Characterizing inhibitors of human AP endonuclease 1.

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1.

Rapid excision of oxidized adenine by human thymine DNA glycosylase.

Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation.

DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex.

The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction.

Structural Insights into the Mechanism of Base Excision by MBD4.

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily.

Excision of 5-Carboxylcytosine by Thymine DNA Glycosylase.

5-Methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are poorly defined, including TDG excision of caC.

Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site.

3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation.

Structural Basis for Excision of 5-Formylcytosine by Thymine DNA Glycosylase.

Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten-eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2'-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically excises fC.

Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues.

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes.

Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein.

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 pocket of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort.

Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2.

Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients.

Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.

Results: We have analyzed the structural features and sequences of proteins having the hot dog fold.

Structural basis for the variation in triclosan affinity to enoyl reductases.

Bacteria synthesize fatty acids in a dissociated type pathway different from that in humans. Enoyl acyl carrier protein reductase, which catalyzes the final step of fatty acid elongation, has been validated as a potential anti-microbial drug target. Triclosan is known to inhibit this enzyme effectively.