in lung cancer and other tumors. The agents entrectinib (RXDX-101), a multi-kinase small molecule inhibitor that selectively inhibits NTRK1, NTRK2, and NTRK3, ROS1 and ALK, and LOXO-101, an ATP-competitive pan-NTRK inhibitor, have shown responses in patients with lung cancer with an acceptable toxicity profile. Although these oncogenic fusions are not very prevalent, the high prevalence of lung cancer makes these findings very relevant and suggests the feasibility of these oncogenes as targets in lung cancer. New data from Ozono and collaborators presented in this issue suggest that BDNF/TrkB signal promotes proliferating migratory and invasive phenotypes and cellular plasticity in squamous cell carcinoma (SCC) of the lung but that it also represents a druggable target that may bring hope to squamous lung cancer patients.

Kai-Xin-San series formulae alleviate depressive-like behaviors on chronic mild stressed mice via regulating neurotrophic factor system on hippocampus.

Kai-xin-san (KXS) is a famous Chinese medicinal formula applied for treating stress-related psychiatric diseases with the symptoms such as depression, forgetfulness and dizziness. In clinic, the composition ratio of KXS is always varied and KXS series formulae are created. Here, we aim to compare the anti-depressive effect of different ratios of KXS and reveal its action mechanism on regulation of neurotrophic factor system. Firstly, daily intra-gastric administration of chemically standardized extracts of KXS series formulae for seven days significantly alleviated the depressive symptoms of chronic unpredictable mild stressed mice displayed by enhanced sucrose consumptions and decreased immobile time of forced swimming coupled with increased locomotor activities. KXS might fulfill this effect by up-regulating the expressions of NGF, BDNF and Trk receptors in hippocampus, which were confirmed by the treatment of corresponding blockers tPA-stop and K252a. The ratio with higher amounts of Ginseng Radix et Rhizoma and Polygalae Radix exerted most profound effect on anti-depression and regulation enzymes in metabolic pathway of neurotrophic factors. These findings suggested that KXS was beneficial for enhancing supplies, up-regulating receptors, and restoring the dysfunction of metabolic pathway of neurotrophic factors, which might account for its anti-depression effect.

Structural characterization of nonactive site, TrkA-selective kinase inhibitors.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis.

OBJECTIVES: Tropomyosin receptor kinase A (TrkA) mediates nociceptor sensitisation by nerve growth factor (NGF), but it is unknown whether selective TrkA inhibition will be an effective strategy for treating osteoarthritis (OA) pain. We determined the effects of a TrkA inhibitor (AR786) on pain behaviour, synovitis and joint pathology in two rat OA models. METHODS: Knee OA was induced in rats by intra-articular monosodium-iodoacetate (MIA) injection or meniscal transection (MNX) and compared with saline-injected or sham-operated controls. Pain behaviour was assessed as weight-bearing asymmetry and paw withdrawal threshold to punctate stimulation. Oral doses (30 mg/kg) of AR786 or vehicle were administered twice daily in either preventive (day -1 to -27) or treatment (day 14-28) protocols. Effect maintenance was evaluated for 2 weeks after treatment discontinuation. Alterations in knee structure (cartilage, subchondral bone and synovium) were examined by macroscopic visualisation of articular surfaces and histopathology. RESULTS: Preventive AR786 treatment inhibited pain behaviour development and therapeutic treatment attenuated established pain behaviour. Weight-bearing asymmetry increased 1 week after treatment discontinuation, but remained less than in vehicle-treated arthritic rats, whereas paw withdrawal thresholds returned to levels of untreated rats within 5 days of treatment discontinuation. AR786 treatment reduced MIA-induced synovitis and did not significantly affect osteochondral pathology in either model. CONCLUSIONS: Blocking NGF activity by inhibiting TrkA reduced pain behaviour in two rat models of OA. Analgesia was observed both using preventive and treatment protocols, and was sustained after treatment discontinuation. Selective inhibitors of TrkA therefore hold potential for OA pain relief.

Topical TrkA Kinase Inhibitor CT327 is an Effective, Novel Therapy for the Treatment of Pruritus due to Psoriasis: Results from Experimental Studies, and Efficacy and Safety of CT327 in a Phase 2b Clinical Trial in Patients with Psoriasis.

Pruritus is an important symptom in psoriasis with no targeted treatment. Tropomyosin-receptor kinase A (TrkA) is associated with pruritus and psoriatic plaque formation. We report the efficacy of a TrkA inhibitor, CT327, on pruritus in psoriasis. A randomised, double-blind, vehicle-controlled Phase 2b clinical trial was conducted in 160 subjects. No effect was found on psoriasis severity using Investigator's Global Assessment (primary endpoint). However, clinically and statistically significant reductions in pruritus were observed in the 108 patient subset reporting at least moderate pruritus at baseline (37.1 mm visual analogue scale improvement (95% CI [-37.5, -6.2], p = 0.0067) for lowest dose; secondary endpoint). Significant modified Psoriasis Area and Severity Index reductions were found in this subset (p < 0.05). Experiments exploring capsaicin-mediated calcium influx, important in pruritus signalling, were performed in sensory neurons. CT327 inhibited capsaicin responses, indicating action at the nerve growth factor-TrkA-TRPV1 pathway. TrkA is a key target in pruritus, and CT327 has potential to become an effective and safe first-in-class treatment.

Maximizing diversity from a kinase screen: identification of novel and selective pan-Trk inhibitors for chronic pain.

We have identified several series of small molecule inhibitors of TrkA with unique binding modes. The starting leads were chosen to maximize the structural and binding mode diversity derived from a high throughput screen of our internal compound collection. These leads were optimized for potency and selectivity employing a structure based drug design approach adhering to the principles of ligand efficiency to maximize binding affinity without overly relying on lipophilic interactions. This endeavor resulted in the identification of several small molecule pan-Trk inhibitor series that exhibit high selectivity for TrkA/B/C versus a diverse panel of kinases. We have also demonstrated efficacy in both inflammatory and neuropathic pain models upon oral dosing. Herein we describe the identification process, hit-to-lead progression, and binding profiles of these selective pan-Trk kinase inhibitors.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Discovery of novel isothiazole inhibitors of the TrkA kinase: structure-activity relationship, computer modeling, optimization, and identification of highly potent antagonists.

The design, synthesis, and biological evaluation of potent inhibitors of the TrkA kinase is presented. A homology model is created to aid in the enhancement of potency and selectivity of isothiazole inhibitors found during a high-throughput screen. Three different syntheses are utilized to make diverse analogs within this series. Aminoheterocycles are found to be good urea surrogates, whereas bicyclic substituents on the C3 thio group were found to be extremely potent TrkA inhibitors in kinase and cell assays.

Effect of electroacupuncture on TRPM7 mRNA expression after cerebral ischemia/reperfusion in rats via TrkA pathway.

The effect of electroacupuncture (EA) on TRPM7 mRNA expression of focal cerebral ischemia in rats and further the role of EA in the relationship between TRPM7 and trkA pathway was investigated. Thirty SD rats were randomly divided into 5 groups : normal group, ischemia/reperfusion group, EA treated group (ischemic rats with EA treatment), TE infusion group (ischemic rats with EA treatment and TE buffer infusion), AS-ODN group (ischemic rats with EA treatment and antisense trkA oligonucleotide infusion). The stroke animal model was established by the modified method of middle cerebral artery occlusion. Antisense trkA oligonucleotide that blocked NGFs effects was injected into cerebroventricle before EA. The TRPM7 mRNA was detected by RT-PCR method. The results showed that there were low TRPM7 mRNA levels in cortex and hippocampus in normal group. Compared with normal group, TRPM7 mRNA expression was increased significantly in ischemia/reperfusion group (P<0.05). A significant reduction in the expression of TR-PM7 mRNA was found in EA treated group in contrast to ischemia/reperfusion group (P<0.05). The expression of TRPM7 mRNA in AS-ODN group was remarkably increased compared with EA treated group and TE infusion group (P<0.05). The results indicated that TRPM7 channels in the ischemic cortex and hippocampus in rats might play a key role in ischemic brain injury. EA could reverse the overexpression of TRPM7 in cerebral ischemia/reperfusion rats. And the inhibitory effect of EA on TRPM7 channels might be through trkA pathway.