An Excimer Clamp for Measuring Damaged Base Excision by the DNA Repair Enzyme NTH1.
Angewandte Chemie (International ed. in English)
Direct measurement of DNA repair enzyme activities is important both for basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Here we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change.This strategy utilizes glycosylation-induced excimer formation of pyrenes, and modified DNA probes incorporating two pyrene deoxynucleotides and a damaged base enable the direct, real-time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to identification of a new small-molecule inhibitor with sub-micromolar potency.
View details for DOI 10.1002/anie.202001516
View details for PubMedID 32109332
Dual Inhibitors of 8-Oxoguanine Surveillance by OGG1 and NUDT1.
ACS chemical biology
Oxidative damage in DNA is one of the primary sources of mutations in the cell. The activities of repair enzymes 8-oxoguanine DNA glycosylase (OGG1) and human MutT Homologue 1 (NUDT1 or MTH1), which work together to ameliorate this damage, are closely linked to mutagenesis, genotoxicity, cancer, and inflammation. Here we have undertaken the development of small-molecule dual inhibitors of the two enzymes as tools to test the relationships between these pathways and disease. The compounds preserve key structural elements of known inhibitors of the two enzymes, and they were synthesized and assayed with recently developed luminescence assays of the enzymes. Further structural refinement of initial lead molecules yielded compound 5 (SU0383) with IC50(NUDT1) = 0.034 muM and IC50(OGG1) = 0.49 muM. The compound SU0383 displayed low toxicity in two human cell lines at 10 muM. Experiments confirm the ability of SU0383 to increase sensitivity of tumor cells to oxidative stress. Dual inhibitors of these two enzymes are expected to be useful in testing multiple hypotheses regarding the roles of 8-oxo-dG in multiple disease states.
View details for DOI 10.1021/acschembio.9b00490
View details for PubMedID 31622553
Ultrafast Oxime Formation Enables Efficient Fluorescence Light-up Measurement of DNA Base Excision.
Journal of the American Chemical Society
DNA glycosylases constitute a biologically and biomedically important group of DNA repair enzymes responsible for initiating base excision repair (BER). Measuring their activities can be useful for studying the mechanisms DNA damage and repair and for practical applications in cancer diagnosis and drug screening. Previous fluorescence methods for assaying DNA glycosylases are often complex and/or limited in scope to a single enzyme type. Here we report a universal base excision reporter (UBER) fluorescence probe design that implements an unprecedentedly rapid oxime reaction (>150 M-1 s-1) with high specificity for the abasic (AP) site of DNA. The molecular rotor design achieves a robust >250-500-fold increase in fluorescence upon reaction with AP sites in DNA. By using the fluorescence reporter in concert with specific DNA lesion-containing substrates, the UBER probe can be used in a coupled assay in principle with any DNA glycosylase. We demonstrate the utility of the UBER probe by assaying five different glycosylases in real time as well as profiling glycosylase activity in cell lysates. We anticipate that the UBER probe will be of considerable utility to researchers studying DNA repair biology owing to its high level of generalizability, ease of use, and compatibility with biologically derived samples.
View details for DOI 10.1021/jacs.9b09812
View details for PubMedID 31774658
Fluorescence Probes of ALKBH2 Measure DNA Alkylation Repair and Drug Resistance Responses.
Angewandte Chemie (International ed. in English)
The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 in vitro or in biological samples. Here we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both in vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and Western blots. Furthermore, the probe provides a viable high throughput assay for drug discovery.
View details for PubMedID 30098084
Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2018; 140 (6): 2105–14
The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 μM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 μM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 μM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.
View details for PubMedID 29376367
Fluorescent Probes of DNA Repair.
ACS chemical biology
DNA repair is now understood to play a key role in a variety of disease states, most notably cancer. Tools for studying DNA have typically relied on traditional biochemical methods which are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such methods. Recently, specific fluorescent probes have been developed to aid in the study of DNA repair. Fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-and-measure format. This review will summarize the distinct classes of probe designs and their potential utility in varied research and preclinical settings.
View details for PubMedID 29156135
- Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome ACS MEDICINAL CHEMISTRY LETTERS 2016; 7 (3): 250-255