Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing.

Core fucosylation is a highly prevalent and significant feature of N-glycosylation in therapeutic monoclonal antibodies produced by mammalian cells where its absence (afucosylation) plays a key role in treatment safety and efficacy. Notably, even slight changes in the level of afucosylation can have a considerable impact on the antibody-dependent cell-mediated cytotoxicity. Therefore, implementing control over afucosylation levels is important in upstream manufacturing to maintain consistent quality across batches of product, since standard downstream processing does not change afucosylation. In this review, the influences and strategies to control afucosylation are presented. In particular, there is emphasis on upstream manufacturing culture parameters and media supplementation, as these offer particular advantages as control strategies over alternative approaches such as cell line engineering and chemical inhibitors. The review discusses the relationship between the afucosylation influences and the underlying cellular metabolism to promote increased process understanding. Also, briefly highlighted is the value of empirical and mechanistic models in evaluating and designing control methods for core fucosylation.

Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization.

Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization. While anti-VGEF therapies have been successful, up to 30% of patients are non-responsive. Therefore, there is a need for new therapeutic targets, and small molecule inhibitors of angiogenesis to complement existing treatments. Apelin and its receptor have recently been shown to play a key role in both developmental and pathological angiogenesis in the eye. Through a cell-based high-throughput screen, we identified 4-aminoquinoline antimalarial drugs as potent selective antagonists of APJ. The prototypical 4-aminoquinoline, amodiaquine was found to be a selective, non-competitive APJ antagonist that inhibited apelin signaling in a concentration-dependent manner. Additionally, amodiaquine suppressed both apelin-and VGEF-induced endothelial tube formation. Intravitreal amodaiquine significantly reduced choroidal neovascularization (CNV) lesion volume in the laser-induced CNV mouse model, and showed no signs of ocular toxicity at the highest doses tested. This work firmly establishes APJ as a novel, chemically tractable therapeutic target for the treatment of ocular neovascularization, and that amodiaquine is a potential candidate for repurposing and further toxicological, and pharmacokinetic evaluation in the clinic.

A Systematic Approach to Identify Biased Agonists of the Apelin Receptor through High-Throughput Screening.

Biased agonists are defined by their ability to selectively activate distinct signaling pathways of a receptor, and they hold enormous promise for the development of novel drugs that specifically elicit only the desired therapeutic response and avoid potential adverse effects. Unfortunately, most high-throughput screening (HTS) assays are designed to detect signaling of G protein-coupled receptors (GPCRs) downstream of either G protein or ß-arrestin-mediated signaling but not both. A comprehensive drug discovery program seeking biased agonists must employ assays that report on the activity of each compound at multiple discrete pathways, particularly for HTS campaigns. Here, we report a systematic approach to the identification of biased agonists of human apelin receptor (APJ). We synthesized 448 modified versions of apelin and screened them against a cascade of cell-based assays, including intracellular cAMP and ß-arrestin recruitment to APJ, simultaneously. The screen yielded potent and highly selective APJ agonists. Representative hits displaying preferential signaling via either G-protein or ß-arrestin were subjected to a battery of confirmation assays. These biased agonists will be useful as tools to probe the function and pharmacology of APJ and provide proof of concept of our systematic approach to the discovery of biased ligands. This approach is likely universally applicable to the search for biased agonists of GPCRs.

In Vivo Rapid Assessment of Compound Exposure (RACE) for Profiling the Pharmacokinetics of Novel Chemical Probes.

The Rapid Assessment of Compound Exposure (RACE) assay is an easy and efficient method for estimating the pharmacokinetic parameter of exposure (AUC: area under the curve) of novel chemical probe compounds in mice. RACE is a truncated and compressed version of a traditional comprehensive in vivo pharmacokinetics study. The method uses a single standard formulation, dose, route of administration, and a small cohort of mice (n = 4). Standardized protocols and an abbreviated sample collection scheme reduce the labor needed to perform both the in-life and bioanalytical phases of the study. The procedure reduces the complexity of data analysis by eliminating all but one calculated pharmacokinetic parameter; estimated exposure (eAUC20-120), a parameter that is sufficient to rank order compounds based on exposure, but is also easily determined by most software using the simple trapezoidal rule. The RACE assay protocol is readily applicable to early/exploratory studies of most compounds, and is intended to be employed by laboratories with limited expertise in pharmacology and pharmacokinetics. Curr. Protoc. Chem. Biol. 4:299-309 © 2012 by John Wiley & Sons, Inc.

Metabolic regulation of an fnr gene knockout Escherichia coli under oxygen limitation.

In addition to our previous study on the effect of fnr gene knockout on the metabolism in Escherichia coli under aerobic conditions (Kumar and Shimizu, Microb Cell Fact 2011), here we further investigated the effect of fnr gene knockout on the metabolism under micro-aerobic condition based on gene expressions, enzyme activities and intracellular metabolic fluxes. The objective of the present research is to clarify the metabolic regulation mechanism on how the culture environment, such as oxygen level, affects the cell metabolism in relation to gene expressions, enzyme activities and fluxes via global regulators such as Fnr and ArcA/B systems. Under micro-aerobic condition, the flux through Pfl and Frd were reduced for the mutant, which are due to fnr gene knockout. The decreased flux through Pfl may have caused accumulation of PYR, which increased the flux through LDH. The fnr gene knockout caused arcA to be downregulated, and thus the TCA cycle was activated, and cyoA and cydB genes were upregulated. The downregulation of arcA caused lpdA and aceE, F to be upregulated where the flux through PDHc increased. The fnr gene knockout indirectly caused cra gene transcript level to be decreased, which in turn caused the glycolysis genes to be upregulated, which correspond to the increase in the specific glucose consumption rate. The fnr gene knockout also caused crp transcript level to be increased, where there might be some relationship between the two due to similar structure and gene sequence. It may be quite important to understand the metabolic regulation mechanism based on different levels of information for the efficient metabolic engineering and control of the culture environment for process optimization.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production.

OBJECTIVE: To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling. METHODS AND RESULTS: Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells. CONCLUSION: Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli.

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved.

STAT3 as a target for inducing apoptosis in solid and hematological tumors.

Studies in the past few years have provided compelling evidence for the critical role of aberrant Signal Transducer and Activator of Transcription 3 (STAT3) in malignant transformation and tumorigenesis. Thus, it is now generally accepted that STAT3 is one of the critical players in human cancer formation and represents a valid target for novel anticancer drug design. This review focuses on aberrant STAT3 and its role in promoting tumor cell survival and supporting the malignant phenotype. A brief evaluation of the current strategies targeting STAT3 for the development of novel anticancer agents against human tumors harboring constitutively active STAT3 will also be presented.

An oxazole-based small-molecule Stat3 inhibitor modulates Stat3 stability and processing and induces antitumor cell effects.

Stat3 is hyperactivated in many human tumors and represents a valid target for anticancer drug design. We present a novel small-molecule Stat3 inhibitor, S3I-M2001, and describe the dynamics of intracellular processing of activated Stat3 within the context of the biochemical and biological effects of the Stat3 inhibitor. S3I-M2001 is an oxazole-based peptidomimetic of the Stat3 Src homology (SH) 2 domain-binding phosphotyrosine peptide that selectively disrupts active Stat3:Stat3 dimers. Consequently, hyperactivated Stat3, which hitherto occurs as "dotlike" structures of nuclear bodies, undergoes an early aggregation into nonfunctional perinuclear aggresomes and a late-phase proteasome-mediated degradation in malignant cells treated with S3I-M2001. Thus, S3I-M2001 inhibited Stat3-dependent transcriptional regulation of tumor survival genes, such as Bcl-xL. Furthermore, Stat3-dependent malignant transformation, survival, and migration and invasion of mouse and human cancer cells harboring persistently activated Stat3 were inhibited by S3I-M2001. Finally, S3I-M2001 inhibited growth of human breast tumor xenografts. The study identifies a novel Stat3 inhibitor, S3I-M2001, with antitumor cell effects mediated in part through a biphasic loss of functional Stat3. The study represents the first on intracellular Stat3 stability and processing following inhibition by a small molecule that has significant antitumor activity.

Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity.

S3I-201 (NSC 74859) is a chemical probe inhibitor of Stat3 activity, which was identified from the National Cancer Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3beta homodimer. S3I-201 inhibits Stat3.Stat3 complex formation and Stat3 DNA-binding and transcriptional activities. Furthermore, S3I-201 inhibits growth and induces apoptosis preferentially in tumor cells that contain persistently activated Stat3. Constitutively dimerized and active Stat3C and Stat3 SH2 domain rescue tumor cells from S3I-201-induced apoptosis. Finally, S3I-201 inhibits the expression of the Stat3-regulated genes encoding cyclin D1, Bcl-xL, and survivin and inhibits the growth of human breast tumors in vivo. These findings strongly suggest that the antitumor activity of S3I-201 is mediated in part through inhibition of aberrant Stat3 activation and provide the proof-of-concept for the potential clinical use of Stat3 inhibitors such as S3I-201 in tumors harboring aberrant Stat3.

Isoform selective inhibition of STAT1 or STAT3 homo-dimerization via peptidomimetic probes: structural recognition of STAT SH2 domains.

The identification of constitutively activated STAT (Signal Transducers and Activators of Transcription) proteins in aberrant cell signaling pathways has led to investigations targeting the selective disruption of specific STAT isoforms directly associated with oncogenisis. We have identified, through the design of a library of peptidomimetic inhibitors, agents that selectively disrupt STAT1 or STAT3 homo-dimerization at low micromolar concentrations. ISS840 has 20-fold higher inhibition of STAT1 homo-dimerization (IC(50) value of 31 microM) relative to STAT3 (IC(50) value of 560 microM).

Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein.

Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G0-G1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-xL and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.

Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli.

The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on (13)C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake. This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant. In general, the changes in enzyme activities more or less correlated with ratios of gene expression, while the changes in metabolic fluxes did not correlate with enzyme activities. For example, high flux ratios were obtained through the oxidative pentose phosphate pathway due to an increased concentration of glucose-6-phosphate rather than to favorable enzyme activity ratios. In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways.