Published papers

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) axis is an attractive therapeutic target for various intractable diseases. Although protein–protein interaction inhibitors against Keap1-Nrf2 have been developed over the past decade, more structural expansion is needed to improve efficacy. In this article, several candidate compounds are designed and synthesized as novel Nrf2 activators and their intracellular Nrf2-activating effects are evaluated. Among the synthesized compounds, a novel naphthalene-1,4-(4-ethoxybenzensulfonamide) bearing a tertiary acetamide side chain at the 2-position strongly activated intracellular Nrf2. Particularly, the pyrrolidine-type acetamide compound showed the strongest intracellular Nrf2 activation. X-ray cocrystallography revealed that this compound can bind to the DC domain of Keap1. Additionally, the pyrrolidine-type acetamide compound induced the mRNA expression of the representative Nrf2 target genes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Moreover, the compound exhibited anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage cell line. Conclusively, these results suggest that the pyrrolidine-type naphthalene-2-acetamide is a promising compound for the development of Nrf2 activators that can be applied to treat inflammatory diseases.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a crucial role in cellular defenses against oxidative stress and inflammation. Under normal conditions, Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase adaptor, binds to Nrf2, facilitating its ubiquitination and subsequent degradation via the proteasome. In this study, we investigated the properties of KMN003, a novel Nrf2 activator specifically designed to stabilize Nrf2 by disrupting its interaction with Keap1. X-ray crystallographic analysis revealed that KMN003 binds to the DGR-Cul3 (DC) domain of Keap1, occupying the Nrf2 interaction site. An AlphaScreen assay further showed that KMN003 effectively inhibits the binding between the Keap1 DC domain and the DLG motif of Nrf2 (IC₅₀ = 300 nM). We also investigated the mechanism of Nrf2 activation by KMN003 and its anti-inflammatory properties using murine macrophage-like RAW264.7 cells. KMN003 significantly reduced the lipopolysaccharide (LPS)-induced production of nitric oxide, CCL2, and tumor necrosis factor-alpha (TNFα) as well as the mRNA expression of inducible nitric oxide synthase, CCL2, and TNFα, which are essential inflammatory markers. KMN003 strongly inhibited nuclear translocation and transcriptional activation of nuclear factor-kappa B (NF-κB), a central regulator of inflammatory gene expression. KMN003 did not affect the LPS-induced phosphorylation of ERK or JNK, but strongly induced p38 phosphorylation in the absence of the LPS stimulation. The inhibition of p38 with SB203580 blocked KMN003-induced Nrf2 transcriptional activation despite promoting Nrf2 accumulation. These results highlight KMN003 as a promising anti-inflammatory drug that selectively stabilizes and activates Nrf2 via the p38 pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via its spike (S) protein, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor. Glycans are thought to influence this interaction by modulating the binding affinity between the S protein and its receptor. In this study, we screened 300 carbohydrate species using a glycan array to identify potential ligands that interact with the S proteins of the Delta and Omicron variants. Among the identified candidates, two aminoglycoside antibiotics, tobramycin and sisomicin, exhibited notable binding to the S protein. Surface plasmon resonance (SPR), circular dichroism (CD), and in silico docking analyses confirmed direct interactions between these aminoglycosides and the S protein, revealing distinct binding characteristics. Nuclear magnetic resonance (NMR) analysis further localized the tobramycin-binding site within the receptor-binding domain (RBD) of the S protein. Tobramycin and sisomicin showed a tendency to inhibit SARS-CoV-2 replication in human induced pluripotent stem cell (hiPSC)-derived lung organoids, though the effect did not reach statistical significance. Docking simulations using the trimeric S model suggested that aminoglycosides bind at an inter-subunit interface. These findings demonstrate that aminoglycosides can directly interact with the SARS-CoV-2 S protein and may serve as scaffolds for developing host-independent antiviral agents against SARS-CoV-2 and its variants.

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein alpha subunit with GDP (Galpha.GDP) and the G protein betagamma subunit (Gbetagamma). Ligand binding to GPCRs promotes the GDP-GTP exchange on Galpha, leading to the dissociation of the GTP-bound form of Galpha (Galpha.GTP) and Gbetagamma. Then, Galpha.GTP and Gbetagamma bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Galpha hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gbetagamma. The G proteins are classified with four major families based on the amino acid sequences of Galpha: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Galphai3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Galpha(i3) in complex with a GTP-analogue, GTPgammaS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPgammaS-bound states, as indicated in the crystal structures. The assignments of Galpha(i3).GDP would be useful for the analyses of the dynamics of Galpha(i3) and its interactions with various target molecules.

Magnesium ion (Mg(2+)) is an essential metal element for life, and has many cellular functions, including ATP utilization, activation of enzymes, and maintenance of genomic stability. The intracellular Mg(2+) concentration is regulated by a class of transmembrane proteins, called Mg(2+) transporters. One of the prokaryotic Mg(2+) transporters, MgtE, is a 450-residue protein, and functions as a dimer. We previously reported that MgtE exhibits the channel-like electrophysiological property, i.e., it permeates Mg(2+) according to the electrochemical potential of Mg(2+). The Mg(2+)-permeation pathway opens in response to the decrease of the intracellular Mg(2+) concentration, while it is completely closed at the intracellular Mg(2+) concentration of 10 mM. The crystal structures of the MgtE dimer revealed that the Mg(2+)-sensing cytoplasmic region consists of the N and CBS domains. The Mg(2+)-bound state of MgtE adopts a compact, globular conformation, which is stabilized by the coordination of a number of Mg(2+) ions between these domains. On the other hand, in the Mg(2+)-unbound state, these domains are far apart, and fixed by the crystal packing. Therefore, structural analyses in solution were awaited, in order to characterize the Mg(2+)-dependent alteration of the MgtE structure and dynamics relevant to its gating. In this paper, we report the backbone resonance assignments of the dimer of the cytoplasmic region of the MgtE from Thermus thermophilus with a molecular weight of 60 KDa, in the Mg(2+)-unbound state.

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein alpha subunit (Galpha(GDP)) and G protein betagamma subunit (Gbetagamma). Ligand binding to GPCRs promotes the GDP-GTP exchange on Galpha, leading to the dissociation of the GTP-bound form of Galpha (Galpha(GTP)) and Gbetagamma. Then, Galpha(GTP) and Gbetagamma bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Galpha hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gbetagamma. G proteins are classified with four major families based on the amino acid sequences of Galpha: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Galpha(i3), a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPgammaS.

HisJ is a histidine binding subunit of the histidine permease, which exists in the outer membrane of Gram-negative bacteria. In order to incorporate the periplasmic histidine into the cell, HisJ captures histidine in the periplasm, and transfers the histidine to the transmembrane complex of histidine permease that is an ABC transporter. We established the backbone resonance assignments of (1)H/(13)C/(15)N-labeled HisJ from Escherichia coli, in the histidine-bound and unbound states.

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. A variety of ligands, including heterotrimeric G protein betagamma subunits (G betagamma), bind to the cytoplasmic regions of GIRK and modulate its activity. We established the backbone resonance assignments of (2)H/(13)C/(15)N-labeled cytoplasmic regions of mouse GIRK1, which form a tetramer with a molecular weight of 96 K.

We describe an NMR-based approach, the amino acid selective cross-saturation (ASCS) method, to identify the pairs of the interface residues of protein-protein complexes. ASCS uses a "cross-saturation (CS)-donor" protein, in which only one amino acid is selectively (1)H-labeled in a (2)H-background, and a "CS-acceptor" protein with uniform (2)H, (15)N labeling. Irradiation of the (1)H-labeled amino acid, which exists only in the donor, decreases the intensity of the (1)H- (15)N HSQC signals of the acceptor residues proximal to the (1)H-labeled CS-source residue(s) through the CS phenomenon. Given the three-dimensional structure of each protein in the complex, but not the complex structure, the combinatorial analysis of multiple ASCS results specify the CS-source residue(s), based on the spatial complementarity between the CS-source residues on the CS donor and the cross-saturated amide protons on the acceptor. NMR investigations of the labeling selectivity and efficiency in an E. coli host, which are critical for ASCS, revealed that Ala, Arg, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, and Tyr are selectively labeled with a high (1)H/(2)H ratio. The observation of the ASCS was then confirmed using the known structure of the yeast ubiquitin (Ub) and yeast ubiquitin hydrolase 1 (YUH1). Conversely, reasonable candidates for the CS-source residues were suggested by the analysis of the ASCS results, with reference to the individual structures of YUH1 and Ub. The pairwise distance information between the CS-source residues and the cross-saturated amide groups obtained by ASCS will be useful for modeling protein-protein complexes.

In cancer cells, Ras protein mutations activate a signaling cascade that phosphorylates kinases, transcription, and translation factors, driving cancer cell proliferation. One such factor, FOXO3a, promotes apoptosis-related gene transcription. However, in many cancer cells, FOXO3a is phosphorylated and is bound to 14-3-3ζ at phosphorylation sites. The 14-3-3ζ binding displaces phosphorylated FOXO3a from DNA, suppressing apoptosis. Since the phosphorylation sites are far from the DNA-binding domain (DBD) of FOXO3a, the mechanism of displacement remains unclear. Using isothermal titration calorimetry and fluorescence-detection size-exclusion chromatography, we find that 14-3-3ζ strongly displaces DNA from di-phosphorylated FOXO3a (dpFOXO3a), despite similar dissociation constants for dpFOXO3a–14-3-3ζ and dpFOXO3a–DNA. Nuclear magnetic resonance data identify weak, but direct binding of 14-3-3ζ to the DBD, suggesting direct competition. These findings suggest that 14-3-3ζ enhances its competitive ability by dual tethering to the DBD of FOXO3a via phosphorylation sites, effectively displacing DNA.

Developing compounds with novel structures is important for the production of new drugs. From an intellectual perspective, confirming the patent status of newly developed compounds is essential, particularly for pharmaceutical companies. The generation of a large number of compounds has been made possible because of the recent advances in artificial intelligence (AI). However, confirming the patent status of these generated molecules has been a challenge because there are no free and easy-to-use tools that can be used to determine the novelty of the generated compounds in terms of patents in a timely manner; additionally, there are no appropriate reference databases for pharmaceutical patents in the world. In this study, two public databases, SureChEMBL and Google Patents Public Datasets, were used to create a reference database of drug-related patented compounds using international patent classification. An exact structure search system was constructed using InChIKey and a relational database system to rapidly search for compounds in the reference database. Because drug-related patented compounds are a good source for generative AI to learn useful chemical structures, they were used as the training data. Furthermore, molecule generation was successfully directed by increasing and decreasing the number of generated patented compounds through incorporation of patent status (i.e., patented or not) into learning. The use of patent status enabled generation of novel molecules with high drug-likeness. The generation using generative AI with patent information would help efficiently propose novel compounds in terms of pharmaceutical patents. Scientific contribution: In this study, a new molecule-generation method that takes into account the patent status of molecules, which has rarely been considered but is an important feature in drug discovery, was developed. The method enables the generation of novel molecules based on pharmaceutical patents with high drug-likeness and will help in the efficient development of effective drug compounds.

A useful calmodulin (CaM) antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), was invented by Hidaka et al. in 1978 (J. Pharmacol. Exp. Ther. 207, 8-15). Here, we have designed new CaM antagonists on the basis of the three-dimensional structure of Ca2+/CaM complexed with W-7. Eleven new compounds all share a similar architecture, in which two W-7 molecules are linked between their aminohexyl termini by a linker with different functionalities. A wide range of inhibitory activities against Ca2+/CaM-dependent protein kinase I (CaM kinase I) has been observed with these self-crosslinked W-7 analogs, (W-7)2. In vitro competitive CaM kinase I assays using CaM kinase I and nuclear magnetic resonance studies indicated that one (W-7)2 molecule binds to one CaM molecule as expected, with the two chloronaphthalene rings of (W-7)2 being anchored separately to the N- and C-terminal hydrophobic pockets of Ca2+/CaM. The most potent compound, N,N'-bis[6-(5-chloro-1-naphthalenesulfonyl)-amino-1-hexyl]-p-xylen e-diamine ((W-7)2 - 10), inhibits CaM kinase I activity at an IC50 value of 0.23 microM; about 75 times more effectively than W-7. The length and basicity of the linker sequence in (W-7)2 significantly contribute to inhibitory activity. The present study opens an avenue for developing powerful CaM antagonists that could be used at low doses in vivo.

Transcriptional regulation is coupled with numerous intracellular signaling processes often mediated by second messengers. Now, growing evidence points to the importance of Ca(2+), one of the most versatile second messengers, in activating or inhibiting gene transcription through actions frequently mediated by members of the EF-hand superfamily of Ca(2+)-binding proteins. Calmodulin and calcineurin, representative members of this EF-hand superfamily, indirectly regulate transcription through phosphorylation/dephosphorylation of transcription factors in response to a Ca(2+) increase in the cell. Recently, a novel EF-hand Ca(2+)-binding protein called DREAM has been found to interact with regulatory sequences of DNA, thereby acting as a direct regulator of transcription. Finally, S100B, a dimeric EF-hand Ca(2+)-binding protein, interacts with the tumor suppressor p53 and controls its transcriptional activity. In light of the structural studies reported to date, this review provides an overview of the structural basis of EF-hand Ca(2+)-binding proteins linked with transcriptional regulation.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

Aggregation of the 43 kDa TAR DNA-binding protein (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP-43 N-terminal domain dimerisation has been suggested to ameliorate TDP-43 aggregation. However, the relationship between these factors and the solubility of TDP-43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP-43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP-43-preferable UG-repeats, we uncovered two distinct mechanisms for modulating TDP-43 solubility by RNA binding: One is N-terminal domain dimerisation, and the other is the spatial separation of two TDP-43 molecules. This study provides new molecular insights into the regulation of TDP-43 solubility.

The solution structure of calcium-bound calmodulin (CaM) complexed with an antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), has been determined by multidimensional NMR spectroscopy. The structure consists of one molecule of W-7 binding to each of the two domains of CaM. In each domain, the W-7 chloronaphthalene ring interacts with four methionine methyl groups and other aliphatic or aromatic side-chains in a deep hydrophobic pocket, the site responsible for CaM binding to CaM-dependent enzymes such as myosin light chain kinases (MLCKs) and CaM kinase II. This competitive binding at the same site between W-7 and CaM-dependent enzymes suggests the mechanism by which W-7 inhibits CaM to activate the enzymes. The orientation of the W-7 naphthalene ring in the N-terminal pocket is rotated approximately 40 degrees with respect to that in the C-terminal pocket. The W-7 ring orientation differs significantly from the Trp800 indole ring of smooth muscle MLCK bound to the C-terminal pocket and the phenothiazine ring of trifluoperazine bound to the N or C-terminal pocket. These comparative structural analyses demonstrate that the two hydrophobic pockets of CaM can accommodate a variety of bulky aromatic rings, which provides a plausible structural basis for the diversity in CaM-mediated molecular recognition.

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.