Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Polymeric Nanocarriers with Controlled Chain Flexibility Boost mRNA Delivery In Vivo through Enhanced Structural Fastening.

Messenger RNA (mRNA) shows high therapeutic potential, though effective delivery systems are still needed for boosting its application. Nanocarriers loading mRNA via polyion complexation with block catiomers into core-shell micellar structures are promising systems for enhancing mRNA delivery. Engineering the interaction between mRNA and catiomers through polymer design can promote the development of mRNA-loaded micelles (mRNA/m) with increased delivery efficiency. Particularly, the polycation chain rigidity may critically affect the mRNA-catiomer interplay to yield potent nanocarriers, yet its effect remains unknown. Herein, the influence of polycation stiffness on the performance of mRNA/m by developing block complementary catiomers having polycation segments with different flexibility, that is, poly(ethylene glycol)-poly(glycidylbutylamine) (PEG-PGBA) and PEG-poly(L-lysine) (PEG-PLL) is studied. PEG-PGBA allows more than 50-fold stronger binding to mRNA than the relatively more rigid PEG-PLL, resulting in mRNA/m with enhanced protection against enzymatic attack and polyanions. mRNA/m from PEG-PGBA significantly enhances mRNA in vivo bioavailability and increased protein translation, indicating the importance of controlling polycation flexibility for forming stable polyion complexes with mRNA toward improved delivery.

Thermodynamic and computational analyses reveal the functional roles of the galloyl group of tea catechins in molecular recognition.

Catechins, biologically active polyphenols in green tea, exhibit various biological activities, such as anticancer and antiviral activities, arising from interactions with functional proteins. However, the molecular details of these interactions remain unclear. In this study, we investigated the interactions between human serum albumin (HSA) and various catechins, including some with a galloyl group, by means of isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and docking simulations. Our results indicate that the galloyl group was important for recognition by HSA and was responsible for enthalpic gains derived from a larger buried surface area and more van der Waals contacts. Thus, our thermodynamic and computational analyses suggest that the galloyl group plays important functional roles in the specific binding of catechins to proteins, implying that the biological activities of these compounds may be due in part to the physicochemical characteristics of the galloyl group.

Development of drug discovery screening system by molecular interaction kinetics-mass spectrometry.

RATIONALE: Drug discovery studies invariably require qualitative and quantitative analyses of target compounds at every stage of drug discovery. We have developed a system combining molecular interaction analysis and mass spectrometry (LC-MS) using the principle of nanopore optical interferometry (nPOI) called molecular interaction kinetics-mass spectrometry (MIK-MS). Since nPOI has high binding capacity, the bond-dissociated compound can be directly detected using LC-MS. In this study, we use carbonic anhydrase II (CAII) as a ligand and apply six small compounds as analytes and report the affinity analysis using MIK-MS. METHODS: CAII was immobilized onto a COOH sensor chip using standard amine coupling. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions. An amount of 50 µL of mix solution was injected over the reference channel and sample channel for CAII immobilization. The solutions eluting from the sensor chip were collected from the waste-line of the SKi Pro system every 30 s. Reconstructed elution samples were then injected into the LC-MS/MS system. RESULTS: A mixture containing furosemide, acetazolamide, 4-sulfamoylbenzoic acid, 5-(dimethylamino)-1-naphthalene sulfonamide (DNSA), sulfanilamide and sulpiride (15 µM each) was injected into the CAII-immobilized sensor chip, and the fractions eluted from the SKi Pro system were collected and subjected to selected reaction monitoring LC-MS characterization. Specific results were obtained for acetazolamide, DNSA, furosemide and sulpiride. The results suggest that the association-dissociation curve of a mixed sample can be obtained by one-time MIK-MS analysis. CONCLUSIONS: Six small-molecule binders of CAII were analyzed quantitatively using nPOI and MIK-MS, and the results were compared to published surface plasmon resonance (SPR) results. The nPOI and SPR results show good agreement, confirming the reliability of the analysis. Time-dependent binding results may be obtained by our MS sensorgram approach. Drugs that meet medical needs in a short period are required; this nPOI-LC-MS system is considered an important tool for rapid drug discovery.