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1.
J Med Chem ; 65(3): 2342-2360, 2022 02 10.
Article in English | MEDLINE | ID: mdl-35007061

ABSTRACT

Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds 26 and 30 have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Imidazoles/pharmacology , Transcription Factors/antagonists & inhibitors , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Drug Design , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Molecular Structure , Protein Binding , Protein Domains , Proto-Oncogene Proteins c-myc/metabolism , Structure-Activity Relationship , Transcription Factors/chemistry , Transcription Factors/metabolism
2.
J Fluor Chem ; 261-2622022 Sep.
Article in English | MEDLINE | ID: mdl-37197608

ABSTRACT

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

3.
J Med Chem ; 64(14): 10497-10511, 2021 07 22.
Article in English | MEDLINE | ID: mdl-34236185

ABSTRACT

The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 and 26 are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.


Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Lysine/pharmacology , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , A549 Cells , Cell Cycle Proteins/metabolism , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Lysine/chemistry , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Structure-Activity Relationship , Transcription Factors/metabolism , Triazoles/chemical synthesis , Triazoles/chemistry
4.
Angew Chem Int Ed Engl ; 60(3): 1220-1226, 2021 01 18.
Article in English | MEDLINE | ID: mdl-32975004

ABSTRACT

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.


Subject(s)
Proteins/metabolism , Water/chemistry , Humans , Transcription Factors/chemistry
5.
Molecules ; 25(17)2020 Aug 29.
Article in English | MEDLINE | ID: mdl-32872491

ABSTRACT

As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed 19F (PrOF) NMR and 1H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought to combine these two NMR-based assays into a new screening workflow. This combination of protein- and ligand-observed experiments allows for a time- and resource-efficient multiplexed screen of mixtures of fragments and proteins. PrOF NMR is first used to screen mixtures against two proteins. Hit mixtures for each protein are identified then deconvoluted using 1H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and Plasmodium falciparum (Pf) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, PfGCN5, and fragments binding both proteins, respectively. Select hits were characterized, revealing a broad range of affinities from low µM to mM dissociation constants. Follow-up experiments supported a low-affinity second binding site on PfGCN5. This approach can be used to bias fragment screens towards more selective hits at the onset of inhibitor development in a resource- and time-efficient manner.


Subject(s)
Antigens, Nuclear/chemistry , Drug Discovery/methods , Histone Acetyltransferases/chemistry , Nerve Tissue Proteins/chemistry , Protozoan Proteins/chemistry , Transcription Factors/chemistry , Binding Sites , Humans , Ligands , Magnetic Resonance Imaging , Models, Molecular , Plasmodium falciparum , Protein Binding , Protein Domains , Small Molecule Libraries
6.
Org Biomol Chem ; 18(27): 5174-5182, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32588860

ABSTRACT

Bromodomain-containing proteins regulate transcription through protein-protein interactions with chromatin and serve as scaffolding proteins for recruiting essential members of the transcriptional machinery. One such protein is the bromodomain and PHD-containing transcription factor (BPTF), the largest member of the nucleosome remodeling complex, NURF. Despite an emerging role for BPTF in regulating a diverse set of cancers, small molecule development for inhibiting the BPTF bromodomain has been lacking. Here we cross-validate three complementary biophysical assays to further the discovery of BPTF bromodomain inhibitors for chemical probe development: two direct binding assays (protein-observed 19F (PrOF) NMR and surface plasmon resonance (SPR)) and a competitive inhibition assay (AlphaScreen). We first compare the assays using three small molecules and acetylated histone peptides with reported affinity for the BPTF bromodomain. Using SPR with both unlabeled and fluorinated BPTF, we further determine that there is a minimal effect of 19F incorporation on ligand binding for future PrOF NMR experiments. To guide medicinal chemistry efforts towards chemical probe development, we subsequently evaluate two new BPTF inhibitor scaffolds with our suite of biophysical assays and rank-order compound affinities which could not otherwise be determined by PrOF NMR. Finally, we cocrystallize a subset of small molecule inhibitors and present the first published small molecule-protein structures with the BPTF bromodomain. We envision the biophysical assays described here and the structural insights from the crystallography will guide researchers towards developing selective and potent BPTF bromodomain inhibitors.


Subject(s)
Nerve Tissue Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Antigens, Nuclear/chemistry , Biophysical Phenomena , Magnetic Resonance Spectroscopy , Nerve Tissue Proteins/chemistry , Protein Domains , Surface Plasmon Resonance , Transcription Factors/chemistry
8.
Biochemistry ; 59(20): 1871-1880, 2020 05 26.
Article in English | MEDLINE | ID: mdl-32356653

ABSTRACT

Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.


Subject(s)
Histones/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nucleosomes/metabolism , Transcription Factors/metabolism , Acetylation , Histones/chemistry , Histones/genetics , Humans , Nucleosomes/chemistry , Protein Processing, Post-Translational , Transcription Factors/chemistry , Transcriptional Activation
9.
Org Lett ; 22(10): 3946-3950, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32347732

ABSTRACT

1,4-Thiazepanes and 1,4-thiazepanones represent seven-membered ring systems with highly 3D character and are currently underrepresented in fragment screening libraries. A nuclear magnetic resonance (NMR) fragment screen identified 1,4-acylthiazepanes as new BET (bromodomain and extraterminal domain) bromodomain ligands; however, an efficient and readily diversified synthesis for library development has not been reported. Here we report a one-pot synthesis using α,ß-unsaturated esters and 1,2-amino thiols to form 1,4-thiazepanones as precursors to 1,4-thiazepanes with high 3D character. This reaction proceeds in reasonable time (0.5-3 h) and in good yield and tolerates a broad scope of α,ß-unsaturated esters. Several 1,4-thiazepanes were synthesized by a two-step transformation and were characterized as new BET bromodomain ligands using protein-observed 19F NMR. This synthesis should provide ready access to diverse 3D fragments for screening libraries.


Subject(s)
Esters/chemistry , Proteins/chemistry , Drug Discovery , Ligands , Magnetic Resonance Spectroscopy , Molecular Structure , Protein Domains , Small Molecule Libraries/chemistry
10.
ACS Med Chem Lett ; 10(12): 1648-1654, 2019 Dec 12.
Article in English | MEDLINE | ID: mdl-31857841

ABSTRACT

Fragment-based ligand discovery has been successful in targeting diverse proteins. Despite drug-like molecules having more 3D character, traditional fragment libraries are largely composed of flat, aromatic fragments. The use of 3D-enriched fragments for enhancing library diversity is underexplored especially against protein-protein interactions. Here, we evaluate using 3D-enriched fragments against bromodomains. Bromodomains are highly ligandable, but selectivity remains challenging, particularly for bromodomain and extraterminal (BET) family bromodomains. We screened a 3D-enriched fragment library against BRD4(D1) via 1H CPMG NMR with a protein-observed 19F NMR secondary assay. The screen led to 29% of the hits that are selective over two related bromodomains, BRDT(D1) and BPTF, and the identification of underrepresented chemical bromodomain inhibitor scaffolds. Initial structure-activity relationship studies guided by X-ray crystallography led to a ligand-efficient thiazepane, with good selectivity and affinity for BET bromodomains. These results suggest that the incorporation of 3D-enriched fragments to increase library diversity can benefit bromodomain screening.

11.
ACS Med Chem Lett ; 10(9): 1296-1301, 2019 Sep 12.
Article in English | MEDLINE | ID: mdl-31531200

ABSTRACT

The Bromodomain and Extra Terminal (BET) family of proteins recognize post-translational N-ε-acetylated lysine modifications, regulating transcription as "reader" proteins. Bromodomain inhibitors are interesting targets for the development of potential cancer, inflammation, and heart disease treatments. Several dual kinase-bromodomain inhibitors have been identified by screening kinase inhibitor libraries against BET proteins. Although potentially useful from a polypharmacology standpoint, multitarget binding complicates deciphering molecular mechanisms. This report describes a systematic approach to mitigating kinase activity in a dual kinase-bromodomain inhibitor based on a 1,2,3-triazole-pyrimidine core. By modifying the triazole substituent and altering the pyrimidine core, this structure-activity relationship study enhanced BET activity while reducing the p38α kinase activity >90,000-fold. A BRD4-D1 cocrystal structure indicates that the 1,2,3-triazole is acting as a N-ε-acetylated lysine mimic. A BRD4 sensitive cell line, MM.1S, was used to demonstrate activity in cells, which is further supported by reduced c-Myc expression.

12.
Org Biomol Chem ; 17(7): 2020-2027, 2019 02 13.
Article in English | MEDLINE | ID: mdl-30706071

ABSTRACT

Bromodomain and PHD finger containing protein transcription factor (BPTF) is an epigenetic protein involved in chromatin remodelling and is a potential anticancer target. The BPTF bromodomain has one reported small molecule inhibitor (AU1, rac-1). Here, advances made on the structure-activity relationship of a BPTF bromodomain ligand are reported using a combination of experimental and molecular dynamics simulations leading to the active enatiomer (S)-1. Additionally, a ligand deconstruction analysis was conducted to characterize important pharmacophores for engaging the BPTF bromodomain. These studies have been enabled by a protein-based fluorine NMR approach, highlighting the versatility of the method for selectivity, ligand deconstruction, and ligand binding. To enable future analysis of biological activity, cell growth analyses in a panel of cancer cell lines were carried out using CRISPR-Cas9 and (S)-1 to identify cell-based model systems that are sensitive to BPTF inhibition.


Subject(s)
Nerve Tissue Proteins/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridines/pharmacology , Small Molecule Libraries/pharmacology , Transcription Factors/antagonists & inhibitors , Antigens, Nuclear , Cell Proliferation , Crystallography, X-Ray , Humans , Ligands , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship
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