Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity

Abstract

The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.

Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY

“…These insights can be used to guide future design of MutY/MUTYH specific probes to monitor the activity, or lack thereof, of MutY/MUTYH variants. 53 Moreover, the SARs presented here can be applied toward the development of MutY/MUTYH specific inhibitors that may find utility in cancer therapeutics. 54…”

Sustained delivery of gemcitabine via in situ injectable mussel-inspired hydrogels for the local therapy of pancreatic cancer

“…Concretely, the broad peaks within the range of 6.72-6.82 ppm were attributable to the aromatic proton (m, 3H, C 6 H 3 (OH) 2 ) of the catechol group. 13,31 The peaks ranging from 4.28 to 4.64 ppm were ascribed to CMC protons. The peak of the methylene proton was obtained at 2.86 ppm (s, 2H, C 6 H 3 (OH) 2 -CH 2 -).…”

“…Herein, two types of polysaccharide derivatives with different precursor ratios (denoted as OCMC-DA 4 /CMCS 2 -Gem) were synthesized according to our preceding work. 31 Gemcitabine hydrochloride (99%) was purchased from Nanjing Drum Tower Hospital (Nanjing, China). The morphology, structure, and mechanical strength of the drug-loaded material were characterized by solid-state nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy in the attenuated total reflectance mode (FTIR-ATR), field emission scanning electron microscopy (FE-SEM), thermogravimetric analysis (TGA), and rheology testing analysis.…”

“…The more detailed synthetic procedure and commonly used characterization instruments are shown in our previous report or ESI. † 31…”

“…32 In comparison with the drug-loaded hydrogel system, the drug-free hydrogel formulation went through a similar change when the gel was formed according to our previously reported findings. 31 The optimal gelation time (B46 s) for convenient handling and precise controllability would have a certain guiding significance for the application of localized targeted drug delivery in clinical practice. Besides, Fig.…”