REarranged During Transfection Kinase Inhibitors Research Articles

Generating Potential RET-Specific Inhibitors Using a Novel LSTM Encoder-Decoder Model.

The receptor tyrosine kinase RET (rearranged during transfection) plays a vital role in various cell signaling pathways and is a critical factor in the development of the nervous system. Abnormal activation of the RET kinase can lead to several cancers, including thyroid cancer and non-small-cell lung cancer. However, most RET kinase inhibitors are multi-kinase inhibitors. Therefore, the development of an effective RET-specific inhibitor continues to present a significant challenge. To address this issue, we built a molecular generation model based on fragment-based drug design (FBDD) and a long short-term memory (LSTM) encoder-decoder structure to generate receptor-specific molecules with novel scaffolds. Remarkably, our model was trained with a molecular assembly accuracy of 98.4%. Leveraging the pre-trained model, we rapidly generated a RET-specific-candidate active-molecule library by transfer learning. Virtual screening based on our molecular generation model was performed, combined with molecular dynamics simulation and binding energy calculation, to discover specific RET inhibitors, and five novel molecules were selected. Further analyses indicated that two of these molecules have good binding affinities and synthesizability, exhibiting high selectivity. Overall, this investigation demonstrates the capacity of our model to generate novel receptor-specific molecules and provides a rapid method to discover potential drugs.

Precision medicine in endocrinology exemplified by medullary thyroid cancer

Medullary thyroid cancer (MTC) is a prime example for precision medicine in endocrinology and underlines the immediate benefits of basic, translational and healthcare research for patients with a rare disease in clinical . A mutation in the rearranged during transfection (RET) proto-oncogene that codes for a transmembrane receptor protein tyrosine kinase, leads to constitutive activation of the kinase, which is the decisive pathomechanism for the disease. The MTC occurs in a sporadic (somatic RET mutation) or hereditary form (RET germline mutation, multiple endocrine neoplasia types 2 and 3). For germline mutation carriers the timing of preventive thyroidectomy depends on the RET genotype. For advanced metastasized RET-mutant MTC, selective RET kinase inhibitors are available, which are currently considered to be game changers in the treatment. Based on the specific tumor marker calcitonin, MTC can be identified at an early stage during the differential diagnosis of thyroid nodules. The preoperative calcitonin level even enables statements on the degree of dissemination of the disease and on the probability of a cure through surgery. A new development is the consideration of desmoplasia as a histopathological biomarker for the metastatic potential of a MTC, which could possibly modify the operative approach as well as the future MTC nomenclature. Furthermore, the postoperative calcitonin level and the calcitonin doubling time are highly valid prognostic markers for tumor burden and biological aggressiveness of MTC and therefore decisive for patient follow-up. Biochemical, molecular and histological markers enable a risk-adapted surgical treatment and together with new targeted systemic treatments have contributed to a paradigm shift in the diagnostics, prognosis and treatment of MTC in recent years. Endocrine precision medicine for MTC therefore enabled a change from the previous purely symptom-oriented to a modern preventive and individualized treatment.

Preclinical Pharmacokinetics and in vitro Metabolism of FHND5071, a Novel Selective RET Kinase Inhibitor.

Rearranged during transfection (RET) is a transmembrane receptor tyrosine kinase that plays a crucial role in tumorigenesis. FHND5071, a potent and selective RET kinase inhibitor, could exert antitumor effects by inhibiting RET autophosphorylation. The present work aims to profile the pharmacokinetics of FHND5071 in in vivo and in vitro experiments as a ground work for further clinical research. The absorption, distribution, metabolism, and excretion properties of FHND5071 were examined, along with metabolite production and cytochrome P450 (CYP) phenotyping assay. Additionally, plasma protein binding and pharmacokinetics in mice were investigated. Microsomal stability assay corroborated moderate to high clearance of FHND5071, and the use of UPLC-Q-TOF-MS identified a total of six metabolites and suggested a possible metabolic pathway involving oxidation, demethylation, and N-dealkylation. Primary contributors to the CYP-mediated metabolism of FHND5071 were found to be CYP2C8 and CYP3A4, and FHND5071 displayed low permeability and acted as a substrate for the P-glycoprotein (P-gp). FHND5071 had a moderate to high binding in plasma and exhibited a moderate absorption degree (absolute bioavailability > 60%) The distribution of FHND5071 in mouse tissues was rapid (mostly peaking at 1-4 h) and wide (detectable in almost all tissues and organs), with the highest exposure in the spleen. A small fraction of FHND5071 was excreted via the urine and feces, and a presumed metabolic pathway involving 20 metabolites in mice is proposed. Pharmacokinetic characteristics of FHND5071 were systemically profiled, which may lay the foundation for further clinical development as a drug candidate.

Selpercatinib: A Review in Advanced RET Fusion-Positive NSCLC.

Selpercatinib (Retevmo®/Retsevmo®) is an orally-administered, selective inhibitor of rearranged during transfection (RET) kinase approved for the treatment of advanced RET fusion-positive non-small cell lung cancer (NSCLC). In a pivotal phase 1/2 clinical trial in this population, selpercatinib treatment was associated with robust and durable responses, including intracranial responses, in patients previously treated with platinum-based chemotherapy, as well as in treatment-naïve patients. Selpercatinib had a manageable tolerability profile and an acceptable safety profile; adverse events could generally be managed with dose reductions and only a small proportion of patients discontinued selpercatinib due to treatment-related adverse events. The most common treatment-related adverse events that were grade 3-4 in severity were hypertension, elevated alanine aminotransferase and elevated aspartate aminotransferase. Thus, currently available evidence suggests that selpercatinib is a promising new RET-targeted therapy option for patients with advanced RET fusion-positive NSCLC.

1-Methyl-3-((4-(quinolin-4-yloxy)phenyl)amino)-1H-pyrazole-4-carboxamide derivatives as new rearranged during Transfection (RET) kinase inhibitors capable of suppressing resistant mutants in solvent-front regions

REarranged during Transfection (RET) is a validated target for anticancer drug discovery and two selective RET inhibitors were approved by US FDA in 2020. However, acquired resistance mediated by secondary mutations in the solvent-front region of the kinase (e.g. G810C/S/R) becomes a major challenge for selective RET inhibitor therapies. Herein, we report a structure-based design of 1-methyl-3-((4-(quinolin-4-yloxy)phenyl)amino)-1H-pyrazole-4-carboxamide derivatives as new RET kinase inhibitors which are capable of suppressing the RETG810 C/R resistant mutants. One of the representative compounds, 8q, potently suppressed wild-type RET kinase with an IC50 value of 13.7 nM. It also strongly inhibited the proliferation of BaF3 cells stably expressing various oncogenic fusions of RET kinase with solvent-front mutations, e.g. CCDC6-RETG810C, CCDC6-RETG810R, KIF5B-RETG810C and KIF5B-RETG810R, with IC50 values of 15.4, 53.2, 54.2 and 120.0 nM, respectively. Furthermore, 8q dose-dependently inhibited the activation of RET and downstream signals and obviously triggered apoptosis in Ba/F3-CCDC6-RETG810 C/R cells. The compound also exhibited significant anti-tumor efficacy with a tumor growth inhibition (TGI) value of 66.9% at 30 mg/kg/day via i. p. in a Ba/F3-CCDC6-RETG810C xenograft mouse model. Compound 8q may be utilized as a lead compound for drug discovery combating acquired resistance against selective RET inhibitor therapies.

Design and development of photoswitchable DFG-Out RET kinase inhibitors

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase that is required for development of multiple human tissues, but which is also an important contributor to human cancers. RET activation through rearrangement or point mutations occurs in thyroid and lung cancers. Furthermore, activation of wild type RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET is therefore an attractive therapeutic target for small-molecule kinase inhibitors. Non-invasive control of RET signaling with light offers the promise of unveiling its complex spatiotemporal dynamics in vivo. In this work, photoswitchable DFG-out RET kinase inhibitors based on heterocycle-derived azobenzenes were developed, enabling photonic control of RET activity. Based on the binding mode of DFG-out kinase inhibitors and using RET kinase as the test model, we developed a photoswitchable inhibitor with a quinoline “head” constituting the azoheteroarene. This azo compound was further modified by three different strategies to increase the difference in biological activity between the E-isomer and the light enriched Z-isomer. Stilbene-based derivatives were used as model compounds to guide in the selection of substituents that could eventually be introduced to the corresponding azo compounds. The most promising quinoline-based compound showed more than a 15-fold difference in bioactivity between the two isomers in a biochemical assay. However, the same compound showed a decreased Z/E (IC50) ratio in the cellular assay, tentatively assigned to stability issues. The corresponding stilbene compound gave a Z/E (IC50) ratio well above 100, consistent with that measured in the biochemical assay. Ultimately, a 7-azaindole based photoswitchable DFG-out kinase inhibitor was shown to display more than a 10-fold difference in bioactivity between the two isomers, in both a biochemical and a cell-based assay, as well as excellent stability even under reducing conditions.

BOS172738, a highly potent and selective RET inhibitor, for the treatment of RET-altered tumors including RET-fusion+ NSCLC and RET-mutant MTC: Phase 1 study results.

3008 Background: RET (REarranged during Transfection) gene alterations (mutations and fusions) leading to constitutive kinase activity are identified as drivers of disease progression in multiple tumor types, including non-small cell lung cancer (NSCLC) and medullary thyroid cancer (MTC). BOS172738 is an investigational, potent, selective oral RET kinase inhibitor. This next-generation inhibitor was designed with nanomolar potency against RET and >300-fold selectivity against vascular endothelial growth factor receptor 2, to maximize the potential therapeutic window. Methods: NCT03780517 is a phase 1 study consisting of a dose-escalation and dose-expansion phase. During the escalation, 67 patients with RET-altered advanced solid tumors received once-daily oral doses of BOS172738 (10-150 mg). Intra-patient dose escalation was allowed as was over-accrual to dose levels deemed to be safe. Study endpoints were safety (CTCAE v. 4.03), tolerability and confirmed overall response rate (ORR; RECIST 1.1). The data cutoff was Dec. 11, 2020. Results: BOS172738 exhibited a favorable safety profile (n=67) for long-term administration with most treatment-emergent adverse events (TEAEs) classified as grade ≤2 and deemed unrelated to drug. The most common TEAEs were creatinine phosphokinase (CPK) increase (54%), dyspnea (34%), facial edema, aspartate aminotransferase elevation, anemia (25% each), neutropenia, diarrhea (22% each), fatigue (21%), and constipation (20%). BOS172738 was not associated with hypertension or significant hepatoxicity. BOS172738 demonstrated broad anti-tumor activity with an investigator-assessed ORR of 33% (n=18/54), a NSCLC ORR of 33% (n=10/30), MTC ORR of 44% (n=7/16, including 1 complete response) and one patient with RET fusion+ pancreatic cancer reported a partial response. Responders included patients with brain metastases with one patient whose brain lesion decreased by 43%. The median duration of response has not been reached. Many patients remain on study, including the longest of 659 days, at data cutoff. BOS172738 exhibited dose-dependent exposure (AUC, Cmax), rapid absorption (median Tmax 1 to 4.5 h), and an extended half-life (approximately 65 hours) maximizing target coverage. Conclusions: BOS172738 is a highly potent and selective RET inhibitor with a differentiated safety profile and clinical activity against RET-altered tumors, including patients with brain metastases. BOS172738 continues to be evaluated in patients with NSCLC, MTC, and in patients previously treated with other selective RET inhibitors. Clinical trial information: NCT03780517.

First-in-Human Study for a Selective Rearranged During Transfection Tyrosine Kinase Inhibitor, GSK3352589, to Investigate the Safety, Tolerability, and Pharmacokinetics in Healthy Volunteers.

Rearranged during transfection (RET), a neuronal growth factor receptor tyrosine kinase, regulates the development of sympathetic, parasympathetic, motor, and sensory neurons in the enteric nervous system. The intended site of action for GSK3352589 is intestinal tissues. GSK3352589 is an RET kinase inhibitor that was administered in double-blind, randomized, placebo-controlled single-dose (SD) and repeat-dose (RD) studies in healthy subjects to investigate its safety/tolerability and pharmacokinetics. In the SD study (n = 28), GSK3352589 was dosed from 2 to 400 mg, including a food effect arm (25 mg). In the RD study (n = 40), GSK3352589 was dosed for 14 days with food twice daily from 5 to 200mg. With single (fed and fasted) and repeat (fed) doses, bioavailability was low and less than dose-proportional. There was a food effect with 25 mg once daily but may not be clinically relevant. Elimination half-life was ≥17 hours at SD ≥ 15 mg. Accumulation ratios for Cmax , AUCt , AUCtau , and AUC24 after twice-daily dosing to steady state ranged from 1.2 to 1.8 for all doses except the 200-mg dose, which had ratios between 1.9 and 2.7. Administration of GSK3352589 after SD and RD was well tolerated with no safety concerns in healthy subjects.

Molecular Modelling Studies on Pyrazole Derivatives for the Design of Potent Rearranged during Transfection Kinase Inhibitors.

RET (rearranged during transfection) kinase, one of the receptor tyrosine kinases, plays a crucial role in the development of the human nervous system. It is also involved in various cell signaling networks responsible for the normal cell division, growth, migration, and survival. Previously reported clinical studies revealed that deregulation or aberrant activation of RET signaling can cause several types of human cancer. For example, medullary thyroid carcinoma (MTC) and multiple endocrine neoplasia (MEN2A, MEN2B) occur due to sporadic mutation or germline RET mutation. A number of RET kinase inhibitors have been approved by the FDA for the treatment of cancer, such as cabozantinib, vandetanib, lenvatinib, and sorafenib. However, each of these drugs is a multikinase inhibitor. Hence, RET is an important therapeutic target for cancer drug design. In this work, we have performed various molecular modelling studies, such as molecular docking and dynamics simulation for the most active compound of the pyrazole series as RET kinase inhibitors. Furthermore, molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) free energy calculation and 3-dimensional quantitative structure–activity relationship (3D-QSAR) were performed using g_mmpbsa and SYBYL-X 2.1 package. The results of this study revealed the crucial binding site residues at the active site of RET kinase and contour map analysis showed important structural characteristics for the design of new highly active inhibitors. Therefore, we have designed ten RET kinase inhibitors, which showed higher inhibitory activity than the most active compound of the series. The results of our study provide insights to design more potent and selective RET kinase inhibitors.

Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer.

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

Efficacy and Tolerability of Pyrazolo[1,5-a]pyrimidine RET Kinase Inhibitors for the Treatment of Lung Adenocarcinoma.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

Roles of the RET Proto-oncogene in Cancer and Development.

RET (REarranged during Transfection)is activated by DNA rearrangement of the 3′ fragment of the receptor tyrosine kinase gene, namely, RET proto-oncogene, with the 5′ fragment of various genes with putative dimerization domains, such as a coiled coil domain, that are necessary for constitutive activation. RET rearrangements have been detected in a variety of human cancers, including thyroid, lung, colorectal, breast, and salivary gland cancers. Moreover, point mutations in RET are responsible for multiple endocrine neoplasia types 2A and 2B, which can develop into medullary thyroid cancer and pheochromocytoma. Substantial effort is currently being exerted in developing RET kinase inhibitors. RET is also responsible for Hirschsprung’s disease, a developmental abnormality in the enteric nervous system. Gene knockout studies have demonstrated that RET plays essential roles in the development of the enteric nervous system and kidney as well as in spermatogenesis. Studies regarding RET continue to provide fascinating challenges in the fields of cancer research, neuroscience, and developmental biology.

Exploring the Potential of RET Kinase Inhibition for Irritable Bowel Syndrome: A Preclinical Investigation in Rodent Models of Colonic Hypersensitivity.

Abdominal pain represents a significant complaint in patients with irritable bowel syndrome (IBS). While the etiology of IBS is incompletely understood, prior exposure to gastrointestinal inflammation or psychologic stress is frequently associated with the development of symptoms. Inflammation or stress-induced expression of growth factors or cytokines may contribute to the pathophysiology of IBS. Here, we aimed to investigate the therapeutic potential of inhibiting the receptor of glial cell line-derived neurotrophic factor, rearranged during transfection (RET), in experimental models of inflammation and stress-induced visceral hypersensitivity resembling IBS sequelae. In RET-cyan fluorescent protein [(CFP) RetCFP/+] mice, thoracic and lumbosacral dorsal root ganglia were shown to express RET, which colocalized with calcitonin gene-related peptide. To understand the role of RET in visceral nociception, we employed GSK3179106 as a potent, selective, and gut-restricted RET kinase inhibitor. Colonic hyperalgesia, quantified as exaggerated visceromotor response to graded pressures (0-60 mm Hg) of isobaric colorectal distension (CRD), was produced in multiple rat models induced 1) by colonic irritation, 2) following acute colonic inflammation, 3) by adulthood stress, and 4) by early life stress. In all the rat models, RET inhibition with GSK3179106 attenuated the number of abdominal contractions induced by CRD. Our findings identify a role for RET in visceral nociception. Inhibition of RET kinase with a potent, selective, and gut-restricted small molecule may represent a novel therapeutic strategy for the treatment of IBS through the attenuation of post-inflammatory and stress-induced visceral hypersensitivity.

First-in-Human Studies for a Selective RET Tyrosine Kinase Inhibitor, GSK3179106, to Investigate the Safety, Tolerability, and Pharmacokinetics in Healthy Volunteers.

Rearranged during transfection (RET), a neuronal growth factor receptor tyrosine kinase, regulates the development of the sympathetic, parasympathetic, motor, and sensory neurons in the enteric nervous system. GSK3179106 is a RET kinase inhibitor that was administered in double-blind, randomized, placebo-controlled single-dose and repeat-dose studies in healthy subjects to investigate its pharmacokinetics and safety/tolerability. In the single-dose study (n = 16), GSK3179106 was dosed from 10 mg to 800 mg, including a food effect arm. In the repeat-dose study (n = 46), GSK3179106 was dosed for 14 days with food once daily (QD) from 5 mg to 100 mg and twice daily (BID) at 100mg and 200mg. With single fasted doses, bioavailability was low and less than dose proportional. A significant food effect was observed with a 100-mg QD dose. Drug exposure after QD and BID repeat dosing with food showed dose dependency up to 100 mg but was not dose proportional. There were no significant differences in exposure between 100-mg and 200-mg BID doses of GSK3179106. Accumulation was observed with both QD and BID dosing. Single doses up to 800 mg and repeat doses up to 400 mg were well tolerated with no safety concerns in healthy subjects.

Abstract 4784: TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth

Abstract The rearranged during transfection (RET) gene is a well-known proto-oncogene and encodes a single-pass transmembrane receptor tyrosine kinase. RET fusions and point mutations are oncogenic drivers in NSCLC, medullary thyroid cancer and other solid tumors, and therefore potential targets for cancer therapy. Multikinase inhibitors targeting the RET mutations have been tested in clinical trials with moderate efficacy in terms of tumor shrinkage and PFS.Moreover, multikinase inhibitors are characterized by poor tolerability due to off-target kinase inhibitory activities. To widen the therapeutic index, a selective RET kinase inhibitor is highly desirable. TAS0286/HM05 is a novel highly selective and potent RET kinase inhibitor.RET kinase assay was performed by homogeneous time-resolved fluorescence (HTRF) method. In-vitro proliferation studies and pharmacodynamics analyses were conducted in cancer cell lines with the RET fusions and RET activating mutations. The antitumor efficacy of TAS0286/HM05 was evaluated using mice xenograft models implanted with cancer cells with various RET gene abnormality. TAS0286/HM05 was orally administered for 14 or 28 consecutive days after grouping. In-vitro and in-vivo RET phosphorylation was detected using western blot analysis. The IC50 value for RET kinase of TAS0286/HM05 was below 1 nM. TAS0286/HM05 showed highly selective RET inhibitory activity among 283 kinases. In cellular assay, TAS0286/HM05 strongly suppressed phosphorylation of RET expressed in cells with various RET fusions and activating mutations, and inhibited cell proliferation at around 10 nM. At this concentration, apoptosis was also observed. The potency was higher than other multikinase inhibitors with RET inhibitory activity. Furthermore, in in-vivo efficacy studies, TAS0286/HM05 significantly inhibited the growth of tumors harboring various RET fusions and activating mutations at a range of 20 to 100 mg/kg/day without any body weight loss. The antitumor efficacy of TAS0286/HM05 was more potent than pre-existing multikinase inhibitors at their maximum tolerated dose. In particular, TAS0286/HM05 dramatically induced tumor regression of 40% within 15 days in animals implanted with LC-2/ad cells, a human lung adenocarcinoma cell line with CCDC6-RET fusion gene. TAS0286/HM05 is a novel and highly selective RET inhibitor with prominent tolerability. The potency of TAS0286/HM05 against tumors with RET abnormalities was stronger than currently marketed multikinase inhibitors being tested in NSCLC patients with RET fusions. TAS0286/HM05 is a promising agent for future clinical development in patients with RET gene abnormalities. Citation Format: Hidenori Fujita, Isao Miyazaki, Masanori Kato, Yukari Yamada, Keiji Ishida, Tomonori Haruma, Haruka Nagasaki, Kenjiro Ito, Akihiro Hashimoto, Yasuo Kodama, Kaoru Funabashi, Emanuela Lovati, Kazutaka Miyadera, Kenichi Matsuo, Yoshikazu Iwasawa. TAS0286/HM05, a novel highly selective RET inhibitor, prominently inhibits various RET defective tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4784.

Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a Clinical Candidate for the Treatment of IBS.

Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.

Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade.

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors.

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.