Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with aminoindane and phenylpyrrolidine P4 motifs

The rational design, syntheses and evaluation of potent sulfonamidopyrrolidin-2-one-based factor Xa inhibitors incorporating aminoindane and phenylpyrrolidine P4 motifs are described. These series delivered highly potent anticoagulant compounds with excellent oral pharmacokinetic profiles; however, significant time dependant P450 inhibition was an issue for the aminoindane series, but this was not observed with the phenylpyrrolidine motif, which produced candidate quality molecules with potential for once-daily oral dosing in humans.     

Tumor Stress-Induced Phosphoprotein1 (STIP1) as a Prognostic Biomarker in Ovarian Cancer

Stress-induced phosphoprotein 1 (STIP1) has been recently identified as a released biomarker in human ovarian cancer. In addition, STIP1 secreted by human ovarian cancer cells has been shown to promote tumor cell proliferation by binding to ALK2 (activin A receptor, type II-like kinase 2) and activating the SMAD-ID3 signaling pathways. In this study, a total of 330 ovarian cancer tumor samples were evaluated for STIP1 expression by immunohistochemistry and analyzed for a possible correlation with patient characteristics and survival. The quantification of immunoreactivity was accomplished by applying an immunohistochemical scoring system (histoscore). Patients with high-level STIP1 expression (histoscore ≥169) had a significantly worse survival (high STIP1, mean survival time = 76 months; low STIP1, mean survival time = 112 months; P<0.0001). Moreover, STIP1 histoscores were significantly higher in high-grade tumors (grade 3) than in low-grade (grade 1–2) malignancies (P<0.0001), suggesting that STIP1 may be a proxy for tumor aggressiveness. The results of multivariable analysis revealed that high STIP1 histoscores, advanced stages, histologic types, and the presence of residual disease (≥2 cm) were independent predictors of poor prognosis. The addition of STIP1 histoscores improved the prediction of overall and progression-free survival rates in the multivariable Cox proportional hazard model. The treatment of ovarian cancer cells with recombinant STIP1 stimulated cell proliferation and migration, but co-treatment with anti-STIP1 antibodies abrogated this effect. Our findings suggest that STIP1 expression may be related to prognosis and that the STIP1 pathway may represent a novel therapeutic target for human ovarian cancer.     

Immunologic hypo- or non-responder in natural dengue virus infection

Serologically defined primary dengue virus infection and/or subsequent homologous serotype infection is known to be associated with less severe disease as compared with secondary subsequent heterologous serotype infection. In geographical locales of high dengue endemicity, almost all individuals in the population are infected at some point in time and should therefore are at high risk of secondary infection. Interestingly, dengue viremia in healthy blood donors whose sera apparently lack detectable levels of specific antibody to dengue viral antigens has been reported. The incidence rate of potential immunologic hypo- or non-responders following natural primary dengue virus infection in dengue endemic regions, who do become immune responders only after repeated exposure, has not been described. These are the patients who may be diagnosed as primary infection in the subsequent infection, but actually are secondary infection. This concept has important implications with regards to the hypothesis of immunological enhancement of dengue pathogenesis, which has largely been advanced based on empirical observations and/or from in vitro experimental assays. The fact that dengue naïve travelers can suffer from severe dengue upon primary exposure while visiting dengue endemic countries underscores one of the major problems in explaining the role of immune enhancement in the pathogenesis of severe dengue virus infection. This evidence suggests that the mechanism(s) leading to severe dengue may not be associated with pre-existing enhancing antibody. Consequently, we propose a new paradigm for dengue virus infection classification. These include a) patients with naïve primary infection, b) those that are serologically defined primary in dengue endemic zones and c) those who are serologically defined secondary dengue virus infection. We submit that clarity with regards to such definitions may help facilitate the delineation of the potential mechanisms of severe dengue virus infection.     

Interactome-wide Analysis Identifies End-binding Protein 1 as a Crucial Component for the Speck-like Particle Formation of Activated Absence in Melanoma 2 (AIM2) Inflammasomes

Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for interleukin 1β production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in nasopharyngeal carcinoma cells treated with specific stimuli of these interactomes (H₂O₂, poly (dA:dT), and EBV noncoding RNA, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-apoptosis-associated speck-like protein containing caspase activation and recruitment domain antibodies after stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of end binding protein 1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of interleukin 1β secretion. We confirmed that end binding protein 1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that end binding protein 1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In nasopharyngeal carcinoma tissues, immunohistochemical staining showed that end binding protein 1 expression was elevated and significantly correlated with AIM2 and ASC expression in nasopharyngeal carcinoma tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that end binding protein 1 is crucial for the speck-like particle formation that represents activated inflammasomes.Nasopharyngeal carcinoma (NPC)¹ is a malignancy of the head and the neck that is highly prevalent in Southern China and Southeast Asia (1). Both environmental and genetic risk factors are considered to be important for the development of NPC (2, 3); among them, Epstein-Barr virus (EBV) infection of the epithelium is the most important known factor (1). In addition to the EBV-encoded oncoprotein-mediated blockade of intracellular mechanisms in EBV-associated tumors (1), chronic inflammation is considered to be an important oncogenic factor in NPC (4). Interleukin 1 beta (IL-1β), which is an inflammatory cytokine that has oncogenic effects in many tumors (5), can be detected in NPC tumor tissues (6, 7). IL-1β secretion is mediated by cytosolic protein complexes called inflammasomes, which induce IL-1β secretion by activating catalytic caspase 1 (8). However, no previous study has examined inflammasome components in NPC tumor cells or the mechanisms of inflammasome regulation in NPC.Inflammasomes are cytoplasmic receptors that act in innate immunity to recognize intracellular pathogen-associated molecular pattern (PAMP) or danger signal-associated molecular pattern (DAMP). A number of inflammasomes have been identified in recent years (9), and they can be classified into different subgroups according to their recognizing PAMP. These groups include the NOD-like receptors (NLR), which sense intracellular pathogens (e.g. bacteria, fungi, and parasites) and activate pro-caspase 1 with or without an adaptive protein called apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) (10). Activated caspase 1 then induces IL-1β secretion through direct cleavage of pro-IL-1β (8). Among the NLR family members, the NLRP3 inflammasome recognizes both pathogens and danger signals such as ATP or reactive oxygen species (ROS) generation (11, 12). Members of the two other subgroups, absence in melanoma 2 (AIM2) and retinoic acid-inducible gene I (RIG-I), sense cytoplasmic double-strand DNA and 5′-triphoshphate RNA, respectively, and then recruit ASC to activate pro-caspase 1 (13, 14). Although inflammasomes are important for pathogen defense in immune cells, recent studies have shown that inflammasomes also participate in tumorigenesis in colon cancer and melanoma (15–17). A previous report showed that EBV noncoding RNAs (EBERs) are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected B lymphocytes (18). This report is consistent with our recent unpublished observation that RIG-I is activated by EBERs in NPC cells. We additionally show that NLRP3 is triggered by tumor microenvironmental factors, such as ATP and ROS, and the clinical drug cisplatin; AIM2 recognizes EBV genomic DNA and is activated by irradiation in NPC cells. Although these inflammasomes play important role in NPC, the regulation and the interactome of these inflammasome complexes are not fully understood.On activation by PAMP or DAMP, the activated inflammasomes tend to aggregate in the cytosol as speck-like particles (13). Biochemical and cell biological data have indicated that the core components of the inflammasome comprise the receptor, ASC, and pro-caspase 1, but an increasing number of proteins have been identified as interacting with these complexes. For example, heat-shock protein 90 (HSP90) is essential for the function of the NLRP3 and RIG-I inflammasomes (19, 20). NLRC5, another member of the NLR family, is involved in the NLRP3 inflammasome and is required for its activity (21). Rac1, a small Rho GTPase family member, is reportedly required for NLRP3 inflammasome activation during C. pneumoniae infection (22). The S. Typhimurium effector, SopE, activates caspase 1 through Rac1 activity (23), whereas Yersinia bacteria prevent caspase 1 activation by inhibiting Rac1 activity via the effector protein, YopE (24). Notably, Rac1 regulates cytoskeletal rearrangement (25), suggesting that cytoskeletal components may participate in inflammasome activation.End-binding protein 1 (EB1), an adenomatous polyposis coli (APC)-binding protein, regulates microtubule polymerization by recruiting the plus-end tracking protein (+TIP) complex to the plus end of microtubules (26). The interaction of EB1 and the +TIP complex depends on the C-terminal (CT) domain of EB1, whereas the calponin homology (CH) domain of EB1 binds to the microtubule (26). Many studies have shown that EB1 participates in different biological processes, including mitosis, migration and signal transduction (27–29), and also that it plays an oncogenic role in cancer by affecting cell growth or migration (30, 31). However, although EB1 is known to be a cytoskeletal component that is regulated by the small GTPase, RhoA (28), its role in inflammasome activation has not yet been explored.Here, we used the isobaric tags for relative and absolute quantification (iTRAQ) approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cell lines treated with their specific stimuli, H₂O₂, poly (dA:dT), and EBER, respectively. We characterized the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cells by proteomic analysis, and report for the first time that EB1 can directly bind to the AIM2 inflammasome and is essential for speck-like particle formation in NPC cells. Finally, we suggest some possible mechanisms for EB1-associated AIM2 inflammasome activation via microtubule polymerization and RhoA activity.     

Structure and property based design of factor Xa inhibitors: biaryl pyrrolidin-2-ones incorporating basic heterocyclic motifs

Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating basic biaryl P4 groups, producing highly potent inhibitors with significant anticoagulant activities and encouraging oral pharmacokinetic profiles.     

An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.     

Cdc42-dependent formation of the ZO-1/MRCKβ complex at the leading edge controls cell migration

Zonula occludens (ZO)-1 is a multi-domain scaffold protein known to have critical roles in the establishment of cell-cell adhesions and the maintenance of stable tissue structures through the targeting, anchoring, and clustering of transmembrane adhesion molecules and cytoskeletal proteins. Here, we report that ZO-1 directly binds to MRCKβ, a Cdc42 effector kinase that modulates cell protrusion and migration, at the leading edge of migrating cells. Structural studies reveal that the binding of a β hairpin from GRINL1A converts ZO-1 ZU5 into a complete ZU5-fold. A similar interaction mode is likely to occur between ZO-1 ZU5 and MRCKβ. The interaction between ZO-1 and MRCKβ requires the kinase to be primed by Cdc42 due to the closed conformation of the kinase. Formation of the ZO-1/MRCKβ complex enriches the kinase at the lamellae of migrating cells. Disruption of the ZO-1/MRCKβ complex inhibits MRCKβ-mediated cell migration. These results demonstrate that ZO-1, a classical scaffold protein with accepted roles in maintaining cell-cell adhesions in stable tissues, also has an active role in cell migration during processes such as tissue development and remodelling.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.