Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Anti-fibrotic effects of a methylenedioxybenzene compound, CW209292 on dimethylnitrosamine-induced hepatic fibrosis in rats.

A series of methylenedioxybenzene compounds were synthesized and found to have hepatoprotective effects in chemical-induced hepatotoxicity models. The purpose of the present study was to investigate the anti-fibrotic effects of a synthetic methylenedioxybenzene compound, CW209292, using the dimethylnitrosamine (DMN)-induced chronic liver injury model in rats. Liver injuries were induced in Sprague Dawley rats by injection of DMN (intraperitoneally, 10 microl/kg) 3 times per week for 4 weeks. The rats were treated with CW209292 (per os, 25 or 75 mg/kg/d) for 4 weeks. Treatment of rats with DMN for 4 weeks resulted in significant decreases in serum albumin levels, whereas concomitant treatment with CW209292 prevented these decreases. CW209292 treatment also shortened prothrombin time prolonged by DMN, providing evidence that the agent was active in preserving liver function against DMN insult. DMN treatment caused marked increases in plasma bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), and hyaluronic acid levels; CW209292 treatment reversed these increases. CW209292 also significantly reduced hepatic hydroxyproline content as well as hepatic fibrosis and inflammation in histological examination. Additionally, immunochemically detectable hepatic collagen type IV and alpha-smooth muscle actin levels were decreased by CW209292 treatment. Proliferation of hepatic stellate cells isolated from DMN-treated rats was inhibited by CW209292. Furthermore, tumor growth factor (TGF)-beta1 mRNA expression was increased in DMN-treated rats, whereas CW209292 treatment prevented these increases. These results suggest that CW209292 exhibits anti-fibrotic effects in Sprague Dawley rats with DMN-induced hepatic fibrosis by blocking the mRNA expression of TGF-beta1 and subsequent inhibition of the proliferation of hepatic stellate cells.

Comparison of in vivo nephrotoxicity in the rabbit by a pyrrolidinyl-thio Carbapenem CW-270031.

CW-270031 is a novel synthesized carbapenem antibiotic with a broad antimicrobial activity. Carbapenem antibiotics are well known for their nephrotoxicity. In this study, we evaluated the nephrotoxicity potential of this compound in rabbits, which are known for being more sensitive than other animals to renal insult. CW-270031 was administered to NZW male rabbits via an ear vein (200 mg/kg, single injection). Blood samples were collected on 2, 3, and 4 days after treatment. Urea nitrogen and creatinine in plasma were quantified. Four days after the treatment, all animals were autopsied and histopathological examinations were performed on their kidneys, revealing that cephaloridine and imipenem were highly nephrotoxic, and cefazolin had mild renal toxicity, whereas CW-270031 as well as meropenem and tienam had no toxicity to the kidney. The present findings suggest that CW-270031 is a potential carbapenem antibiotic with no nephrotoxicity.

Inhibition of dimethylnitrosamine-induced liver fibrosis by [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] (oltipraz) in rats: suppression of transforming growth factor-beta1 and tumor necrosis factor-alpha expression.

Oltipraz is a cancer chemopreventive agent active against a wide variety of chemical carcinogens. In spite of the intense chemoprevention and toxicology studies on oltipraz, no information is available on its antifibrotic efficacy. In the present study, the effects of oltipraz on dimethylnitrosamine (DMN)-induced liver fibrogenesis were assessed in rats. As part of mechanistic studies, the expression of transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha) was monitored. Treatment of rats with DMN (10 microl/kg body weight, i.p., three times per week for 4 weeks) resulted in marked increases in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (gamma-GT) activities. DMN also caused an increase in the plasma bilirubin content, whereas total plasma protein and albumin levels were rather decreased. Oltipraz (50 mg/kg body weight, p.o., three times per week for 4 weeks) inhibited the increases in plasma ALT, AST, gamma-GT and bilirubin by DMN. DMN increased liver fibrosis as histopathologically assessed by Van Gieson's staining and Masson's trichrome staining (fibrosis score, 3.7; Knodell score, 16), which was reduced by oltipraz treatment (fibrosis score, 2.5; Knodell score, 8.0). Reverse transcription-polymerase chain reaction analysis revealed that oltipraz inhibited an increase in the TGF-beta1 mRNA by DMN. Oltipraz was also active in reducing the production of plasma TNF-alpha by DMN or lipopolysaccharide (LPS), which would contribute to its cytoprotective effect. These results demonstrated that oltipraz inhibited hepatocyte injury and impairment of liver function induced by DMN, and reduces DMN-induced liver fibrosis possibly through suppression of TGF-beta1 and TNF-alpha production.

YY162 prevents ADHD-like behavioral side effects and cytotoxicity induced by Aroclor1254 via interactive signaling between antioxidant potential, BDNF/TrkB, DAT and NET.

Methylphenidate (MP) has become the primary drug of choice for treatment of attention-deficit/hyperactivity disorder (ADHD). However, its psychotropic effects severely hamper long-term clinical use. We evaluated the effects of YY162, which consists of terpenoid-strengthened Ginkgo biloba and ginsenoside Rg3, on the ADHD-like condition induced by Aroclor1254, because both components have been suggested to modulate oxidative stress, dopaminergic neurotransmission, and brain-derived neurotrophic factor (BDNF) signaling, which may be critical targets for understanding the pathogenesis of ADHD. YY162 attenuated the increase in reactive oxygen species (ROS) and decrease in BDNF levels induced by Aroclor1254 in SH-SY5Y neuroblastoma cells. YY162 significantly attenuated Aroclor1254-induced ADHD-like behavior and oxidative stress in ICR mice. Furthermore, YY162 attenuated reductions in p-TrkB, BDNF, dopamine transporter (DAT) and norepinephrine transporter (NET) expression. These attenuating effects of YY162 were comparable to those of MP. Importantly, K252a, a TrkB antagonist, counteracted the protective effects of YY162. Our results suggest that YY162 possesses significant protective activities against ADHD-like conditions with negligible behavioral side effects, and that interactive signaling between antioxidant potential and BDNF/TrkB receptor for the positive modulation of the DAT and NET is important for YY162-mediated neuroprotective activity.

Peptidomimetic small-molecule compounds promoting cardiogenesis of stem cells.

Embryonic stem (ES) cells may be used as an alternative source of functionally intact cardiomyocytes for ischemic heart disease. Several natural and synthetic small molecules have been identified as useful tools for controlling and manipulating stem cell renewal and differentiation. Currently, there is an urgent requirement for novel small molecules that specifically induce differentiation of stem cells into cardiomyocytes. To identify compounds that promote cardiomyogenesis of stem cells, cell-based screening of a peptidomimetic small-molecule library was carried out. A series of ß-turn peptidomimetic compounds, including CW209E, increased the expression of α-MHC promoter-driven enhanced green fluorescent protein (EGFP) and ratio of beating embryoid bodies (EBs) without inducing cytotoxicity in mouse embryonic stem cells. CW209E also increased the number of beating EBs in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Thus, this chemical compound should be useful for elucidation of the molecular pathway of cardiogenesis and generation of cardiomyocytes ex vivo, which can be further applied for experimental or clinical cell therapy for ischemic heart diseases.

Potential inhibition of PDK1/Akt signaling by phenothiazines suppresses cancer cell proliferation and survival.

3'-Phosphoinositide-dependent kinase-1 (PDK1) has been identified for its ability to phosphorylate and activate Akt. Accumulated studies have shown that the activation of the PDK1/Akt pathway plays a pivotal role in cell survival, proliferation, and tumorigenesis. Therefore, the PDK1/Akt pathway is believed to be a critical target for cancer intervention. In this paper, we report the discovery of a new function of phenothiazines, widely known as antipsychotics, inhibiting PDK1/Akt pathway. Upon epidermal growth factor (EGF) stimulation, phenothiazines specifically suppressed the kinase activity of PDK1 and the phosphorylation level of Akt. The inhibition of PDK1/Akt kinase resulted in suppression of EGF-induced cell growth and induction of apoptosis in human ovary cancer cells. In particular, phenothiazines were highly selective for downstream targets of PDK1/Akt and did not inhibit the activation of phosphatidylinositol 3-kinase (PI3K), EGFR, or extracellular signal-regulated kinase 1/2 (ERK1/2). In particular, phenothiazines effectively suppressed tumor growth in nude mice of human cancer cells. Taken together, these findings provide strong evidence for novel function of phenothiazines, pharmacologically targeting PDK1/Akt for anticancer drug discovery.

CW-270033, a novel pyrrolidinyl-thio carbapenem, has potent antimicrobial activity in vitro and in vivo.

CW-270033, an injectable carbapenem, is a novel, synthesized pyrrolidinyl-thio carbapenem. In the present study, the in-vitro and in-vivo antibacterial activities of CW-270033 against wild-type strains and clinical isolates were compared with those of imipenem and meropenem. CW-270033 was more active than imipenem against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) clinical isolates, but was slightly less active than meropenem. Against the Gram-positive clinical isolates methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), CW-270033 was slightly more active than meropenem, but was less active than imipenem. CW-270033 displayed potent in-vivo activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus SMITH in the mouse systemic infection model; the efficacy of CW-270033 in this model was 2--7 fold higher than that of meropenem. This activity was comparable to the in-vitro activity of CW-270033. An intravenous injection of CW-270033 showed that the half-life of CW-270033 in serum in mice was about 20 min, which was about two times that of meropenem. CW-270033 was also found to be resistant to hydrolysis by the mouse renal dehydropeptidase I (DHP-I) enzyme.

A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.