Akito Hata

Design, Synthesis, and Evaluation of the Highly Selective and Potent G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure

A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative 5 and 1,2,4-triazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an N-benzylcarboxamide moiety with highly potent activity toward GRK2 and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, and 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the discovery of 115h (GRK2 IC50 = 18 nM), which was obtained the cocrystal structure with human GRK2 and an inhibitor of GRK2 that potentiates β-adrenergic receptor (βAR)-mediated cAMP accumulation and prevents internalization of βARs in β2AR-expressing HEK293 cells treated with isoproterenol. Therefore, 115h appears to be a novel class of therapeutic for heart failure treatment.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response

Significance l-asparaginase (ASNase) is a critical component of treatment protocols for acute lymphoblastic leukemia (ALL). Although the cure rates have dramatically improved, the prognosis for patients with recurrent ALL remains poor. General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. As inhibitors targeting GCN2 have been lacking, the potential of GCN2 inhibitors as cancer therapeutic agents remains unclear. Here we report potent GCN2 inhibitors that exhibit synergistic antiproliferative effects with ASNase in asparagine synthetase-low cancer. Our findings enhance the molecular understanding of the disrupted amino acid response caused by GCN2 inhibition under limited asparagine availability. Combined treatment with GCN2 inhibitors and ASNase shows promise for achieving improved outcomes in ALL and other types of cancer. General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Maintenance ramucirumab monotherapy after intolerable toxicities following docetaxel plus ramucirumab

We recently reported the efficacy and safety of docetaxel (DOC) plus ramucirumab (RAM) with primary prophylactic pegylated (PEG)-granulocyte-colony stimulating factor (G-CSF) for pretreated non-small cell lung cancer (NSCLC) [1]. DOC+RAM exhibited high response and disease control rates as a salvage-line chemotherapy [2, 3]. However, some cases receiving this regimen suffered from intolerable toxicities such as oral mucositis, anorexia, malaise, nail change, and/ or peripheral edema. Even if clinical benefit is obtained, such intolerable toxicities occasionally make it difficult to continue DOC+RAM therapy. In such cases, we actively adopt maintenance RAM monotherapy without DOC. In our cohort, 10 (19%) of 52 patients receiving DOC+RAM with PEG-G-CSF moved to maintenance RAM monotherapy after intolerable toxicities following clinical response (1 complete response, 5 partial response, and 4 stable disease). Reasons for these movements were all adverse events (AEs) ≥grade 2: 4 oral mucositis; 3 anorexia; 2 malaise; 2 numbness; 2 nail change; and 2 peripheral edema (some overlapping). Median cycles of DOC+RAM before RAM monotherapy was 6 (range, 3-6). Median cycles of RAM monotherapy was 4 (range, 1-19). Among these 10 patients, median progressionfree survival (PFS) from DOC+RAM initiation was 7.0 (range, 3.2-21.0+) months (Figure 1), and that of RAM monotherapy was 3.0 (range, 0.8-15.5+) months (Figure 2). Median overall survival (OS) was not reached. All intolerable toxicities during DOC+RAM were improved after moving to RAM monotherapy. Three (30%) of the 10 patients are on treatment without progression. AEs observed during RAM monotherapy were 4 grade 2 hypertension and 1 grade 3 proteinuria, representing an extremely high tolerability. To the best of our knowledge, this is the first report to suggest the effectiveness of maintenance RAM monotherapy following DOC+RAM in pretreated NSCLC. Ramucirumab is a recombinant monoclonal antibody of the IgG1 class that binds to vascular endothelial growth factor receptor-2 (VEGFR-2) and blocks the activation of the receptor, preventing tumor angiogenesis, growth, and proliferation. In salvage-line settings of gastric cancer, RAM monotherapy demonstrated survival benefit over placebo [4]. This evidence implies prognostic contribution by RAM monotherapy for advanced cancers. Similar results for another VEGF inhibitor, bevacizumab (BEV) were reported in a pivotal phase III study. The E4599 trial comparing carboplatin plus paclitaxel with or without BEV for non-squamous NSCLC showed superior RR, PFS, and OS in the triple combination therapy [5]. In the study, maintenance BEV monotherapy was continued after induction triple combination therapy. This maintenance BEV monotherapy might have affected prolonged PFS and OS, while reducing toxicities. In patients suffering from intolerable toxicities following response to DOC+RAM, movement to RAM monotherapy could reduce severe AEs, improve quality of life, and prolong PFS. Further studies are warranted Letter to the Editor

Does afatinib plus bevacizumab combination therapy induce positive conversion of T790M in previously-negative patients?

Third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are markedly effective for T790M-positive patients. To confer their clinical benefit to more patients, a novel therapy to induce positive conversion in T790M-negative patients may be possible. We retrospectively reviewed medical records of patients who had received rebiopsy after completion of ABC-study: a prospective phase II study of Afatinib plus Bevacizumab Combination (ABC)-therapy after acquired resistance to EGFR-TKI. Between October 2014 and September 2016, 32 eligible patients were enrolled in ABC-study at our institutes. Eighteen patients were T790M-negative and 14 were T790M-positive before ABC-therapy. Rebiopsy was performed on 13 T790M-negative and 5 T790M-positive patients after progression of ABC-therapy. In 8 (62%) of 13 T790M-negative patients, T790M status changed from negative to positive after ABC-therapy. Seven of these 8 patients underwent osimertinib therapy. The response rate and median time to treatment failure were 86% and 12.2 months, respectively. There were no adverse events ≥grade 3, nor any treatment-related deaths. On the other hand, T790M remained positive after ABC-therapy in all 5 previous T790M-positive patients. ABC-therapy could induce positive conversion of T790M even in previously-negative patients. We hypothesize that ABC-therapy could provoke “clonal selection”, which purifies T790M-positive cancer cells in heterogeneous tumors. Further studies are warranted to confirm this phenomena.

CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells

The CDK8/19 kinase module comprises a subcomplex that interacts with the Mediator complex and regulates gene expression through phosphorylation of transcription factors and Mediator subunits. Mediator complex subunits have been increasingly implicated in cancer and other diseases. Although high expression of CDK8/19 has been demonstrated in prostate cancer, its function has not been thoroughly examined. Here we report that CDK8/19 modulates the gene expression of cell cycle regulators and thereby maintains the proper G1/S transition in prostate cancer cells. We show that highly selective CDK8/19 inhibitors exerted anti-proliferative activity in prostate cancer cells both in vitro and in vivo. In CDK8/19 inhibitor-sensitive prostate cancer cells, the compounds reduced the population of G1 phase cells and elevated that of S phase cells through the modulation of G1/S transition regulators at the level of mRNA expression. Furthermore, the premature G1/S transition induced a DNA damage response that was followed by ATR-dependent and caspase-independent cell death. These findings suggest a novel role of CDK8/19 in transcription-mediated cell cycle control, albeit with possible contribution of other proteins inhibited by the compounds. Our data provide a rationale for further investigation of CDK8/19 inhibitors as a new therapeutic approach to prostate cancer.

Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors

Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

Docetaxel Plus RAmucirumab With Primary Prophylactic Pegylated Granulocyte-ColONy Stimulating Factor Support for Elderly Patients With Advanced Non–small-cell Lung Cancer: A Multicenter Prospective Single Arm Phase II Trial: DRAGON Study (WJOG9416L)

&NA; We exhibit our ongoing multicenter, prospective, single‐arm, phase II trial of docetaxel plus ramucirumab with primary prophylactic pegylated‐granulocyte‐colony stimulating factor (PEG‐G‐CSF) support for chemotherapy‐naive elderly patients with advanced non–small‐cell lung cancer (NSCLC) (University Hospital Medical Information Network database: UMIN000030598). Docetaxel monotherapy is the Japanese standard of care for chemotherapy‐naive elderly patients with advanced NSCLC. Docetaxel plus ramucirumab showed superior survival benefit over docetaxel monotherapy in the second‐line setting for NSCLC. A Japanese phase II study comparing docetaxel plus ramucirumab and docetaxel monotherapy in the second‐line setting showed febrile neutropenia (FN) incidence of approximately one‐third in the docetaxel plus ramucirumab arm. Docetaxel plus ramucirumab could be a promising candidate for elderly patients with NSCLC, but such high FN incidence is a clinically critical concern. To overcome this problem, we adopt a routine primary prophylactic PEG‐G‐CSF with docetaxel plus ramucirumab therapy. We hypothesize that primary prophylactic PEG‐G‐CSF reduces FN and maximizes the efficacy of docetaxel plus ramucirumab in Japanese elderly patients with NSCLC. Intravenous docetaxel (60 mg/m2, day 1) plus ramucirumab (10 mg/kg, day 1) with subcutaneous PEG‐G‐CSF (3.6 mg, day 2) every 3 weeks is administered until progression. The primary endpoint is overall response rate (ORR). We decided the threshold ORR to be 20%, and the expected ORR 35%. Taking statistical points (&agr;/&bgr; errors: 0.05/0.80) and ineligible patients into account, the sample size was set at 65. When the study results are promising, we will conduct a phase III trial to compare docetaxel plus ramucirumab with PEG‐G‐CSF support versus docetaxel monotherapy for chemotherapy‐naive elderly patients with NSCLC.

Afatinib Plus Bevacizumab Combination After Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small Cell Lung Cancer: Multicenter, Single-Arm, Phase 2 Trial (ABC Study)

Preclinical studies suggested that the addition of bevacizumab could overcome acquired resistance (AR) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The aim of this study was to evaluate the clinical efficacy and safety of a combination of afatinib and bevacizumab after AR.