Screening inhibitors for blocking UHRF1-methylated DNA interaction with capillary electrophoresis.

Epigenetic inheritance in mammals relies in part on propagation of DNA methylation patterns throughout development. UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) is required for maintenance the methylation pattern. It was reported that UHRF1 is overexpressed in a number of cancer types, and its depletion has been established to inhibit growth and invasion of cancer cells. It has been considered as a new therapeutic target for cancer. In the present work, we described a method for screening inhibitors for blocking the formation of UHRF1-methylated DNA (mDNA) complex by using nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM). A recombinant UHRF1 with the SRA domain (residues 408-643), a fluorescently labeled double strand mDNA (12 mer) and a known inhibitor mitoxantrone were employed for proof of concept. The method allows to measure the dissociation constant (Kd) of the UHRF1-mDNA complex as well as the rate kinetic constant for complex formation (kon) and dissociation (koff). A small chemical library composed of 60 natural compounds were used to validate the method. Sample pooling strategy was employed to improve the screening throughput. The merit of the method was confirmed by the discovery of two natural products proanthocyanidins and baicalein as the new inhibitors for blocking the formation of UHRF1-mDNA complex. Our work demonstrated that CE represents a straightforward and robust technique for studying UHRF1-mDNA interaction and screening of the inhibitors.

A gold foil covered fused silica capillary tip as a sheathless interface for coupling capillary electrophoresis-mass spectrometry.

A method for the preparation of an on-column ESI emitter used as the sheathless interface for coupling capillary electrophoresis (CE) with mass spectrometry (MS) was developed. The emitter was directly fabricated at the outlet end of the separation capillary which was etched with HF solution to a symmetrical tip. The tip was covered with a small piece of gold foil which was fixed by epoxy resin glue for electrical contact. Such a prepared ESI emitter can produce a stable ESI signal over the wide range of flow rate from 50 nL/min to 800 nL/min. The performance of the CE-MS with the sheathless interface was evaluated by using the separation of four alkaloids. It was found that the strong electroosmotic flow produced by the multiple polyelectrolyte coating on the capillary is necessary for maintaining a stable MS signal. Effect of the running buffer composition, concentration and the CE separation voltages on the ESI signal strength were investigated. The absolute detection limits for the alkaloids was determined as fmol level. Moreover, the CE-MS was applied for the analyses of trypsin digestion of cytochrome C and small molecular organic anions. The emitter performed very stable with a lifetime of at least 180 h.

Screening of inhibitors against histone demethylation jumonji domain-containing protein 3 by capillary electrophoresis.

Jumonji domain-containing proteins (JMJDs) play an important role in the epigenetic regulation of gene expression. Aberrant regulation of histone modification has been observed in the progression of a variety of diseases, such as neurological disorders and cancer. Therefore, discovery of selective modulators of JMJDs is very attractive in new drug discovery. Herein, a simple capillary electrophoresis (CE) method was developed for screening of inhibitors against JMJD3. A known JMJD3 inhibitor GSK-J1, 5-carboxyfluorescein labeled substrate peptide with an amino acid sequence of KAPRKQLATKAARK(me3)SAPATGG (truncated from histone H3), as well as a small chemical library composed of 37 purified natural compounds and 30 natural extracts were used for method development and validation. The separation of substrate from its demethylated product was achieved by addition of polycation hexadimethrine bromide (HDB) in the running buffer. The enzyme activity was thus assayed accurately through separating the demethylated product from the substrate and then measuring the peak area of the product. The enzyme inhibition can be read out by comparing the peak area of the demethylated product obtained in the present of inhibitors and that of the negative control in the absence of any inhibitor. The merit of the method is proved by discovering two new JMJD3 inhibitors: salvianic acid A and puerarin 6''-O-xyloside.

Determining the affinity of anti-mitotic compounds binding to colchicine binding site of tubulin by affinity probe capillary electrophoresis.

The colchicine binding site of tubulin is often used to screen the anti-mitotic compounds, which are widely used as anti-cancer therapies. In the present work, an affinity probe capillary electrophoresis (APCE) method was developed for determining the affinity of anti-mitotic compounds. To this end, a fluorescently labeled affinity probe, 5-carboxyfluorescein-colchicine (F-colchicine), was prepared for the affinity competition experiment. The probe can form a stable complex with tubulin with the binding stoichiometry of 0.75, and the dissociation constant Kd of the complex was determined as 5.7 × 10-5 mol/L. In the affinity competition experiment, F-colchicine was incubated with tubulin and the test compound in the solution. The F-colchicine-tubulin complexes and free F-colchicine were quickly separated by CE and the concentration of free F-colchicine was accurately determined with the laser induced fluorescence detection. The affinity constant of the tested compound can be measured with the affinity competition binding curve. The enantiomers of the anti-mitotic compound were evaluated by using the method. The binding affinity of the enantiomers displayed an enantioselective manner. Compared to other affinity binding assay methods, our method is more straightforward, more accurate, and more cost-effective.

Novel Triapine Derivative Induces Copper-Dependent Cell Death in Hematopoietic Cancers.

Triapine, an iron chelator that inhibits ribonucleotide reductase, has been evaluated in clinical trials for cancer treatment. Triapine in combination with other chemotherapeutic agents shows promising efficacy in certain hematologic malignancies; however, it is less effective against many advanced solid tumors, probably due to the unsatisfactory potency and pharmacokinetic properties. In this report, we developed a triapine derivative IC25 (10) with potent antitumor activity. 10 Preferentially inhibited the proliferation of hematopoietic cancers by inducing mitochondria reactive oxygen species production and mitochondrial dysfunction. Unlike triapine, 10 executed cytotoxic action in a copper-dependent manner. 10-Induced up-expression of thioredoxin-interacting protein resulted in decreased thioredoxin activity to permit c-Jun N-terminal kinase and p38 activation and ultimately led to the execution of the cell death program. Remarkedly, 10 showed good bioavailability and inhibited tumor growth in mouse xenograft models. Taken together, our study identifies compound 10 as a copper-dependent antitumor agent, which may be applied to the treatment of hematopoietic cancers.