Synthesis and structure-activity relationship of histone deacetylase (HDAC) inhibitors with triazole-linked cap group

Histone deacetylase (HDAC) inhibition is a recent, clinically validated therapeutic strategy for cancer treatment. Small molecule HDAC inhibitors identified so far fall in to three distinct structural motifs: the zinc-binding group (ZBG), a hydrophobic linker, and a recognition cap group. Here we report the suitability of a 1,2,3-triazole ring as a surface recognition cap group-linking moiety in suberoylanilide hydroxamic acid-like (SAHA-like) HDAC inhibitors. Using “click” chemistry (Huisgen cycloaddition reaction), several triazole-linked SAHA-like hydroxamates were synthesized. Structure–activity relationship revealed that the position of the triazole moiety as well as the identity of the cap group markedly affected the in vitro HDAC inhibition and cell growth inhibitory activities of this class of compounds.     

Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection

Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.     

Biochemical Characterization of Hpa2 and Hpa3, Two Small Closely Related Acetyltransferases from Saccharomyces cerevisiae

Based on their sequences, the Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here, we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses showed that Hpa3 translation initiates primarily from Met-19 rather than the annotated start site, Met-1, with a minor product starting at Met-27. When expressed in Escherichia coli and assayed in vitro, Hpa2 and Hpa3 (from Met-19) acetylated histones and polyamines. Whereas Hpa2 acetylated histones H3 and H4 (at H3 Lys-14, H4 Lys-5, and H4 Lys-12), Hpa3 acetylated only histone H4 (at Lys-8). Additionally, Hpa2, but not Hpa3, acetylated certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate d-amino acids, and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo in acetylating histones and other small proteins.     

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.     

Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.     

(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ε-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.     

Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E₂, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE₂ had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.     

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen

Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of α1(I) and α2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58-66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon α1(I) and α2(I) chains but only one 3Hyp residue in skin α1(I) and none in α2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissue-specific manner.