Cryo-EM structure of Influenza A virus NS1 and antiviral protein kinase PKR complex.

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.

Structural basis for T cell immunoglobulin and mucin protein 3 and Toxascaris leonina galectin complex.

T-cell immunoglobulin and mucin protein 3 (Tim-3), also known as Hepatitis A virus cellular receptor 2, has been discovered to have a negative regulatory effect on murine T-cell responses. Galectin-9 exhibits various biological effects, including cell aggregation, eosinophil chemoattraction, activation, and apoptosis, observed in murine thymocytes, T-cells, and human melanoma cells. Such approach demonstrated that Galectin-9 acts as a binding partner on Tim-3 and mediates the T-cell inhibitory effects. Tl-gal is a homologous protein to galectin-9, isolated from the adult stage of the canine gastrointestinal nematode parasite Toxascaris leonina. However, molecular mechanism between Tim-3 and galectin-9 is still remain unknown. Here, we describe the cryo-electron microscopy and X-ray structures and interactions of the Tim-3 and Tl-gal complex as well as their biochemical and biophysical characterization. In the structure, Ser46 residue of Tl-gal NCRD was bound to Asp25 residue of hTim-3. Compared to our previous study, the binding site of the complex is the same as the sugar binding site (the Ser46 residue) of Tl-gal. In addition, analysis of the complex structure revealed that the four Tl-gal molecules were in an open form packing and one mTim-3 peptide was bound to one Tl-gal molecule. These observations suggest that how Tl-gal binds hTim3 is essential to understanding the molecular mechanism for the Tim-3-galectin 9 interaction that regulates immune responses. This could potentially serve as a therapeutic target for inflammatory diseases.

Structural study of novel vaccinia virus E3L and dsRNA-dependent protein kinase complex.

E3L (RNA-binding protein E3) is one of the key IFN resistance genes encoded by VV and consists of 190 amino acids with a highly conserved carboxy-terminal double-stranded RNA-binding domain (dsRBD). PKR (dsRNA-dependent protein kinase) is an IFN-induced protein involved in anti-cell and antiviral activity. PKR inhibits the initiation of translation through alpha subunit of the initiation factor eIF2 (eIF2α) and mediates several transcription factors such as NF-κB, p53 or STATs. Activated PKR also induces apoptosis in vaccinia virus infection. E3L is required for viral IFN resistance and directly binds to PKR to block activation of PKR. In this work, we determined the three-dimensional complex structure of E3L and PKR using cryo-EM and determined the important residues involved in the interaction. In addition, PKR peptide binds to E3L and can increase protein levels of phosphorus-PKR and phosphorus-eIF2α-induced cell apoptosis through upregulation of phosphorus-PKR in HEK293 cells. Taken together, structural insights into E3L and PKR will provide a new optimization and development of vaccinia virus drugs.

Structural basis of the oncogenic KRAS mutant and GJ101 complex.

KRAS mutations occur in a quarter of all human cancers. When activated in its GTP-bound form, RAS stimulates diverse cellular systems, such as cell division, differentiation, growth, and apoptosis through the activations of various signaling pathways, which include mitogen-activated protein kinase (MAPK), phosphoinositide 3 kinases (PI3K), and RAL-GEFs pathways. We found that GJ101 (65LYDVA69) binds directly to the KRAS mutant (G12V) and showed tumor-suppressive activity. In addition, the GJ101 peptide inhibited KRAS mutant as determined by a [α-32P] guanosine triphosphate (GTP) binding assay and suppressed pancreatic cell line in a cell proliferation assay. Herein, the complex structure of KRAS and GJ101 was clarified by X-ray crystallography. Isothermal titration calorimetry showed that GJ101 binds highly with KRAS mutant and the complex structure of KRAS G12V.GJ101 complex presented that the residue of Q61 directly interacted with L65 of GJ101. Overall, the results suggest GJ101 be considered a developmental starting point for KRAS G12V inhibitor.

DED Interaction of FADD and Caspase-8 in the Induction of Apoptotic Cell Death.

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

Oncogenic KRAS: Signaling and Drug Resistance.

RAS proteins play a role in many physiological signals transduction processes, including cell growth, division, and survival. The Ras protein has amino acids 188-189 and functions as GTPase. These proteins are switch molecules that cycle between inactive GDP-bound and active GTP-bound by guanine nucleotide exchange factors (GEFs). KRAS is one of the Ras superfamily isoforms (N-RAS, H-RAS, and K-RAS) that frequently mutate in cancer. The mutation of KRAS is essentially performing the transformation in humans. Since most RAS proteins belong to GTPase, mutated and GTP-bound active RAS is found in many cancers. Despite KRAS being an important molecule in mostly human cancer, including pancreatic and breast, numerous efforts in years past have persisted in cancer therapy targeting KRAS mutant. This review summarizes the biological characteristics of these proteins and the recent progress in the exploration of KRAS-targeted anticancer, leading to new insight.

Molecular Characteristics of RAGE and Advances in Small-Molecule Inhibitors.

Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily. RAGE binds and mediates cellular responses to a range of DAMPs (damage-associated molecular pattern molecules), such as AGEs, HMGB1, and S100/calgranulins, and as an innate immune sensor, can recognize microbial PAMPs (pathogen-associated molecular pattern molecules), including bacterial LPS, bacterial DNA, and viral and parasitic proteins. RAGE and its ligands stimulate the activations of diverse pathways, such as p38MAPK, ERK1/2, Cdc42/Rac, and JNK, and trigger cascades of diverse signaling events that are involved in a wide spectrum of diseases, including diabetes mellitus, inflammatory, vascular and neurodegenerative diseases, atherothrombosis, and cancer. Thus, the targeted inhibition of RAGE or its ligands is considered an important strategy for the treatment of cancer and chronic inflammatory diseases.

Targeting Lactate Dehydrogenase A with Catechin Resensitizes SNU620/5FU Gastric Cancer Cells to 5-Fluorouracil.

Resistance to anticancer therapeutics occurs in virtually every type of cancer and becomes a major difficulty in cancer treatment. Although 5-fluorouracil (5FU) is the first-line choice of anticancer therapy for gastric cancer, its effectiveness is limited owing to drug resistance. Recently, altered cancer metabolism, including the Warburg effect, a preference for glycolysis rather than oxidative phosphorylation for energy production, has been accepted as a pivotal mechanism regulating resistance to chemotherapy. Thus, we investigated the detailed mechanism and possible usefulness of antiglycolytic agents in ameliorating 5FU resistance using established gastric cancer cell lines, SNU620 and SNU620/5FU. SNU620/5FU, a gastric cancer cell harboring resistance to 5FU, showed much higher lactate production and expression of glycolysis-related enzymes, such as lactate dehydrogenase A (LDHA), than those of the parent SNU620 cells. To limit glycolysis, we examined catechin and its derivatives, which are known anti-inflammatory and anticancer natural products because epigallocatechin gallate has been previously reported as a suppressor of LDHA expression. Catechin, the simplest compound among them, had the highest inhibitory effect on lactate production and LDHA activity. In addition, the combination of 5FU and catechin showed additional cytotoxicity and induced reactive oxygen species (ROS)-mediated apoptosis in SNU620/5FU cells. Thus, based on these results, we suggest catechin as a candidate for the development of a novel adjuvant drug that reduces chemoresistance to 5FU by restricting LDHA.

Molecular Characteristics of Amyloid Precursor Protein (APP) and Its Effects in Cancer.

Amyloid precursor protein (APP) is a type 1 transmembrane glycoprotein, and its homologs amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are highly conserved in mammals. APP and APLP are known to be intimately involved in the pathogenesis and progression of Alzheimer's disease and to play important roles in neuronal homeostasis and development and neural transmission. APP and APLP are also expressed in non-neuronal tissues and are overexpressed in cancer cells. Furthermore, research indicates they are involved in several cancers. In this review, we examine the biological characteristics of APP-related family members and their roles in cancer.

Understand KRAS and the Quest for Anti-Cancer Drugs.

The KRAS oncogene is mutated in approximately ~30% of human cancers, and the targeting of KRAS has long been highlighted in many studies. Nevertheless, attempts to target KRAS directly have been ineffective. This review provides an overview of the structure of KRAS and its characteristic signaling pathways. Additionally, we examine the problems associated with currently available KRAS inhibitors and discuss promising avenues for drug development.

Structure and Activities of the NS1 Influenza Protein and Progress in the Development of Small-Molecule Drugs.

The influenza virus causes human disease on a global scale and significant morbidity and mortality. The existing vaccination regime remains vulnerable to antigenic drift, and more seriously, a small number of viral mutations could lead to drug resistance. Therefore, the development of a new additional therapeutic small molecule-based anti-influenza virus is urgently required. The NS1 influenza gene plays a pivotal role in the suppression of host antiviral responses, especially by inhibiting interferon (IFN) production and the activities of antiviral proteins, such as dsRNA-dependent serine/threonine-protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase (OAS)/RNase L. NS1 also modulates important aspects of viral RNA replication, viral protein synthesis, and virus replication cycle. Taken together, small molecules that target NS1 are believed to offer a means of developing new anti-influenza drugs.

Structural basis of the p53 DNA binding domain and PUMA complex.

PUMA (p53-upregulated modulator of apoptosis) is localized in mitochondria and a direct target in p53-mediated apoptosis. p53 elicits mitochondrial apoptosis via transcription-dependent and independent mechanisms. p53 is known to induce apoptosis via the transcriptional induction of PUMA, which encodes proapoptotic BH3-only members of the Bcl-2 protein family. However, the transcription-independent mechanisms of human PUMA remain poorly defined. For example, it is not known whether PUMA interacts directly with the DNA binding domain (DBD: residues 92-293) of p53 in vitro. Here, the structure of the complex between the DBD of p53 and PUMA peptide was elucidated by X-ray crystallography. Isothermal titration calorimetry showed that PUMA peptide binds strongly with p53 DBD, and the crystal structure of p53-PUMA peptide complex revealed it contains four molecules of p53 DBD and one PUMA peptide per asymmetric unit in space group P1. PUMA peptide bound to the N-terminal residues of p53 DBD. A cell proliferation assay demonstrated PUMA peptide inhibited the growth of a lung cancer cell line. These results contribute to understanding of the mechanism responsible for p53-mediated apoptosis.

Hemistepsin A suppresses colorectal cancer growth through inhibiting pyruvate dehydrogenase kinase activity.

Most cancer cells primarily produce their energy through a high rate of glycolysis followed by lactic acid fermentation even in the presence of abundant oxygen. Pyruvate dehydrogenase kinase (PDK) 1, an enzyme responsible for aerobic glycolysis via phosphorylating and inactivating pyruvate dehydrogenase (PDH) complex, is commonly overexpressed in tumors and recognized as a therapeutic target in colorectal cancer. Hemistepsin A (HsA) is a sesquiterpene lactone isolated from Hemistepta lyrata Bunge (Compositae). Here, we report that HsA is a PDK1 inhibitor can reduce the growth of colorectal cancer and consequent activation of mitochondrial ROS-dependent apoptotic pathway both in vivo and in vitro. Computational simulation and biochemical assays showed that HsA directly binds to the lipoamide-binding site of PDK1, and subsequently inhibits the interaction of PDK1 with the E2 subunit of PDH complex. As a result of PDK1 inhibition, lactate production was decreased, but oxygen consumption was increased. Mitochondrial ROS levels and mitochondrial damage were also increased. Consistent with these observations, the apoptosis of colorectal cancer cells was promoted by HsA with enhanced activation of caspase-3 and -9. These results suggested that HsA might be a potential candidate for developing a novel anti-cancer drug through suppressing cancer metabolism.

Ilimaquinone Induces the Apoptotic Cell Death of Cancer Cells by Reducing Pyruvate Dehydrogenase Kinase 1 Activity.

In cancer cells, aerobic glycolysis rather than oxidative phosphorylation (OxPhos) is generally preferred for the production of ATP. In many cancers, highly expressed pyruvate dehydrogenase kinase 1 (PDK1) reduces the activity of pyruvate dehydrogenase (PDH) by inducing the phosphorylation of its E1α subunit (PDHA1) and subsequently, shifts the energy metabolism from OxPhos to aerobic glycolysis. Thus, PDK1 has been regarded as a target for anticancer treatment. Here, we report that ilimaquinone (IQ), a sesquiterpene quinone isolated from the marine sponge Smenospongia cerebriformis, might be a novel PDK1 inhibitor. IQ decreased the cell viability of human and murine cancer cells, such as A549, DLD-1, RKO, and LLC cells. The phosphorylation of PDHA1, the substrate of PDK1, was reduced by IQ in the A549 cells. IQ decreased the levels of secretory lactate and increased oxygen consumption. The anticancer effect of IQ was markedly reduced in PDHA1-knockout cells. Computational simulation and biochemical assay revealed that IQ interfered with the ATP binding pocket of PDK1 without affecting the interaction of PDK1 and the E2 subunit of the PDH complex. In addition, similar to other pyruvate dehydrogenase kinase inhibitors, IQ induced the generation of mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in the A549 cells. The apoptotic cell death induced by IQ treatment was rescued in the presence of MitoTEMPO, a mitochondrial ROS inhibitor. In conclusion, we suggest that IQ might be a novel candidate for anticancer therapeutics that act via the inhibition of PDK1 activity.

A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants.

Kirsten-RAS (KRAS) has been the target of drugs because it is the most mutated gene in human cancers. Because of the low affinity of drugs for KRAS mutations, it was difficult to target these tumor genes directly. We found a direct interaction between KRAS G12V and tumor suppressor novel H-REV107 peptide with high binding affinity. We report the first crystal structure of an oncogenic mutant, KRAS G12V-H-REV107. This peptide was shown to interact with KRAS G12V in the guanosine diphosphate (GDP)-bound inactive state and to form a stable complex, blocking the activation function of KRAS. We showed that the peptide acted as an inhibitor of mutant KRAS targets by [α-32P] guanosine triphosphate (GTP) binding assay. The H-REV107 peptide inhibited pancreatic cancer and colon cancer cell lines in cell proliferation assay. Specially, the H-REV107 peptide can suppress pancreatic tumor growth by reduction of tumor volume and weight in xenotransplantation mouse models. Overall, the results presented herein will facilitate development of novel drugs for inhibition of KRAS mutations in cancer patients.

6'-Sialylgalactose inhibits vascular endothelial growth factor receptor 2-mediated angiogenesis.

Angiogenesis should be precisely regulated because disordered neovascularization is involved in the aggravation of multiple diseases. The vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2) axis is crucial for controlling angiogenic responses in vascular endothelial cells (ECs). Therefore, inactivating VEGFR-2 signaling may effectively suppress aberrant angiogenesis and alleviate related symptoms. In this study, we performed virtual screening, identified the synthetic disaccharide 6'-sialylgalactose (6SG) as a potent VEGFR-2-binding compound and verified its high binding affinity by Biacore assay. 6SG effectively suppressed VEGF-A-induced VEGFR-2 phosphorylation and subsequent in vitro angiogenesis in HUVECs without inducing cytotoxicity. 6SG also inhibited VEGF-A-induced extracellular-regulated kinase (ERK)/Akt activation and actin stress fiber formation in HUVECs. We demonstrated that 6SG inhibited retinal angiogenesis in a mouse model of retinopathy of prematurity and tumor angiogenesis in a xenograft mouse model. Our results suggest a potential therapeutic benefit of 6SG in inhibiting angiogenesis in proangiogenic diseases, such as retinopathy and cancer.

Machilin A Inhibits Tumor Growth and Macrophage M2 Polarization Through the Reduction of Lactic Acid.

Lactate dehydrogenase A (LDHA) is an important enzyme responsible for cancer growth and energy metabolism in various cancers via the aerobic glycolytic pathway. Here, we report that machilin A (MA), which acts as a competitive inhibitor by blocking the nicotinamide adenine dinucleotide (NAD) binding site of LDHA, suppresses growth of cancer cells and lactate production in various cancer cell types, including colon, breast, lung, and liver cancers. Furthermore, MA markedly decreased LDHA activity, lactate production, and intracellular adenosine triphosphate (ATP) levels induced by hypoxia-induced LDHA expression in cancer cells, and significantly inhibited colony formation, leading to reduced cancer cell survival. In mouse models inoculated with murine Lewis lung carcinoma, MA significantly suppressed tumor growth as observed by a reduction of tumor volume and weight; resulting from the inhibition of LDHA activity. Subsequently, the suppression of tumor-derived lactic acid in MA-treated cancer cells resulted in decrease of neovascularization through the regulation of alternatively activated macrophages (M2) polarization in macrophages. Taken together, we suggest that the reduction of lactate by MA in cancer cells directly results in a suppression of cancer cell growth. Furthermore, macrophage polarization and activation of endothelial cells for angiogenesis were indirectly regulated preventing lactate production in MA-treated cancer cells.

Structure and Function of the Influenza A Virus Non-Structural Protein 1.

The influenza A virus is a highly infectious respiratory pathogen that sickens many people with respiratory disease annually. To prevent outbreaks of this viral infection, an understanding of the characteristics of virus-host interaction and development of an anti-viral agent is urgently needed. The influenza A virus can infect mammalian species including humans, pigs, horses and seals. Furthermore, this virus can switch hosts and form a novel lineage. This so-called zoonotic infection provides an opportunity for virus adaptation to the new host and leads to pandemics. Most influenza A viruses express proteins that antagonize the antiviral defense of the host cell. The non-structural protein 1 (NS1) of the influenza A virus is the most important viral regulatory factor controlling cellular processes to modulate host cell gene expression and double-stranded RNA (dsRNA)-mediated antiviral response. This review focuses on the influenza A virus NS1 protein and outlines current issues including the life cycle of the influenza A virus, structural characterization of the influenza A virus NS1, interaction between NS1 and host immune response factor, and design of inhibitors resistant to the influenza A virus.

Huzhangoside A Suppresses Tumor Growth through Inhibition of Pyruvate Dehydrogenase Kinase Activity.

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.

A Novel Lactate Dehydrogenase Inhibitor, 1-(Phenylseleno)-4-(Trifluoromethyl) Benzene, Suppresses Tumor Growth through Apoptotic Cell Death.

The Warburg effect, wherein cancer cells prefer glycolysis rather than oxidative phosphorylation even under normoxic conditions, is a major characteristic of malignant tumors. Lactate dehydrogenase A (LDHA) is the main enzyme regulating the Warburg effect, and is thus, a major target for novel anti-cancer drug development. Through our ongoing screening of novel inhibitors, we found that several selenobenzene compounds have inhibitory effects on LDHA activity. Among them, 1-(phenylseleno)-4-(trifluoromethyl) benzene (PSTMB) had the most potent inhibitory effect on the enzymatic activity of LDHA. The results from biochemical assays and computational modeling showed that PSTMB inhibited LDHA activity. In addition, PSTMB inhibited the growth of several tumor cell lines, including NCI-H460, MCF-7, Hep3B, A375, HT29, and LLC. In HT29 human colon cancer cells, PSTMB dose-dependently inhibited the viability of the cells and activity of LDHA, without affecting the expression of LDHA. Under both normoxic and hypoxic conditions, PSTMB effectively reduced LDHA activity and lactate production. Furthermore, PSTMB induced mitochondria-mediated apoptosis of HT29 cells via production of reactive oxygen species. These results suggest that PSTMB may be a novel candidate for development of anti-cancer drugs by targeting cancer metabolism.