Elevated expression of transglutaminase 2 (TGase 2, EC 2.3.2.13, protein-glutamine γ-glutamyltransferase, gene name TGM2) is known as one of the most upregulated genes during epithelial-mesenchymal transition (EMT) in ovarian cancer. Despite initial complete responses to conventional chemotherapy, ovarian cancer often recurs with metastasis, presenting a significant clinical challenge. Drug-resistant ovarian cancer cells exhibit markedly higher levels of TGase 2 compared to normal ovarian epithelium, which is associated with EMT activation, enabling them to evade chemotherapy effects. Intracellular TGase 2 is recognized as a key factor in maintaining the mesenchymal phenotype. Therefore, while EMT expression can be effectively reversed by inhibiting TGase 2, the underlying mechanism of this effect remains unclear. We found that TGase 2 promotes EMT by directly binding to glycogen synthase kinase-3β (GSK3β), promoting the stabilization of β-catenin. Domain mapping revealed that the N-terminus of TGase 2 interacts with the mid-region of GSK3β, leading to the autophagic degradation of GSK3β. Pharmacological disruption of this N-terminal interaction by streptonigrin, in combination with standard chemotherapy, extended overall survival in a xenograft model of ovarian cancer. This study identified TGase 2 as a pivotal regulator of EMT-driven metastasis and drug resistance.

The VHL-containing cullin-RING E3 ubiquitin ligase (CRL2VHL) complex is an E3 ligase commonly used in targeted protein degradation (TPD). Hydroxyproline-based ligands that mimic VHL substrates have been developed as anchor molecules for proteolysis-targeting chimeras (PROTACs) in TPD. To expand the chemical space for VHL ligands, we conducted fragment screening using VHL–ELOB–ELOC (VBC) proteins. We found that certain 7-hydroxycoumarin derivatives (7HCs), rather than VHL, would bind to the ELOC component of the VBC complex. The 7HC binding site overlapped with the CUL2 binding interface on ELOC but did not overlap with the CUL5 binding interface, suggesting that 7HCs may influence the formation of CRL2 but not CRL5. Although the binding affinities of these 7HCs to the VBC complex were relatively low, they represent novel and promising foundational agents for the development of chemical probes or inhibitors that target ELOC-containing CRLs.

The elongin BC complex (ELOBC) interacts with BC-box-containing proteins and plays a role in various cellular processes, including transcriptional regulation and ubiquitination. Elongin BC and polycomb repressive complex 2-associated protein (EPOP) contains a BC-box motif in its N-terminal region and influences cancer cell proliferation and differentiation. A previous study showed that a BC-box containing an EPOP-derived peptide suppresses cancer cell growth and induces apoptosis by disrupting the interaction between the ELOBC and its partner proteins. Here, we report the crystal structure of the EPOP BC-box peptide bound to the ELOBC and compare it with the structures of other BC-box-containing proteins in complex with the ELOBC. The overall structure of interactions between the BC-box and the ELOC was similar across different complexes, indicating a conserved binding mode. Our structural analysis revealed that the strictly conserved leucine residue (Leu40) within the BC-box of EPOP, which was previously suggested to be critical for interactions between the BC-box and the ELOC, was deeply embedded in the hydrophobic pocket of the ELOC protein. This study provided structural insights into BC-box-mediated protein-protein interactions and may serve as a fundamental resource for developing small molecules that modulate the interactions between ELOBC and BC-box-containing proteins.

The proximal domains of mitochondria and the endoplasmic reticulum (ER) are linked by tethering factors on each membrane, allowing the efficient transport of substances, including lipids and calcium, between them. However, little is known about the regulation and function of mitochondria-ER contacts (MERCs) dynamics under mitochondrial damage. In this study, we apply NanoBiT technology to develop the MERBiT system, which enables the measurement of reversible MERCs formation in living cells. Analysis using this system suggests that induction of mitochondrial ROS increases MERCs formation via RMDN3 (also known as PTPIP51)-VAPB tethering driven by RMDN3 phosphorylation. Disruption of this tethering caused lipid radical accumulation in mitochondria, leading to cell death. The lipid radical transfer activity of the TPR domain in RMDN3, as revealed by an in vitro liposome assay, suggests that RMDN3 transfers lipid radicals from mitochondria to the ER. Our findings suggest a potential role for MERCs in cell survival strategy by facilitating the removal of mitochondrial lipid radicals under mitochondrial damage.

As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners. It is still unclear how RKIP switches between monomeric and dimeric forms. Here, we identified the role of cysteine 133 in RKIP structural dynamics using recombinant human RKIP (rhRKIP) proteins purified from Escherichia coli BL21(DE3) cells. Mutation of alanine or serine instead of cysteine in RKIP proteins did not affect the biochemical characteristics, while dynamic light scattering and liquid chromatography (LC) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) suggested distinct peaks in solution, which were identified via LC–MS/MS analyses, and further clarified the role of cysteine in RKIP dimerization. rhRKIP dimer formation was abrogated by a 32-aa peptide mimicking the region between two RKIP proteins for dimerization. In addition, the 32-aa peptide and its short derivatives were investigated for effects on cancer cell viability. Taken together, our findings suggest that it may be possible to regulate RKIP function by controlling its dynamics with reducing agents, which could aid the targeting of cancer cells.

In eukaryotic cells, mitochondria and the endoplasmic reticulum (ER) form close contacts at mitochondria-associated ER membranes (MAMs), which are involved in diverse cellular processes. The outer mitochondrial membrane protein Fis1, known for its role in mitochondrial fission, has been reported to interact with the ER-resident protein Bap31. Here, we present crystal structures of the cytosolic domain of human Fis1 in two distinct conformations, along with a co-crystal structure of Fis1 bound to the C-terminal region of the Bap31_vDED domain. One Fis1 structure resembles monomeric yeast Fis1 and features a characteristic N-terminal "Fis1 arm" conformation, which may indicate an autoinhibitory function. In the co-complex, the Bap31_vDED region engages the convex surface of Fis1's tetratricopeptide repeat (TPR) domain. These findings provide structural insight into the interaction between Fis1 and Bap31 at ER-mitochondria contact sites.

Background Non-small cell lung cancer (NSCLC) is associated with abnormal activation of the epidermal growth factor receptor (EGFR) due to overexpression or mutations. While EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, are used to treat NSCLC, resistance often develops, due to additional EGFR mutations or activation of alternative signaling pathways. Therefore, novel drugs to overcome EGFR-TKI resistance are needed for effective treatment of NSCLC. Narciclasin (Ncs) is a cytotoxic alkaloid from Narcissus species and exhibit antitumor and anti-inflammatory activities. Methods Cell viability assay was assessed using trypan blue staining and the Live/Dead viability assay. The growth inhibitory effects of Ncs were evaluated by WST-1 assay and cell cycle analysis across multiple NSCLC cell lines, including expressing A549 and H1299 (wt-EGFR), gefitinib-resistant H1975 (L858R/T790M-EGFR), gefitinib-sensitive PC-9 (exon 19 deleted-EGFR), and gefitinib-resistant PC-9 derivative, PC-9-GR. Ncs binding to wt-EGFR and mutant EGFRs was simulated with molecular docking models. Ncs effects on EGFR kinase activity was evaluated in vitro kinase assay using wt-EGFR and L858R/T790M-EGFR. Anti-tumor effects of Ncs in vivo were assessed using C. elegans tumor model expressing L858R/T790M-EGFR and mouse model xenografted with A549 and H1975. Histological analysis was conducted to measure EGFR, p-EGFR, and p-STAT3 levels in tumor tissues. Results Ncs exhibited significant growth inhibitory effects on various NSCLC cell lines, including, A549, H1299 and PC-9 cells (gefitinib-sensitive), and H1975 and PC-9-GR cells (gefitinib-resistant). Notably, Ncs dramatically reduced cell growth with IC50 of 22 nM in H1975 cells expressing gefitinib -resistant EGFR mutant, much lower than any other cell lines. Ncs dramatically induced G2/M arrest in H1975 cells. Ncs binds to both wt-EGFR and mutant EGFRs in molecular docking models and preferentially inhibited the kinase activity of L858R/T790M-EGFR compared to wt-EGFR. In a C. elegans tumor model, Ncs reduced the tumor-mimicking multivulva phenotype. Ncs treatment resulted in decreased tumor growth in mice xenografted with A549 and H1975 cells and lowered levels of EGFR, p-EGFR, and p-STAT3 in tumor tissues. Conclusions Our results suggest that Ncs exerts anti-tumor activity by inhibiting EGFR activity and downstream signaling. This effect is particularly evident in cases with EGFR mutations that confer resistance to TKIs, including gefitinib, supporting the potential of Ncs as a therapeutic agent for TKI-resistant NSCLC.

The activatable fluorescence probe Q-Atezol demonstrates great potential as an exceptional sensor for assessing PD-L1 expression in three-dimensional cell structures and for in vivo applications.

Small-cell lung cancer (SCLC) is highly lethal because the tumors grow and metastasize rapidly. Effective treatments for SCLC are lacking currently. A recent study demonstrated that the E1A binding protein P300 (EP300) KIX domain has pro-tumorigenic activity and is selectively involved in the development and growth of SCLC. These findings suggest the possibility of developing small-molecule inhibitors of EP300 KIX as new targeted therapies for SCLC. In this study, we reported the crystal structure of the human EP300 KIX domain at 2.9 & Aring; resolution except for a flexible loop and C-terminal end. The overall structure was almost identical to that of the cAMP response element-binding protein (CBP) KIX. Nine EP300 KIX residues were different from those of CBP KIX. Among these non-strictly conserved residues, Ala627, which corresponds to Asp647 in CBP KIX, reduces the negative surface potential. Asn581 and Arg613 contributed to the formation of additional hydrogen bonds in the EP300 KIX structure. Further structural analysis revealed that the hydrophobic residues that form the allosteric network in CBP KIX were well conserved in the EP300 KIX structure. This study lays the groundwork for structure-based drug design for SCLC.

본 논문은 ‘비(非)중생자의 선행’에 대한 이중적 이해가 필요함을 제시한다. 첫째, ‘비(非)중생자의 선행’은 특별은총의 관점에서 본질적으로 죄이다. 그것은 하나님께서 받으실 만한 요건을 갖고 있지 않기 때문이다. 그것은 성경적 의미에서 선행을 참으로 선행되게 하는 요소가 빠져 있기 때문이다. 즉 선행은 하나님을 향한 사랑과 믿음에서 솟아나는 행위이어야 하며, 하나님의 영광을 목적으로 해야 하며, 하나님의 명령을 규정으로 삼는 행위이어야 한다. 비중생자의 선행에는 이러한 요소들이 전혀 없다. 따라서 하나님이 받으실 수 없고, 오히려 하나님을 불쾌하게 만들 뿐이다. 둘째, 일반은총의 결과로 ‘비(非)중생자의 선행’은 가능하다. 비중생자의 선행이 비록 하나님께서 받으실 수 없는 선행이지만 사람을 유익하게 할 수는 있다. 그들의 선행을 ‘시민적 의’(civil righteousness) 혹은 ‘사회적 선행’으로 이해해야 한다. 이러한 선행이 가능한 것은 ‘일반은총’ 때문이다. 특별은총은 아니더라도 인간은 은총이 있어야지만 선을 행할 수 있다. 일반은총이 죄를 없애지는 못하지만, 죄의 억제하는 기능을 한다. 그리고 비중생자로 하여금 ‘사회적 선행’을 행하도록 한다. 셋째, 비중생자의 선행에 대한 이중적 이해가 필요하다. 특별은총의 관점과 일반은총의 관점에서 이해할 뿐만 아니라, 비중생자의 선행이 선행이 될 수 없는 근본적인 이유를 알아야 한다. 그것이 ‘행위 자체’가 아닌 ‘행위자와 그의 상태’에 있음을 인식해야 한다. 이러한 두 관점이 없으면 우리는 자칫 하나님께 범죄할 수 있기 때문이다. “… 그렇다고 해서 선한 행위를 무시한다면 그것은 하나님께 더 큰 죄가 되고, 그분을 더욱 불쾌하시게 만들게 된다”(웨스트민스터신앙고백 제16장 7항 마지막 문장).

BRD4 contains two tandem bromodomains (BD1 and BD2) that recognize acetylated lysine for epigenetic reading, and these bromodomains are promising therapeutic targets for treating various diseases, including cancers. BRD4 is a well-studied target, and many chemical scaffolds for inhibitors have been developed. Research on the development of BRD4 inhibitors against various diseases is actively being conducted. Herein, we propose a series of [1,2,4]triazolo[4,3-b]pyridazine derivatives as bromodomain inhibitors with micromolar IC50 values. We characterized the binding modes by determining the crystal structures of BD1 in complex with four selected inhibitors. Compounds containing [1,2,4] triazolo[4,3-b]pyridazine derivatives offer promising starting molecules for designing potent BRD4 BD inhibitors.

Bromodomain and extra-terminal domain (BET) proteins have been considered as potent candidates for anti-cancer drug development. As epigenetic readers, they modulate gene expression by recognizing acetylated lysine residues on histones. Therefore, the pharmacological inhibition of BET proteins has been extensively studied. Herein, we report the novel chemical scaffold of N-(pyridin-2-yl)-1H-benzo[d][1,2,3]triazol-5-amine as BET inhibitors using high-throughput screening assay. Through the analysis of structure-activity relationships, we developed a potent novel compound, which exhibited a better IC50 value about 2-fold compared to iBet762 against the BRD4 bromodomain (BD). The addition of a sulfonyl group to the pyridine ring enhanced the inhibitory activity. Structural studies showed a clear electron density map for the inhibitor and revealed the structural basis for the critical role of the sulfonyl group in the interaction with BRD4.

HucR is a MarR family protein of Deinococcus radiodurans, which binds tightly to the intergenic region of HucR and the uricase gene to inhibit their expression. Urate (or uric acid) antagonizes the repressor function of HucR by binding to HucR to impede its association with the cognate DNA. The previously reported crystal structure of HucR was without the bound urate showing significant structural homology to other MarR structures. In this paper, we report the crystal structure of HucR determined with the urate bound. However, despite the fact that the urate is found at a site well-known to harbor ligands in other MarR family proteins, the overall HucR structure indicates that no significant change in structure takes place with the urate bound. Structure analysis further suggests that the urate interaction in HucR is mediated by histidine/glutamate side chains and ordered water molecules stabilized by various residues. Such interaction is quite unique compared to other known structural interactions between urate and its binding proteins. Furthermore, structural comparison of the apo- and the urate bound forms allows us to hypothesize that the Trp20-mediated water network in the apo-form stabilizes the proper HucR fold for cognate DNA binding, and that urate binding, also via Trp20, and the consequent reorganization of water molecules in the binding pocket, likely disrupts the DNA binding configuration to result in the attenuated DNA binding.

Myeloid epithelial reproductive proto-oncogene tyrosine kinase (MERTK) plays an essential role in modulating cancer immune tolerance by regulating macrophage efferocytosis. Studies are underway to develop small-molecule chemicals that inhibit MERTK as cancer immunotherapeutic agents, but these efforts are in their early stages. This study identified BMS794833, whose primary targets are MET and VEGFR2, as a potent MERTK inhibitor and developed a real-time efferocytosis monitoring system. The X-ray cocrystal structure revealed that BMS794833 was in contact with the ATP-binding pocket and the allosteric back pocket, rendering MERTK inactive. Homogeneous time-resolved fluorescence kinetic and Western blotting analyses showed that BMS794833 competitively inhibited MERTK activity in vitro and inhibited the autophosphorylation of MERTK in macrophages. We developed a system to monitor MERTK-dependent efferocytosis in real time, and using this system, we confirmed that BMS794833 significantly inhibited the efferocytosis of differentiated macrophages. Finally, BMS794833 significantly inhibited efferocytosis in vivo in a mouse model. These data show that BMS794833 is a type II MERTK inhibitor that regulates macrophage efferocytosis. In addition, the real-time efferocytosis monitoring technology developed in this study has great potential for future applications.

In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.

Epigenetic regulation is known to play a key role in progression of anti-cancer therapeutics. Lysine acetylation is an important mechanism in controlling gene expression. There has been increasing interest in bromodomain owing to its ability to modulate transcription of various genes as an epigenetic ‘reader.’ Herein, we report the design, synthesis, and X-ray studies of novel aristoyagonine (benzo[6,7]oxepino[4,3,2-cd]isoindol-2(1H)-one) derivatives and investigate their inhibitory effect against Brd4 bromodomain. Five compounds 8ab, 8bc, 8bd, 8be, and 8bf have been discovered with high binding affinity over the Brd4 protein. Co-crystal structures of these five inhibitors with human Brd4 bromodomain demonstrated that it has a key binding mode occupying the hydrophobic pocket, which is known to be the acetylated lysine binding site. These novel Brd4 bromodomain inhibitors demonstrated impressive inhibitory activity and mode of action for the treatment of cancer diseases.

2-oxoglutarate and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) expression is upregulated in a variety of cancers and has been related to poor prognosis. However, despite this significance to cancer progression, the precise oncogenic mechanism of OGFOD1 is not understood. We demonstrated that OGFOD1 plays a role in enhancing the transcriptional activity of RNA polymerase II in breast cancer cells. OGFOD1 directly binds to the C-terminal domain of RNA polymerase II to alter phosphorylation status. The elimination of OGFOD1 resulted in decreased tumor development. Additionally, cell cycle-dependent kinase 7 and cell cycle-dependent kinase 9, critical enzymes for activating RNA polymerase II, phosphorylated serine 256 of OGFOD1, whereas a non-phosphorylated mutant OGFOD1 failed to enhance transcriptional activation and tumor growth. Consequently, OGFOD1 helps promote tumor growth by enhancing RNA polymerase II, whereas simultaneous phosphorylation of OGFOD1 by CDK enzymes is essential in stimulating RNA polymerase II-mediated transcription both in vitro and in vivo, and expression of target genes.

The present invention relates to a pharmaceutical composition for preventing, treating or alleviating cancer, containing an activation Inhibitor of PLK1 as an active ingredient, and a compound according to the present invention selectively binds to PBD of PLK1, thereby having advantages of high selectivity and binding affinity for PLK1 and low toxicity. Therefore, a PLK1 activation inhibitor compound according to the present invention can be effectively used as an anticancer agent by inhibiting the growth of various cancer cells, and can be expected to exhibit synergistic effects with existing developed anticancer agents through co-administration, in addition to individual administration thereof.

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.