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1.
J Biol Chem ; 291(25): 13014-27, 2016 Jun 17.
Article in English | MEDLINE | ID: mdl-27056325

ABSTRACT

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.


Subject(s)
CREB-Binding Protein/antagonists & inhibitors , E1A-Associated p300 Protein/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , T-Lymphocytes, Regulatory/drug effects , Acetylation/drug effects , CREB-Binding Protein/chemistry , CREB-Binding Protein/metabolism , Cell Differentiation/drug effects , Cell Line , Cells, Cultured , E1A-Associated p300 Protein/chemistry , E1A-Associated p300 Protein/metabolism , Forkhead Transcription Factors/metabolism , Histones/metabolism , Humans , Molecular Docking Simulation , Protein Structure, Tertiary/drug effects , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/metabolism , Transcriptome/drug effects
2.
ACS Med Chem Lett ; 7(2): 145-50, 2016 Feb 11.
Article in English | MEDLINE | ID: mdl-26985289

ABSTRACT

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.

3.
Biochemistry ; 53(38): 6063-77, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25184411

ABSTRACT

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N(5)-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.


Subject(s)
Actinobacteria/enzymology , Bacterial Proteins/metabolism , Crystallography, X-Ray , Flavin-Adenine Dinucleotide/metabolism , Mixed Function Oxygenases/metabolism , Actinobacteria/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalytic Domain , Crystallization , Gene Expression Regulation, Bacterial , Mixed Function Oxygenases/genetics , Models, Molecular , NADP/metabolism , Oxidation-Reduction , Protein Conformation
4.
Biochemistry ; 51(1): 382-90, 2012 Jan 10.
Article in English | MEDLINE | ID: mdl-22148158

ABSTRACT

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.


Subject(s)
DNA Damage , DNA Glycosylases/chemistry , DNA Glycosylases/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Catalysis , Crystallography, X-Ray , DNA Adducts/chemistry , DNA Glycosylases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Genome, Human , Humans , Mutagenesis , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/metabolism , Plasmids , Protein Conformation
5.
J Biol Chem ; 286(15): 13205-13, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21349833

ABSTRACT

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.


Subject(s)
Cytosine/analogs & derivatives , DNA Glycosylases/chemistry , DNA, Neoplasm/chemistry , Neoplasm Proteins/chemistry , Catalytic Domain , Cytosine/chemistry , Cytosine/metabolism , DNA Damage/physiology , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Humans , Hydrogen Bonding , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/genetics , Structure-Activity Relationship
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