Bisubstrate-Type Chemical Probes Identify GRP94 as a Potential Target of Cytosine-Containing Adenosine Analogs.
Ontology highlight
ABSTRACT:
SUBMITTER: Pechalrieu D
PROVIDER: S-EPMC7336334 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Hsp90 isoform-selective inhibition is highly desired as it can potentially avoid the toxic side-effects of pan-inhibition. The current study developed selective inhibitors of one such isoform, Grp94, predicated on the chimeric and pan-Hsp90 inhibitor, radamide (RDA). Replacement of the quinone moiety of RDA with a phenyl ring (2) was found to be better suited for Grp94 inhibition as it can fully interact with a unique hydrophobic pocket present in Grp94. An extensive SAR for this scaffold showed that substitutions at the 2- and 4-positions (8 and 27, respectively) manifested excellent Grp94 affinity and selectivity. Introduction of heteroatoms into the ring also proved beneficial, with a 2-pyridine derivative (38) exhibiting the highest Grp94 affinity (K(d)=820 nM). Subsequent cell-based assays showed that these Grp94 inhibitors inhibit migration of the metastatic breast cancer cell line, MDA-MB-231, as well as exhibit an anti-proliferative affect against the multiple myeloma cell line, RPMI 8226.
| S-EPMC4142655 | biostudies-literature
Project description:Developing selective enzyme inhibitors allows for the expansion of molecular toolboxes to investigate functions and activities of target enzymes. The histone acetyltransferase 1 (HAT1) is among the first histone acetyltransferase (HAT) enzymes that were discovered in the mid-1990s; however, it remains one of the poorly studied enzymes in comparison with the other HATs. Although HAT1 has been linked to various disease states, no inhibitors have been reported to target HAT1. Here, we designed a set of peptide-CoA conjugates as bisubstrate inhibitors of HAT1 with submicromolar potency. In particular, the bisubstrate inhibitor H4K12CoA exhibited a low Ki value of 1.1 nM for HAT1. In addition, H4K12CoA was shown to be a competitive inhibitor with respect to both AcCoA and H4 peptide, suggesting a unique kinetic mechanism of HAT1 catalysis. Creating these submicromolar inhibitors offers mechanistic tools to better understand how HAT1 recognizes substrates and cofactors, as well as provides chemical leads to further develop therapeutic agents to target this important enzyme for disease therapy.
| S-EPMC6533141 | biostudies-literature
Project description:Post translational modifications (PTMs, e.g., phosphorylation, acetylation and methylation) of histone play important roles in regulating many fundamental cellular processes such as gene transcription, DNA replication and damage repair. While 'writer' and 'eraser' enzymes modify histones by catalyzing the addition and removal of histone PTMs, 'reader' proteins recognize these modified histones and 'translate' the PTMs by executing distinct cellular programs. Therefore, identification of the regulating enzymes and binding partners of histone PTMs is essential for understanding their regulatory mechanisms and cellular functions. Here we report the development of diazirine-based photoaffinity probes for identification of 'readers' and 'erasers' of histone PTMs. When compared with previously described benzophenone-based photoaffinity probes, the present probes demonstrate significantly improved photo-cross-linking rates, yields and specificities for capturing proteins that recognize a trimethylation mark on histone H3 lysine 4 (H3K4Me3). Furthermore, we show that the diazirine-based probes can also be used to identify enzymes that catalyse the removal of histone lysine acetylation and malonylation. This study provides new chemical tools for examining PTM-mediated protein-protein interactions and broadens the scope of our photo-cross-linking strategy from finding histone PTM 'readers' to identifying dynamic and transient interactions between PTMs and their 'erasers'.
| S-EPMC5811097 | biostudies-literature
Project description:Targeting epigenetic proteins is a rapidly growing area for medicinal chemistry and drug discovery. Recent years have seen an explosion of interest in developing small molecules binding to bromodomains, the readers of acetyl-lysine modifications. A plethora of co-crystal structures has motivated focused fragment-based design and optimization programs within both industry and academia. These efforts have yielded several compounds entering the clinic, and many more are increasingly being used as chemical probes to interrogate bromodomain biology. High selectivity of chemical probes is necessary to ensure biological activity is due to an on-target effect. Here, we review the state-of-the-art of bromodomain-targeting compounds, focusing on the structural basis for their on-target selectivity or lack thereof. We also highlight chemical biology approaches to enhance on-target selectivity.
| S-EPMC5321501 | biostudies-literature
Project description:We prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions. We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] and hydroxyl radicals. We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2-3 bases on the 3'-side of the mismatch. Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints. MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps. None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II). We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine). RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage. However, YGAR inhibits DNases I and II cleavage over 4-6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases. These changes suggest that this mismatch has a more pronounced effect on the local DNA structure. These changes are discussed in terms of the structural and dynamic effects of each mismatch.
| S-EPMC1223340 | biostudies-other
Project description:Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell-Chance or "hit-and-run" catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail.
| S-EPMC3076313 | biostudies-literature
Project description:We recently identified a class of pimelic diphenylamide histone deacetylase (HDAC) inhibitors that show promise as therapeutics in the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease. Here, we describe chemical approaches to identify the HDAC enzyme target of these inhibitors. Incubation of a trifunctional activity-based probe with a panel of class I and class II recombinant HDAC enzymes, followed by click chemistry addition of a fluorescent dye and gel electrophoresis, identifies HDAC3 as a unique high-affinity target of the probe. Photoaffinity labeling in a nuclear extract prepared from human lymphoblasts with the trifunctional probe, followed by biotin addition through click chemistry, streptavidin enrichment, and Western blotting also identifies HDAC3 as the preferred cellular target of the inhibitor. Additional inhibitors with different HDAC specificity profiles were synthesized, and results from transcription experiments in FRDA cells point to a unique role for HDAC3 in gene silencing in Friedreich's ataxia.
| S-EPMC2909763 | biostudies-literature
Project description:E3 ligases constitute a large and diverse family of proteins that play a central role in regulating protein homeostasis by recruiting substrate proteins via recruitment domains to the proteasomal degradation machinery. Small molecules can either inhibit, modulate or hijack E3 function. The latter class of small molecules led to the development of selective protein degraders, such as PROTACs (PROteolysis TArgeting Chimeras), that recruit protein targets to the ubiquitin system leading to a new class of pharmacologically active drugs and to new therapeutic options. Recent efforts have focused on the E3 family of Baculovirus IAP Repeat (BIR) domains that comprise a structurally conserved but diverse 70 amino acid long protein interaction domain. In the human proteome, 16 BIR domains have been identified, among them promising drug targets such as the Inhibitors of Apoptosis (IAP) family, that typically contain three BIR domains (BIR1, BIR2, and BIR3). To date, this target area lacks assay tools that would allow comprehensive evaluation of inhibitor selectivity. As a consequence, the selectivity of current BIR domain targeting inhibitors is unknown. To this end, we developed assays that allow determination of inhibitor selectivity in vitro as well as in cellulo. Using this toolbox, we have characterized available BIR domain inhibitors. The characterized chemical starting points and selectivity data will be the basis for the generation of new chemical probes for IAP proteins with well-characterized mode of action and provide the basis for future drug discovery efforts and the development of PROTACs and molecular glues.
| S-EPMC9204419 | biostudies-literature
Project description:The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.
| S-EPMC2223900 | biostudies-literature
Project description:The intracellular localization and target of the napyradiomycin congeners CNQ525.510B and A80815C were explored using an immunoaffinity fluorescence (IAF) approach. Semi-synthetic methods were used to prepare probes from napyradiomycin CNQ525.510B and derivative A80815C. The results of confocal microscopy indicated that probes from both natural products localized predominantly within the endoplasmic reticulum (ER) of HCT-116 human colon carcinoma cells. Parallel immunoaffinity precipitation efforts using a monoclonal antibody designed against the IAF tag, resulted in the isolation of an Hsp90 family member. This protein was identified as human Grp94 (hGrp94), by its specific mass spectral signature. This observation was validated by Western blot analyses and by the result of an in vitro Grp94 binding assay. The fact that the napyradiomycins CNQ525.510B and A80815C bind to hGrp94, and their associated probes localize within the ER, suggest the use of these materials as molecular probes for monitoring ER-based chaperone function.
| S-EPMC3893886 | biostudies-literature