Rearranged During Transfection Kinase Research Articles

Molecular dynamics study of enhanced autophosphorylation by S904F mutation of the RET kinase domain

The aberrant kinase activity of RET (rearranged during transfection), a transmembrane tyrosine kinase, is associated with human cancer. A point mutation caused by the replacement of solvent-front hydrophilic S904, located on the activation loop (A-loop), with a bulky hydrophobic phenylalanine residue can induce resistance to the type I kinase inhibitor vandetanib. A possible mechanism of this drug resistance is the release of a cis-autoinhibited conformation of RET for autophosphorylation, which activates RET kinase. Because the association between S904F mutation and enhanced autophosphorylation is unclear, we conducted molecular modeling analysis to compare unphosphorylated apo wild-type and S904F mutant structures. The structural compactness of the A-loop promoted ATP binding. When the A-loop is extended, the αC helix moves toward the glycine-rich loop, resulting in the protrusion of F735. The extruded F735 connects with E734 and R912 and constrains the ATP pocket entrance. Contrarily, a contracted A-loop pulls the αC helix away from the glycine-rich loop, burying F734 and making the ATP pocket accessible. The mutated F904 stabilizes the contracted A-loop and releases the autoinhibited conformation of RET, thereby facilitating autophosphorylation. We also simulated two ATP-bound systems. The binding free energies of ATP, estimated through the molecular mechanics with a generalized Born and surface area solvation approach, revealed that the S904F mutant was bound more tightly than was the wild type with the ATP. The findings support the premise of autophosphorylation promotion in the S904F mutant.

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.

Kidney malformations and Hirschsprung's disease in carriers of cysteine mutations in exon 10 of the RET proto-oncogene.

This investigation aimed at characterizing penetrance and interrelation of the loss-of-function phenotypes, Hirschsprung's disease and kidney malformations, in carriers of exon 10 REarranged during Transfection (RET) mutations. Comparative analysis of penetrance of and interdependencies between Hirschsprung's disease and kidney malformations among carriers of mutations in RET codons 609, 611, 618, and 620. Hirschsprung's disease and kidney malformations (kidney agenesis, ureteropelvicalyceal dilatation, or polycystic kidney disease) each affected 3.6% (4 patients) of 112 carriers of RET mutations in exon 10. This percentage increased to 13% (4 patients) and 9% (3 patients) of 32 p.Cys620 carriers and 22% (4 patients) and 17% (3 patients) of 18 p.Cys620Arg carriers, respectively. Overall, Hirschsprung's disease (17%, 4 of 24 carriers; P = 0.002) and kidney malformations (13%, 3 of 24 carriers; P = 0.030) were associated with arginine-for-cysteine substitutions. Two of the six patients, originating from different p.Cys620Arg families, harbored both Hirschsprung's disease and kidney malformations. Hirschsprung's disease and kidney malformations are the more penetrant, the closer the cysteine mutations are located to the transmembrane domain (codon 636-657) of the RET kinase receptor.

Pyrazoloadenine Inhibitors of the RET Lung Cancer Oncoprotein Discovered by a Fragment Optimization Approach.

A fragment-based drug-discovery approach was used on a pyrazoloadenine fragment library to uncover new molecules that target the RET (REarranged during Transfection) oncoprotein, which is a driver oncoprotein in ∼2 % of non-small-cell lung cancers. The fragment library was screened against the RET kinase and LC-2/ad (RET-driven), KM-12 (TRKA-driven matched control) and A549 (cytotoxic control) cells to identify selective scaffolds that could inhibit RET-driven growth. An unsubstituted pyrazoloadenine fragment was found to be active on RET in a biochemical assay, but reduced cell viability in non-RET-driven cell lines (EC50 =1 and 3 μM, respectively). To increase selectivity for RET, the pyrazoloadenine was modeled in the RET active site, and two domains were identified that were probed with pyrazoloadenine fragment derivatives to improve RET affinity. Scaffolds at each domain were merged to generate a novel lead compound, 8 p, which exhibited improved activity and selectivity for the RET oncoprotein (A549 EC50 =5.92 μM, LC-2/ad EC50 =0.016 μM, RET IC50 =0.000326 μM).

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.

Enhanced antitumor effect of alectinib in combination with cyclin-dependent kinase 4/6 inhibitor against RET-fusion–positive non–small cell lung cancer cells

ABSTRACT Rearranged during transfection (RET) fusion–positive non–small cell lung cancer (NSCLC) accounts for 1% of lung adenocarcinoma. Although small molecule agents with RET kinase inhibitory activity such as alectinib, vandetanib, and cabozantinib have been clinically evaluated in RET-fusion–positive NSCLC, an effective monotherapy regimen has not been established. We explored agents to use in combination with alectinib to enhance the antitumor effect of alectinib against RET-fusion cells. Cell proliferation under co-treatment with alectinib plus each of six chemotherapeutic agents or six molecularly targeted agents was evaluated in vitro. The combination effect was analyzed by IC50 isobologram and combination index using LC-2/ad and Ba/F3-KIF5B-RET cells. The in vivo combination effect was investigated in a Ba/F3-KIF5B-RET xenograft model. The phosphorylation levels of proteins regulating proliferation were measured by immunoblotting. Palbociclib, a CDK4/6 inhibitor, showed the greatest synergy against LC-2/ad cells in the isobologram analysis and combination index. This synergistic effect was also observed against Ba/F3-KIF5B-RET cells. Another CDK4/6 inhibitor, abemaciclib, also showed a synergistic effect. In vivo, the combination of alectinib plus palbociclib showed a more enhanced antitumor effect than each single agent in a mouse xenograft model with transplanted Ba/F3-KIF5B-RET cells. This combination suppressed the phosphorylation of S6 and Rb more intensely than did either single agent in both LC-2/ad and Ba/F3-KIF5B-RET cell lines, both in vitro and in vivo. Combination therapy with alectinib plus the CDK4/6 inhibitor enhanced the antitumor effect against RET-fusion–positive cells in vitro and in vivo.

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.

RET kinase alterations in targeted cancer therapy.

The rearranged during transfection (RET) gene encodes a protein tyrosine kinase. RET alterations by point mutations and gene fusions were found in diverse cancers. RET fusions allow abnormal expression and activation of the oncogenic kinase, whereas only a few of RET point mutations found in human cancers are known oncogenic drivers. Earlier studies of RET-targeted therapy utilized multi-targeted protein tyrosine kinase inhibitors (TKIs) with RET inhibitor activity. These multi-targeted TKIs often led to high-grade adverse events and were subject to resistance caused by the gatekeeper mutations. Recently, two potent and selective RET TKIs, pralsetinib (BLU-667) and selpercatinib (LOXO-292), were developed. High response rates to these selective RET inhibitors across multiple forms of RET alterations in different types of cancers were observed in clinical trials, demonstrating the RET dependence in human cancers harboring these RET lesions. Pralsetinib and selpercatinib were effective in inhibiting RETV804L/M gatekeeper mutants. However, adaptive mutations that cause resistance to pralsetinib or selpercatinib at the solvent front RETG810 residue have been found, pointing to the need for the development of the next-generation of RET TKIs.

Leveraging Atropisomerism to Obtain a Selective Inhibitor of RET Kinase with Secondary Activities toward EGFR Mutants.

Unstable atropisomerism is innate in many common scaffolds in drug discovery, commonly existing as freely rotating aryl-aryl bonds. Such compounds can access the majority of dihedral conformations around the bond axis; however, most small molecules bind their target within a narrow range of these available conformations. The remaining accessible conformations can interact with other proteins leading to compound promiscuity. Herein, we leverage atropisomerism to restrict the accessible low-energy dihedral conformations available to a promiscuous kinase inhibitor and achieve highly selective and potent inhibitors of the oncogenic target rearranged during transfection (RET) kinase. We then evaluate our lead inhibitor against kinases that were predicted to bind compounds in a similar conformational window to RET, discovering a potent inhibitor of drug-resistant epidermal growth factor receptor (EGFR) mutants including L858R/T790M/C797S EGFR. Leveraging atropisomerism to restrict accessible conformational space should be a generally applicable strategy due to the prevalence of unstable atropisomerism in drug discovery.

Abstract 2209: Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase

Abstract The rearranged during transfection (RET) tyrosine kinase is mostly altered by fusion and it makes RET as a attractive therapeutic target for medullary thyroid cancer and non-small cell lung cancer (NSCLC). Multi-kinase inhibitors (mKIs) such as cabozantinib and vandetanib expressed poor tolerability and limited efficacy due to off-target kinase inhibitory activity in academia and clinical cases. As for selective RET inhibitor, compounds such as BLU-667 and LOXO-292 are under development, currently in clinical trials. We are currently developing potent and selective RET inhibitors to address unmet medical needs for RET-driven cancers. Our team has validated its high potency and selectivity via kinase profiling and in-vitro assay results. We also anticipate our compound to overcome certain mutation, such as V804M. Our compounds strongly suppress phosphorylation of RET in tumor cells with various RET fusions and mutations by inhibiting proliferation at below 10 nM. This potency result is higher than those of other mKIs with RET inhibitory activity. The inhibitory concentration of our compounds to VEGFR2 is more than dozens of fold compared with RET. In addition, our compounds reveal low cytotoxicity in Ba/F3 naïve cells in the 1 µM concentration. These findings lead us to conclude that our compounds can be potent and highly selective inhibitors of RET kinase, a potential novel therapeutic agents for RET-driven cancers. Citation Format: Hua Li, SeoHyun Jo, Woomi Do, Hyunkyung Kim, Hwan Kim, Kyung-ah Seo, Ji-hoon Oh, Jieun Choi, Jaeyoung Ahn, Jung Beom Son, Nam Doo Kim. Novel therapeutic agents for RET-driven cancers, which is highly potent and selective inhibitor to RET kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2209.

Rearranged During Transfection Fusions in Non-Small Cell Lung Cancer.

Identifying and targeting specific oncogenic drivers has become standard of care in the routine management of patients with lung cancer. Research is ongoing to expand the number of drug targets that can offer clinically meaningful outcomes. Rearranged during transfection (RET) fusions are the latest oncogenic driver alterations that show potential as a drug target. RET fusions occur in 1–2% of non-small cell lung cancer (NSCLC) cases. They are more commonly associated with younger age, female gender, non-smokers and Asian ethnicity. The RET kinase is abnormally activated through fusion with a partner protein such as KIF5B, CCDC6 or NCOA4. This leads to downstream intracellular signalling and enhancement of gene transcription and cell proliferation. The effectiveness of multi-kinase inhibitors in RET positive NSCLC has been explored in early phase and retrospective studies. From these studies, the most effective agents identified include cabozantanib and vandetanib. Overall response rates (ORR) vary from 18–47% across studies. In general, these agents have a manageable toxicity profile, although there are a number of off-target toxicities. Similar to the increased activity in ALK rearranged disease, pemetrexed has demonstrated superior response rates in this patient group and should be considered. Selective RET inhibitors, including LOXO-292 and BLU-667, are progressing in clinical trials. LOXO-292 has demonstrated an impressive ORR of 77% in RET positive solid tumours. It is anticipated this agent will be an effective targeted therapeutic option for patients with RET positive lung cancer.

Receptor‐guided 3D‐QSAR Study of Anilinoquinazolines as RET Receptor Tyrosine Kinase Antagonists

A RET (Rearranged during transfection) kinase belongs to a receptor tyrosine kinase family. It plays a major role in development, maturation, survival, and maintenance of cells and tissues. Oncogenic activation of RET has been reported in numerous cancers. Thus, it is a significant therapeutic target for cancer. Present work covers docking and 3D‐QSAR techniques executed on anilinoquinazoline derivatives as RET kinase antagonists. Docking study recognized important active site amino acid residues like Leu730, Gly731, Gly733, Glu734, Ala807, Gly810, Ser811, and Asp874 which inhibits the RET kinase. In 3D‐QSAR technique, receptor‐guided CoMFA (q2 = 0.723, NOC = 5, r2 = 0.980) as well as CoMSIA (q2 = 0.767, NOC = 6, r2 = 0.967) models were produced. The stabilities of these models were evaluated using diverse validation techniques. Contour maps showed that steric and hydrogen bond donor substitutions are favored at R1 and R2 positions whereas positive and negative substitutions are favored at the R1 position to enhance the potency. In addition, the substitution of H‐bond accepting group to the region which is close to both phenyl and piperazine is favored. Hence, the outcome of this study could be beneficial to design more potent inhibitors for RET kinase.

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.

Challenging clinically unresponsive medullary thyroid cancer: Discovery and pharmacological activity of novel RET inhibitors

It is now known that “gain of function” mutations of RET (REarranged during Transfection) kinase are specific and key oncogenic events in the onset of thyroid gland cancers such as the Medullary Thyroid Carcinoma (MTC). Although a number of RET inhibitors exist and are capable of inhibiting RET variants, in which mutations are outside the enzyme active site, the majority becomes dramatically ineffective when mutations are within the protein active site (V804L and V804M). Pursuing a receptor-based virtual screening against the kinase domain of RET, we found that compound 5 is able to inhibit efficiently both wild type and V804L mutant RET. Compound 5 was able to significantly reduce proliferation of both commercially available TT cell lines and surgical thyroid tissues obtained from patients with MTC and displayed a suitable drug-like profile, thus standing out as a promising candidate for further development towards the treatment of clinically unresponsive MTC.