Discovery of ASP6918, a KRAS G12C inhibitor: Synthesis and structure-activity relationships of 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivatives as covalent inhibitors with good potency and oral activity for the treatment of solid tumors.

Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration.

Nutrition provided during the final weeks to patients with non-gastrointestinal solid malignancies.

BACKGROUND & AIMS: Data regarding the delivery of nutrition and nutritional infusions to patients with terminal cancer remain limited; therefore, this real-world analysis investigated differences in nutrition delivery and infusion statuses for patients with terminal-stage cancers. METHODS: Patients who had died following hospitalization for more than seven days due to neoplasms between April 2014 and December 2018 were identified using the medical claims database of Japan. Data regarding oral diets, enteral feeding, infusion volumes, and infusion energies were extracted. The maximum observation period was 28 days prior to patient death. RESULTS: A total of 12,908 patients were included in our analysis. The proportion of patients without dietary or enteral nutrition increased closer to their dates of death. Observations were recorded at 28 (18.9%), 21 (20.9%), 14 (24.6%), 7 (33.0%), and 0 (80.2%) days prior to death. The infusion volumes given to the patients, as well as their energy contents, decreased near death (P-value for the trend <0.001). CONCLUSIONS: Our study revealed the current status of nutritional therapy for patients with terminal cancer in Japan. The number of patients receiving only parenteral nutrition increased near death, while the amount of nutritional intake was low.

Cachexia staging score predicts survival in patients with cancer who receive palliative care.

OBJECTIVE: The aim of this study was to validate the cachexia staging score (CSS), a multidimensional, item-based diagnostic method of cachexia severity, for patients with advanced cancer receiving palliative care. METHODS: Eligible patients were those with cancer who received palliative care during hospitalization between May 2019 and April 2020. All data were collected retrospectively from medical records. Cachexia was graded into four levels according to the CSS. Kaplan-Meier curves were constructed with or without death as the outcome, comparing prognoses among different levels of cachexia with Bonferroni correction. Cox proportional hazards regression analysis was performed to identify factors affecting mortality. RESULTS: The mean age of the 196 patients was 65.8 ± 14 y. Men made up 42% of the study population. Lower body mass index, increased rate of weight loss, increased strength, assistance walking, rising from a chair, climbing stairs, and falls (SARC-F) points, decline in activities of daily living, appetite loss, and abnormal blood biomarkers were significantly more common with increasing severity of cachexia, and survival was shorter (P < 0.001). The hazard ratio (HR) increased with worsening severity of cachexia according to CSS classification (precachexia: HR, 2.78; 95% confidence interval [CI], 0.62-12.46, P = 0.182; cachexia: HR, 4.77; 95% CI, 1.09-20.80; P = 0.038; and refractory cachexia: HR, 11.00; 95% CI, 2.37-51.07; P = 0.002). CONCLUSIONS: The CSS predicted life expectancy in a population of patients receiving palliative care and had excellent prognostic discriminative power to classify patients at different stages of cachexia.

Discovery and biological evaluation of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as covalent inhibitors of KRAS G12C with favorable metabolic stability and anti-tumor activity.

RAS protein plays a key role in cellular proliferation and differentiation. RAS gene mutation is a known driver of oncogenic alternation in human cancer. RAS inhibition is an effective therapeutic treatment for solid tumors, but RAS protein has been classified as an undruggable target. Recent reports have demonstrated that a covalent binder to KRAS protein at a mutated cysteine residue (G12C) is effective for the treatment of solid tumors. Here, we report a series of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as potent covalent inhibitors against KRAS G12C identified throughout structural optimization of an acryloyl amine moiety to improve in vitro inhibitory activity. From an X-ray complex structural analysis, the 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one moiety binds in the switch-II pocket of KRAS G12C. Further optimization of the lead compound (5c) led to the successful identification of 1-[7-[6-chloro-8-fluoro-7-(5-methyl-1H-indazol-4-yl)-2-[(1-methylpiperidin-4-yl)amino]quinazolin-4-yl]-2,7-diazaspiro[3.5]nonan-2-yl]prop-2-en-1-one (7b), a potent compound with high metabolic stabilities in human and mouse liver microsomes. Compound 7b showed a dose-dependent antitumor effect on subcutaneous administration in an NCI-H1373 xenograft mouse model.

Evaluation of skeletal muscle mass using prediction formulas at the level of the 12th thoracic vertebra.

OBJECTIVES: People with cancer have a high risk of cachexia and sarcopenia, which are associated with worse clinical outcomes. We evaluated the prediction accuracy of the Matsuyama et al. and Ishida et al. formulas using computed tomography (CT) slices from the twelfth thoracic vertebra (Th12) level in people with cancer. METHODS: This retrospective study included patients with advanced cancer who underwent thoracic and abdominal CT scans (n = 173). The cross-sectional area (CSA) on CT images was measured at the levels of Th12 and the third lumbar vertebra (L3). The Matsuyama et al. formula used the Th12 CSA, whereas the Ishida et al. formula used only the Th12 CSA of the spinal erectors; thus, the measurements were performed separately. The correlation between predicted and actual L3 CSA was assessed using r and the intraclass correlation coefficient. A prediction-accuracy analysis of the predicted values was also performed. RESULTS: The mean participant age was 66.2 ± 12.8 y; 50.3% of participants were women and 49.7% were men. Strong correlations were observed between the predicted and measured L3 values calculated from the two prediction formulas. The prediction-accuracy analysis using previously reported cutoff values showed that the Ishida et al. method had high sensitivity and the Matsuyama et al. method had high specificity for low skeletal muscle index determined by the predicted and measured L3 skeletal muscle index. CONCLUSIONS: Both the Matsuyama et al. and Ishida et al. formulas had good reliability on CT slices at the Th12 level in people with advanced cancer, indicating that these formulas can be applied in clinical practice.

Assessing skeletal muscle mass based on the cross-sectional area of muscles at the 12th thoracic vertebra level on computed tomography in patients with oral squamous cell carcinoma.

OBJECTIVES: This study aimed to create a formula to estimate the third lumbar vertebra (L3)1 level skeletal muscle cross-sectional area (CSA), known as a standard value to evaluate skeletal muscle mass on computed tomography (CT), using the twelfth thoracic vertebra (Th12) level skeletal muscle CSA on chest CT. MATERIALS AND METHODS: This retrospective observational study included patients aged 40 + years with a diagnosis of oral squamous cell carcinoma (n = 164). Skeletal muscle CSA on CT images was measured using the Th12 and the L3 levels of pretreatment CT scans. The predictive formula was created based on the five-fold cross-validation method with a linear regression model. Correlations between the predicted L3-level CSA and the actual L3-level CSA were evaluated using r and Intraclass Correlation Coefficients (ICC). RESULTS: The predictive formula for L3-level CSA from Th12-level CSA was: CSA at L3 (cm2) = 14.143 + 0.779 * CSA at Th12 (cm2) - 0.212 * Age (y) + 0.502 * Weight (kg) + 13.763 * Sex. Correlations between the predicted and measured L3-level CSA were r = 0.915 [0.886-0.937] and ICC = 0.911 [0.881-0.934]. CONCLUSION: We developed a formula for predicting skeletal muscle mass from the Th12-level CT slice. The predicted L3-level CSA correlated with the measured L3-level CSA.

Determining the optimal value of the Geriatric Nutritional Risk Index to screen older patients with malnutrition risk: A study at a university hospital in Japan.

AIM: The Geriatric Nutritional Risk Index (GNRI) can predict nutritional risk. However, just a few studies have validated the optimal cut-off value of GNRI for nutrition screening in older patients. Hence, this study aimed to determine the optimal value of GNRI to screen the risk of malnutrition among older patients. METHODS: This retrospective cross-sectional study was carried out with 5867 consecutive older adult patients who were admitted to an academic hospital in Japan. Receiver operating characteristic curve analyses were carried out to obtain the optimal cut-off value of GNRI, and the results were compared against the Mini Nutritional Assessment - Short Form and Malnutrition Universal Screening Tool. The validation of the obtained cut-off value was examined on the concordance rate of malnutrition diagnosis based on the European Society of Clinical Nutrition and Metabolism criteria. RESULTS: The mean age of the patients was 76.0 ± 7.0 years. The optimal cut-off value of GNRI for Mini Nutritional Assessment - Short Form ≤11 points was 95.92 (area under the curve 0.827 [0.817-0.838], P < 0.001), and that for Malnutrition Universal Screening Tool ≥1 point was 95.95 (area under the curve 0.788 [0.776-0.799], P < 0.001). By adapting GNRI <96 points as an initial screening cut-off in the European Society of Clinical Nutrition and Metabolism-defined malnutrition process, the concordance rates of comparisons were 98.5% and 98.5% for Mini Nutritional Assessment - Short Form-based and MUST-based diagnosis, respectively. CONCLUSIONS: The study showed GNRI <96 points as the optimal cut-off value for nutritional screening. GNRI might be one of the easy-to-use tools for nutritional screening and for diagnosing malnutrition in older adults. Geriatr Gerontol Int 2020; 20: 811-816.

Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Mutant-Selective Irreversible EGFR Inhibitor, Naquotinib, Inhibits Tumor Growth in NSCLC Models with EGFR-Activating Mutations, T790M Mutation, and AXL Overexpression.

First- and second-generation EGFR tyrosine kinase inhibitors (TKI) are effective clinical therapies for patients with non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, almost all patients develop resistance to these drugs. The EGFR T790M mutation of EGFR is the most predominant mechanism for resistance. In addition, activation of AXL signaling is one of the suggested alternative bypassing pathways for resistance to EGFR-TKIs. Here, we report that naquotinib, a pyrazine carboxamide-based EGFR-TKI, inhibited EGFR with activating mutations, as well as T790M resistance mutation while sparing wild-type (WT) EGFR. In in vivo murine xenograft models using cell lines and a patient-derived xenograft model, naquotinib induced tumor regression of NSCLC with EGFR-activating mutations with or without T790M resistance mutation, whereas it did not significantly inhibit WT EGFR signaling in skin. Furthermore, naquotinib suppressed tumor recurrence during the treatment period of 90 days. In addition, unlike erlotinib and osimertinib, naquotinib inhibited the phosphorylation of AXL and showed antitumor activity against PC-9 cells overexpressing AXL in vitro and in vivo Our findings suggest that naquotinib has therapeutic potential in patients with NSCLC with EGFR-activating mutations, T790M resistance mutation, and AXL overexpression.

Development of a peptide antagonist against fsr quorum sensing of Enterococcus faecalis.

Enterococcus faecalis fsr quorum sensing (QS) involves an 11-residue cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP) that autoinduces two pathogenicity-related extracellular proteases in a cell density-dependent fashion. To identify anti-pathogenic agents that target fsr QS signaling, peptide antagonists of GBAP were created by our unique drug design approach based on reverse alanine scanning. First of all, a receptor-binding scaffold (RBS), [Ala(4,5,6,8,9,11)]Z-GBAP, was created, in which all amino acids within the ring region of GBAP, except for two essential aromatic residues, were substituted to alanine. Next, the substituted alanine residues were changed back to the original amino acid one by one, permitting selection of those peptide combinations exhibiting increased antagonist activity. After three cycles of this reverse alanine scan, [Ala(5,9,11)]Z-GBAP was obtained as a maximally reverted peptide (MRP) holding the strongest antagonist activity. Then, the fifth residue in MRP, which is one of the critical residues to determine agonist/antagonist activity, was further modified by substituting with different types of amino acids including unnatural amino acids. As a result, [Tyr(Bzl)(5), Ala(9,11)]Z-GBAP, named ZBzl-YAA5911, showed the strongest antagonist activity [IC(50) = 26.2 nM and Kd against GBAP receptor (FsrC) = 39.4 nM]. In vivo efficacy of this peptide was assessed with an aphakic rabbit endophthalmitis model. ZBzl-YAA5911 suppressed the translocation of E. faecalis from the aqueous humor into the vitreous cavity by more than 1 order of magnitude and significantly reduced retinal damage. We propose that ZBzl-YAA5911 or its derivatives would be useful as anti-infective agents to attenuate virulence expression in this opportunistic pathogen.

Crystallization and preliminary X-ray analysis of a putative sensor histidine kinase domain: the C-terminal domain of HksP4 from Aquifex aeolicus VF5.

The histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium Aquifex aeolicus VF5, located in the C-terminal half of the protein, was expressed, purified and crystallized. Diffraction-quality crystals were obtained in the presence of adenosine triphosphate (ATP) or adenosine 5'-(ß,γ-imido)triphosphate (AMPPNP) by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals obtained in the presence of ATP and AMPPNP diffracted X-rays to 3.1 and 2.9 Šresolution, respectively, on BL-5A at Photon Factory (Ibaraki, Japan) and were found to belong to the same space group P2(1)2(1)2(1), with unit-cell parameters a=80.2, b=105.5, c=122.0 Šand a=81.5, b=105.5, c=130.9 Å, respectively. Their Matthews coefficients (VM=2.74 and 2.51 Å3 Da(-1), respectively) indicated that both crystals contained four protein molecules per asymmetric unit.

Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat constitutive androstane receptor.

Constitutive active (or androstane) receptor (CAR, NR1I3), a member of the nuclear receptor family, is a major regulator for induction of cytochrome P450 2B (CYP2B) genes by phenobarbital. Phenobarbital-like inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a potent mouse CAR ligand that has been used to study CAR target genes in mice but does not activate human CAR (hCAR) or rat CAR (rCAR). Although 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) was reported to be an hCAR agonistic ligand, activation of hCAR by CITCO in cell-based reporter assay was weak. Therefore, we performed a screening of 50 drugs and chemicals using cell-based reporter assays to identify activators of hCAR. Among them, HMG-CoA reductase inhibitors (cerivastatin, simvastatin, fluvastatin, and atorvastatin) enhanced the hCAR-mediated transcriptional activation of phenobarbital-responsive enhancer module reporter gene by up to 3-fold. Similar activation by HMG-CoA reductase inhibitors was also observed with mouse and rat CARs. On the other hand, pravastatin did not activate hCAR at the concentrations tested (up to 30 microM). The extent of activation by the HMG-CoA reductase inhibitors was stronger than that by CITCO. Cerivastatin, simvastatin, fluvastatin, and atorvastatin induced CYP2B6 mRNA in stable hCAR-expressed FLC7 cells but not in original FLC7 cells. Therefore, we concluded that CAR mediates the effects of HMG-CoA reductase inhibitors on the induction of CYP2B genes, although HMG-CoA reductase inhibitors also activate pregnane X receptor. HMG-CoA reductase inhibitors such as cerivastatin would be useful to study for elucidating molecular and cellular mechanisms of hCAR.