Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.     

Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin

The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim(-/-)) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim(-/-) mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim(-/-) corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1). In cell culture models, WFA exerts G(2)/M cell cycle arrest in a p27(Kip1)- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim(-/-) mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

3-Formylchromone interacts with cysteine 38 in p65 protein and with cysteine 179 in IκBα kinase, leading to down-regulation of nuclear factor-κB (NF-κB)-regulated gene products and sensitization of tumor cells

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.     

CCR5 antagonist TD-0680 uses a novel mechanism for enhanced potency against HIV-1 entry, cell-mediated infection, and a resistant variant

Regardless of the route of transmission, R5-tropic HIV-1 predominates early in infection, rendering C-C chemokine receptor type 5 (CCR5) antagonists as attractive agents not only for antiretroviral therapy but also for prevention. Here, we report the specificity, potency, and underlying mechanism of action of a novel small molecule CCR5 antagonist, TD-0680. TD-0680 displayed the greatest potency against a diverse group of R5-tropic HIV-1 and SIV strains when compared with its prodrug, TD-0232, the Food and Drug Administration-approved CCR5 antagonist Maraviroc, and TAK-779, with EC(50) values in the subnanomolar range (0.09-2.29 nm). Importantly, TD-0680 was equally potent at blocking envelope-mediated cell-cell fusion and cell-mediated viral transmission as well as the replication of a TAK-779/Maraviroc-resistant HIV-1 variant. Interestingly, TD-0232 and TD-0680 functioned differently despite binding to a similar transmembrane pocket of CCR5. Site-directed mutagenesis, drug combination, and antibody blocking assays identified a novel mechanism of action of TD-0680. In addition to binding to the transmembrane pocket, the unique exo configuration of this molecule protrudes and sterically blocks access to the extracellular loop 2 (ECL2) region of CCR5, thereby interrupting the interaction between virus and its co-receptor more effectively. This mechanism of action was supported by the observations of similar TD-0680 potency against CD4-dependent and -independent SIV strains and by molecular docking analysis using a CCR5 model. TD-0680, therefore, merits development as an anti-HIV-1 agent for therapeutic purposes and/or as a topical microbicide for the prevention of sexual transmission of R5-tropic HIV-1.     

Biochemical Characterization of Human Tyrosyl-DNA Phosphodiesterase 2 (TDP2/TTRAP): A Mg2+/Mn2+-DEPENDENT PHOSPHODIESTERASE SPECIFIC FOR THE REPAIR OF TOPOISOMERASE CLEAVAGE COMPLEXES

TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)m(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.     

Islet transplantation in type I diabetes mellitus

For most patients with type I diabetes, insulin therapy and glucose monitoring are sufficient to maintain glycemic control. However, hypoglycemia is a potentially lethal side effect of insulin treatment in patients who are glycemically labile or have hypoglycemia-associated autonomic failure [1]. For those patients, an alternative therapy is beta cell replacement via pancreas or islet transplantation. Pancreas transplants using cadaveric donor organs reduce insulin dependence but carry risks involved in major surgery and chronic immunosuppression. Islet transplantation, in which islets are isolated from donor pancreases and intravenously infused, require no surgery and can utilize islets isolated from pancreases unsuitable for whole organ transplantation. However, islet transplantation also requires immunosuppression, and standard steroid regimens may be toxic to beta cells [2]. The 2000 Edmonton Trial demonstrated the first long-term successful islet transplantation by using a glucocorticoid-free immunosuppressive regimen (sirolimus and tacrolimus). The Clinical Islet Transplantation (CIT) Consortium seeks to improve upon the Edmonton Protocol by using anti-thymocyte globulin (ATG) and TNFα antagonist (etanercept). The trials currently in progress, in addition to research efforts to find new sources of islet cells, reflect enormous potential for islet transplantation in treatment of type I diabetes.     

Current and emerging therapeutic strategies for Fanconi anemia

Fanconi Anemia (FA) is a rare disorder with incidence of 1in 350,000 births. It is characterized by progressive bone marrow failure leading to death of many patients in their childhood while development of cancer at later stages of life in some. The treatment of FA is still a medical challenge. Current treatments of FA include androgen administration, hematopoietic growth factors administration and hematopoietic stem cell transplantation (HSCT). Clinical gene therapy trials are still ongoing. The partial success of current therapies has renewed interest in the search for new treatments. Generation of patient-specific induced pluripotent stem (iPS) has shown promising results for cell and gene based therapy. Small molecule interventions have been observed to delay tumor onset in FA. Tumors deficient in FA pathway can be treated by profiling of DNA repair pathway through synthetic lethality mechanism. Targeting toll-like receptor 8 (TLR8) dependent TNFα overexpression is yet another upcoming therapeutic approach to treat FA patients. In conclusion, in the present scenario of treatments available for FA, a proper algorithm of treatment decisions must be followed for better management of FA patients and to ensure their increased survival. Innovative therapeutic approaches that can prevent both anemia and cancer should be developed for more effective treatment of FA.     

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.¹ Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.² These include RPE65 gene replacement therapy in patients with Leber''s congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt''s disease.^3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.^5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.     

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.^1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.^4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.^1,2The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically¹ or analytically,^2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,^1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.^1,2,7In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N₃) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.