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The brain is a common target organ for breast cancer metastasis, and the risk of brain metastasis is usually high for patients with HER2-positive breast cancer. While T-DXd showed positive results in HER2-positive breast cancer brain metastasis (BCBM), its activity and blood-brain barrier (BBB) permeability in active BCBM need to be further validated (TUXEDO-1 included 15 patients with active brain metastasis). Tucatinib plus Trastuzumab and Capecitabine triplet showed efficacy in patients with brain metastasis, but Tucatinib alone has poor BBB penetration. There is still an unmet medical need for active HER2-positive BCBM. VRN101099 is a highly selective kinase inhibitor of HER2 (Eurofins scanMAX Kinase Profiling, S-score (35) of 0.01). VRN101099 has single- to double-digit nanomolar (IC50) cellular potency in HER2-dependent cancer cells, and Ba/F3 cells expressing HER2 wild type or mutations with selectivity over wildtype EGFR. VRN101099 inhibited proliferation of BT474 with 3.6 nM IC50 but HaCaT with 829.2 nM IC50. VRN101099 binds HER2 kinase by forming a covalent bond to the Cys805 and its irreversible inhibition resulted in a longer target resident time than Tucatinib, confirmed by in vitro washout experiments. Robust in vivo activity of VRN101099 was observed in HER2-positive BT474 xenograft models. Moreover, once daily oral administration of VRN101099 significantly regressed intracranial BT474 tumor, demonstrating greater efficacy than twice daily oral administration of Tucatinib. These results were well explained by the superior brain to plasma exposure of VRN101099 to Tucatinib. Also, VRN101099 showed high exposure in the target organ, the fat pad, which potentiated better clinical translation. These anti-tumor efficacies are correlated with pharmacodynamic responses, as confirmed by decreased HER2, AKT, and ERK phosphorylation. In summary, VRN101099 is a brain penetrant, orally bioavailable, irreversible, and highly selective inhibitor of HER2 with therapeutic potential in HER2-positive BCBM. These data support the clinical development of VRN101099 in HER2-driven cancers. Citation Format: Yikyung Ko, Jihye yoo, Hong-ryul Jung, Hyerim Lim, YeongDeok Lee, Se Hyuk Kim, Dong Guk Shin, Serin Cho, Myung hoe Heo, Haelee Kim, Ha Yeon Cho, Ah Reum Han, Eunhwa Ko, Hwan Geun Choi, Deakwon Kim, Sunghwan Kim. VRN101099, Brain Permeable HER2 Kinase Inhibitor, Shows the Anti-tumor Activity in Preclinical Models of HER2-positive Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-03.

Objectives: This study investigates the effect of parenting stress on mothers of infants and the participation of fathers in raising the infants as perceived by the mothers, and a mother’s satisfaction of her role as a paerent. The infant stage is a period change, and the mothers go through a lot of stress during the stage. We will explore the ways to improve the role satisfaction in parents considering that it is a major change in their lives.Methods: Participants comprised 159 mothers of infants who agreed to participate in the study. They answered questionnaires on parenting stress, fathers’ parenting participation, and satisfaction in parents’ role. Multiple regression analysis was conducted to understand the influence.Results: The results revealed that the parent’s role satisfaction as perceived by a mother was significantly related to her parenting stress and the father’s participation in parenting. The lower the parenting stress and the higher the father’s participation in parenting, the higher was the parent’s role satisfaction. In particular, it was found that the negative interaction between parents and children, and the life factors of fathers, had a direct effect.Conclusion: This study explores how the parenting stress in mothers of infants and the participation of fathers as perceived by mothers affect the mother’s satisfaction with her role as a parent. It is necessary to find ways to reduce the stress of the mothers raising infants in order to increase their role satisfaction, and fathers need to take an active part in nurturing the infant.

Schematic representation for the dual action of AIMP2 via Smurf2 in TGF-β signal pathway.

Identification of TGF-β1-induced phosphorylation site in AIMP2.

Functional significance of TGF-β1-induced phosphorylation of AIMP2.

The effect of AIMP2 on TGF-β receptor I stability, Smad2 phosphorylation and TGF-β target gene expression.

Effect of AIMP2 phosphorylation on its molecular interactions.

Multiple sequence alignment of metazoan AIMP2.

The effect of AIMP2 on nuclear export of Smurf2 via CRM1.

Determination of the peptide regions responsible for the interaction between Smurf2 and AIMP2.

The significance of AIMP2 for Smurf2-dependent downregulation of FBP and the interaction of Smurf2 and FBP.

A possible binding site for AIMP2 in the Smurf2 HECT domain.

AIMP2/p38 is a multifunctional tumor suppressor that normally resides in the cytosol as a scaffold protein of the multi-tRNA synthetase complex (MSC). One of the tumor-suppressive functions of AIMP2 is to facilitate ubiquitin-mediated degradation of FUSE-binding protein (FBP, FUBP1), a transcriptional activator of c-Myc. However, the mechanism by which AIMP2 functions within this pathway and its significance in tumorigenesis are uncertain. Here, we report that Smurf2 is responsible for AIMP2-mediated ubiquitination of FBP, and a mutation in AIMP2 that inhibited its nuclear interaction with Smurf2 enhanced cellular transformation and tumorigenesis in vivo. Treatment of HeLa cells with TGFβ resulted in the phosphorylation of AIMP2 on S156, a residue that is exposed on the embedded GST domain of AIMP2. We further found that phospho-AIMP2 dissociated from the MSC and translocated to the nucleus, where it bound to Smurf2, enhancing ubiquitination of FBP. AIMP2 also inhibited nuclear export of Smurf2 to sustain TGFβ signaling. Collectively, these findings present a novel tumor-suppressive interaction between AIMP2 and Smurf2 and suggest that the disruption of this interaction can lead to oncogenic transformation. Cancer Res; 76(11); 3422–36. ©2016 AACR.

Legend for Supplementary Figures S1-S11. Also includes Supplementary References.

Predicted positioning of tRNA synthetases that interact with AIMP2 in MSC.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

Human pyridoxal 5'-phosphate phosphatase (PLPP), also known as a chronophin, is a phosphatase belonging to subfamily II of the HAD phosphatases, characterized by a large cap domain. As a member of the subfamily, its cap-open conformation is expected for substrate binding. We determined apo and PLP-bound PLPP/chronophin structures showing a cap-closed conformation. The active site, in which a PLP molecule was found, is too small to accommodate a phospho-cofilin peptide, the substrate of chronophin. A conformational change to a cap-open conformation may be required for substrate binding. The core and cap domains are joined through linker peptide hinges that change conformation to open the active site. The crystal structures reveal that a disulphide bond between the cap and core domains restricts the hinge motion. The enzyme displays PLP dephosphorylation activity in the cap-closed conformation with the disulphide bond and even in the crystal state, in which repositioning of the cap and core domains is restricted. Structural analysis suggests that a small substrate such as PLP can bind to the active site through a small movement of a local motif. However, a change to the cap-open conformation is required for binding of larger substrates such as phosphopeptides to the active site.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7

FDA approved three RAF inhibitors for the treatment of tumors containing BRAFV600 mutations, but one of the major drawbacks of these type I RAF inhibitors is to activate MAPK signaling pathway, instead of inhibiting signaling, which is referred to as paradoxical activation. Such undesired paradoxical activation not only leads to renewed tumor growth but also spurs additional cancer growth in non-cancerous wild-type BRAF tissue. Plus, these first-generation RAF inhibitors targeting BRAFV600 mutants are unable to inhibit oncogenic RAF dimers. This has led to the development of type II RAF inhibitors such as belvarafenib and day101 to block the activity of multiple forms of RAF while avoiding paradoxical activation. Two lead-like stage compounds 1 and 2 were specifically designed as type II RAF inhibitors to have activity across RAF isoforms including BRAFV600E, BRAF wild-type, and CRAF, showing higher potency than competitors. Compounds 1 and 2 potently inhibited the growth of BRAFV600E melanoma cells and NRAS or KRAS mutant cancer cells. Compounds 1 and 2 promoted the formation of BRAF/CRAF heterodimers by directly binding to the RAF kinase domain like other type II RAF inhibitors and inhibited phosphorylation of downstream effectors MEK and ERK in a dose-dependent manner in RAS mutant cancer cells, suggesting less paradoxical activation liability. Compound 2 showed superior on-target inhibitory activity for BRAFV600E, BRAF wild-type, and CRAF than belvarafenib through RAF immunoprecipitation (IP) kinase assay. Inhibition of the RAF downstream signaling was also confirmed by quantifying the level of phospho-ERK in NRAS or KRAS mutant cancer cells. In HCT116 (KRASG13D) subcutaneous xenograft model, compound 1 showed tumor growth inhibition efficacy, suggesting a potential to address RAS mutant driven- as well as BRAFV600 mutant driven tumors. Also, combination treatment with MEK inhibitor and/or immune checkpoint inhibitor would further improve the therapeutic activity and expand target indication for unmet medical needs. Citation Format: Kwangwoo Hwang, SeoHyun Jo, Jieun Choi, Ga-young Choi, Jiseon Choi, Ji-Hye Kwon, Dong-Guk Shin, Jiyeon Kim, Se-Hyuk Kim, Haelee Kim, Ha Yeon Cho, Jung Beom Son, Nam Doo Kim, Hwan Geun Choi, Daekwon Kim, Sunghwan Kim. Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB521.

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1-3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the β-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS-GST tetramer complex functions as a frame in the MSC.

AIMP2/p38 is a multifunctional tumor suppressor that normally resides in the cytosol as a scaffold protein of the multi-tRNA synthetase complex (MSC). One of the tumor-suppressive functions of AIMP2 is to facilitate ubiquitin-mediated degradation of FUSE-binding protein (FBP, FUBP1), a transcriptional activator of c-Myc. However, the mechanism by which AIMP2 functions within this pathway and its significance in tumorigenesis are uncertain. Here, we report that Smurf2 is responsible for AIMP2-mediated ubiquitination of FBP, and a mutation in AIMP2 that inhibited its nuclear interaction with Smurf2 enhanced cellular transformation and tumorigenesis in vivo. Treatment of HeLa cells with TGFβ resulted in the phosphorylation of AIMP2 on S156, a residue that is exposed on the embedded GST domain of AIMP2. We further found that phospho-AIMP2 dissociated from the MSC and translocated to the nucleus, where it bound to Smurf2, enhancing ubiquitination of FBP. AIMP2 also inhibited nuclear export of Smurf2 to sustain TGFβ signaling. Collectively, these findings present a novel tumor-suppressive interaction between AIMP2 and Smurf2 and suggest that the disruption of this interaction can lead to oncogenic transformation. Cancer Res; 76(11); 3422–36. ©2016 AACR.

Successful product development and marketing necessitate a study of the consumer concept of culture-specific or deep-positioned branded food. In this study, a new consumer test method was designed based on an authenticity test and used as a reference frame for the target concept without an upsetting story. The response format of this method included the metacognitive certainty response following the sensory authenticity response using the A-Not A test procedure. The method was applied to study the concept of goso flavor, as perceived by 91 female consumers with three commercial soymilk products, having each consumer evaluate each product 45 times over three days. The repeated responses of sensory authenticity were analyzed as mean scores and signal detection theory (SDT) d-prime (d′) values of the product difference. From the metacognitive certainty responses after the sensory authenticity response, a new quantitative group measure of d-prime metacognition (d′MC) was calculated in the SDT context and compared with the other outputs. The measure ranged from negative to positive values, indicating a mismatch to a match for the concept of each product. Data analyses were conducted on both pooled data and segmented data, which was driven from the results of cluster analyses using the mean sensory authenticity scores and SDT C values (estimates of response bias about the concept tested). The results showed that d′MC of each product corresponded to the mean scores and d′ with the advantage of easy interpretation. Overall, d′MC can be a useful group measure for studying the consumer concept towards food and beverages.

Cognitive impairment that can be caused by brain disorders or aging affects quality of life by interfering with abilities to perform activities of daily living (ADL). For this reason, this study developed serious games for the purpose of rehabilitation and proposes an integrated management service model that can recommend the difficulty level of games and manage changes in serious results according to user’s cognitive function ability during rehabilitation. The areas of cognitive training are designed in consideration of game elements in relation to attention, concentration, memory, orientation, executive function and ADL. Based on the analysis results, we established a database for recommendation of game level in the next training session. Attention is further subdivided into selective, sustained and divided attention. Memory is subcategorized into topological memory, associative memory, verbal memory, immediate memory, visual short-term memory, working memory and recall memory. Executive function is classified into inhibitory control, cognitive flexibility and working memory. ADL includes physical ADL and instrumental ADL. This model comprises a total of 19 serious games for cognitive rehabilitation and each game consists of 3 levels of increasing difficulty. Game interface is created with a user-centered design which focuses on visualization for elderly-friendly use. Our integrated management service model for cognitive rehabilitation can provide training on cognitive function in different areas as a single program and recommend the difficulty level of games depending on player’s ability.

A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis . Among response regulators in M. tuberculosis , NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T + 1 and T + 2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Mycobacterium tuberculosis is thought to undergo transformation into its non-replicating persistence state under the influence of hypoxia or nitric oxide (NO). This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT, each of which contains two GAF domains that are responsible for detecting oxygen tension. In this study we determined the crystal structures of the first GAF domain (GAF-A) of DosS, which shows an interaction with a heme. A b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly perpendicular to the β-sheet of the GAF domain. The heme iron was liganded by His-149 at the proximal heme axial position. The iron, in the oxidized form, was six-coordinated with a water molecule at the distal position. Upon reduction, the iron, in ferrous form, was five-coordinated, and when the GAF domain was exposed to atmospheric O2, the ferrous form was oxidized to generate the Met form rather than a ferrous O2-bound form. Because the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen bond network found at the heme site could accelerate the electron transferability and would explain why DosS was unable to bind O2. Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its reduction.

As sodium reduction has become very important in the food industry, various types of unspecified duo-trio tests have been studied to improve its efficiency for studying samples with high sodium content, and a constant-saltier-reference duo-trio test with dual reference, one reference in the first position and the second reference in the middle between the two test stimuli (DTFM), has been recommended. For the duo-trio test, a 'comparison of distance' (COD) strategy has been generally assumed. Yet, theoretically for DTFM, the 2-AFC reminder (2-AFCR) τ-strategy is also possible, which would make DTFM more efficient than the unspecified tetrad test. In this study, the hypothesis was that when subjects are pre-exposed to two types of samples, the 2-AFCR τ-strategy can be adopted in a constant-reference DTFM using a fixed design experiment. In order to test this hypothesis, unspecified tetrad tests involving categorization tasks were used as a means of pre-exposure to the two types of samples for DTFM, and a performance comparison was conducted. Two groups of 39 untrained/naive subjects performed both the unspecified tetrad and DTFM tests in varying orders for the purpose of discriminating two different soup samples of varying sodium content. A comparison of the d' estimate across different methods supported the hypothesis that the more efficient 2-AFCR τ-strategy was appropriate when the tetrad test preceded DTFM, while when DTFM was performed first without pre-categorization of samples, the conventional duo-trio COD strategy was appropriate for the constant-reference DTFM.

Mycobacterium tuberculosis is known to transform into the nonreplicating persistence state under the influence of hypoxia or nitric oxide. DevS-DevR is a two-component regulatory system that mediates the genetic response for the transformation. DevS is a histidine kinase that contains two GAF domains for sensing hypoxia or nitric oxide. The second GAF from M. smegmatis DevS was crystallized using the sitting-drop vapour-diffusion method in the presence of sodium citrate and 2-propanol as precipitants. X-ray diffraction data were collected from crystals containing selenomethionine to a maximum resolution of 2.0 A on a synchrotron beamline. The crystals belong to the hexagonal space group P6(1). The asymmetric unit contains one molecule, corresponding to a packing density of 2.5 A(3) Da(-1). The selenium substructure was determined by the single anomalous dispersion method and structure refinement is in progress.

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O 2 . The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis . The binding of O 2 to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O 2 and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.