Potent hydroxamate-derived compounds arrest endodyogeny of Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonosis that is a worldwide health problem, commonly affecting fetal development and immunodeficient patients. Treatment is carried out with a combination of pyrimethamine and sulfadiazine, which can cause cytopenia and intolerance and does not lead to a parasitological cure of the infection. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins are found in the Toxoplasma gondii genome. Previous work showed the hydroxamate-type KDAC inhibitors Tubastatin A (TST) and Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) were effective against T. gondii. In the present study, the effects of three hydroxamates (KV-24, KV-30, KV-46), which were originally designed to inhibit human KDAC6, showed different effects against T. gondii. These compounds contain a heterocyclic cap group and a benzyl linker bearing the hydroxamic acid group in para-position. All compounds showed selective activity against T. gondii proliferation, inhibiting tachyzoite proliferation with IC50 values in a nanomolar range after 48h treatment. Microscopy analyses showed that after treatment, tachyzoites presented mislocalization of the apicoplast, disorganization of the inner membrane complex, and arrest in the completion of new daughter cells. The number of dividing cells with incomplete endodyogeny increased significantly after treatment, indicating the compounds can interfere in the late steps of cell division. The results obtained in this work that these new hydroxamates should be considered for future in vivo tests and the development of new compounds for treating toxoplasmosis.

Design and synthesis of bioreductive prodrugs of class I histone deacetylase inhibitors and their biological evaluation in virally transfected acute myeloid leukemia cells.

Although histone deacetylase (HDAC) inhibitors show promise in treating various types of hematologic malignancies, they have some limitations, including poor pharmacokinetics and off-target side effects. Prodrug design has shown promise as an approach to improve pharmacokinetic properties and to improve target tissue specificity. In this work, several bioreductive prodrugs for class I HDACs were designed based on known selective HDAC inhibitors. The zinc-binding group of the HDAC inhibitors was masked with various nitroarylmethyl residues to make them substrates of nitroreductase (NTR). The developed prodrugs showed weak HDAC inhibitory activity compared to their parent inhibitors. The prodrugs were tested against wild-type and NTR-transfected THP1 cells. Cellular assays showed that both 2-nitroimidazole-based prodrugs 5 and 6 were best activated by the NTR and exhibited potent activity against NTR-THP1 cells. Compound 6 showed the highest cellular activity (GI50 = 77 nM) and exhibited moderate selectivity. Moreover, activation of prodrug 6 by NTR was confirmed by liquid chromatography-mass spectrometry analysis, which showed the release of the parent inhibitor after incubation with Escherichia coli NTR. Thus, compound 6 can be considered a novel prodrug selective for class I HDACs, which could be used as a good starting point for increasing selectivity and for further optimization.

Quinoline-based thiazolyl-hydrazones target cancer cells through autophagy inhibition.

Heterocyclic pharmacophores such as thiazole and quinoline rings have a significant role in medicinal chemistry. They are considered privileged structures since they constitute several Food and Drug Administration (FDA)-approved drugs for cancer treatment. Herein, we report the synthesis, in silico evaluation of the ADMET profiles, and in vitro investigation of the anticancer activity of a series of novel thiazolyl-hydrazones based on the 8-quinoline (1a-c), 2-quinoline (2a-c), and 8-hydroxy-2-quinolyl moiety (3a-c). The panel of several human cancer cell lines and the nontumorigenic human embryonic kidney cell line HEK-293 were used to evaluate the compound-mediated in vitro anticancer activities, leading to [2-(2-(quinolyl-8-ol-2-ylmethylene)hydrazinyl)]-4-(4-methoxyphenyl)-1,3-thiazole (3c) as the most promising compound. The study revealed that 3c blocks the cell-cycle progression of a human colon cancer cell line (HCT-116) in the S phase and induces DNA double-strand breaks. Also, our findings demonstrate that 3c accumulates in lysosomes, ultimately leading to the cell death of the hepatocellular carcinoma cell line (Hep-G2) and HCT-116 cells, by the mechanism of autophagy inhibition.

Soft drug inhibitors for the epigenetic targets lysine-specific demethylase 1 and histone deacetylases.

Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.

Total synthesis and biological evaluation of histone deacetylase inhibitor WF-3161.

A novel synthesis of the naturally occurring HDAC inhibitor WF-3161 is described. Key steps include the Matteson homologation to generate the stereogenic centres in the side chain, and Pd-catalysed C-H functionalisation to connect the side chain to the peptide backbone. WF-3161 was found to be highly selective for HDAC1, whereas no activity was observed towards HDAC6. High activity was also found against the cancer cell line HL-60.

Melatonin-vorinostat hybrid ligands show higher histone deacetylase and cancer cell growth inhibition than vorinostat.

Anticancer drug conjugates are an emerging approach for future cancer treatment. Here, we report a series of hybrid ligands merging the neurohormone melatonin with the approved histone deacetylase (HDAC) inhibitor vorinostat, using melatonin's amide side chain (3a-e), its indolic nitrogen (5a-d), and its ether oxygen (7a-d) as attachment points. Several hybrid ligands showed higher potency thanvorinostat in both HDAC inhibition and cellular assays on different cultured cancer cell lines. In the most potent HDAC1 and HDAC6 inhibitors, 3e, 5c, and 7c, the hydroxamic acid moiety of vorinostat is linked to melatonin through a hexamethylene spacer. Hybrid ligands 5c and 7c were also found to be potent growth inhibitors of MCF-7, PC-3M-Luc, and HL-60 cancer cell lines. As these compounds showed only weak agonist activity at melatonin MT1 receptors, the findings indicate that their anticancer actions are driven by HDAC inhibition.

Continuous Fluorescent Sirtuin Activity Assay Based on Fatty Acylated Lysines.

Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement.

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

Defined stereoisomers of 2″-amino NAD+ and their activity against human sirtuins and a bacterial (ADP-ribosyl) transferase.

Nicotinamide adenine dinucleotide (NAD+) is an important biomolecule with essential roles at the intersection of energy metabolism, epigenetic regulation and cell signalling. Synthetic analogues of NAD+ are therefore of great interest as chemical tools for medicinal chemistry, chemical biology and drug discovery. Herein, we report the chemical synthesis and full analytical characterisation of three stereoisomers of 2″-amino NAD+, and their biochemical evaluation against two classes of NAD+-consuming enzymes: the human sirtuins 1-3, and the bacterial toxin TccC3. To rationalise the observed activities, molecular docking experiments were carried out with SIRT1 and SIRT2, which identified the correct orientation of the pyrophosphate linkage as a major determinant for activity in this series. These results, together with results from stability tests and a conformational analysis, allow, for the first time, a side-by-side comparison of the chemical and biochemical features, and analytical properties, of different 2″-amino NAD+ stereoisomers. Our findings provide insight into the recognition of co-substrate analogues by sirtuins, and will greatly facilitate the application of these important NAD+ analogues as chemical tool compounds for mechanistic studies with these as well as other NAD+-dependent enyzmes.

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities.

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity.

In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.

Effects of Environmental Conditions on Nephron Number: Modeling Maternal Disease and Epigenetic Regulation in Renal Development.

A growing body of evidence suggests that low nephron numbers at birth can increase the risk of chronic kidney disease or hypertension later in life. Environmental stressors, such as maternal malnutrition, medication and smoking, can influence renal size at birth. Using metanephric organ cultures to model single-variable environmental conditions, models of maternal disease were evaluated for patterns of developmental impairment. While hyperthermia had limited effects on renal development, fetal iron deficiency was associated with severe impairment of renal growth and nephrogenesis with an all-proximal phenotype. Culturing kidney explants under high glucose conditions led to cellular and transcriptomic changes resembling human diabetic nephropathy. Short-term high glucose culture conditions were sufficient for long-term alterations in DNA methylation-associated epigenetic memory. Finally, the role of epigenetic modifiers in renal development was tested using a small compound library. Among the selected epigenetic inhibitors, various compounds elicited an effect on renal growth, such as HDAC (entinostat, TH39), histone demethylase (deferasirox, deferoxamine) and histone methyltransferase (cyproheptadine) inhibitors. Thus, metanephric organ cultures provide a valuable system for studying metabolic conditions and a tool for screening for epigenetic modifiers in renal development.

Structure-Based Design, Docking and Binding Free Energy Calculations of A366 Derivatives as Spindlin1 Inhibitors.

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.

Validation of the Slow Off-Kinetics of Sirtuin-Rearranging Ligands (SirReals) by Means of Label-Free Electrically Switchable Nanolever Technology.

We have discovered the sirtuin-rearranging ligands (SirReals) to be highly potent and selective inhibitors of the NAD+ -dependent lysine deacetylase Sirt2. Using a biotinylated SirReal in combination with biolayer interferometry, we previously observed a slow dissociation rate of the inhibitor-enzyme complex; this had been postulated to be the key to the high affinity and selectivity of SirReals. However, to attach biotin to the SirReal core, we introduced a triazole as a linking moiety; this was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. Herein, we aim to elucidate whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. We used the novel label-free switchSENSE® technology, which is based on electrically switchable DNA nanolevers, to prove that the long residence time of the SirReals is indeed caused by the core scaffold.

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)*.

We have discovered the sirtuin-rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+ -dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis-targeting chimera (PROTAC) enabled small-molecule-induced degradation of Sirt2. Herein, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small-molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells.

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR+ . The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.

Syntheses of Thailandepsin†B Pseudo-Natural Products: Access to New Highly Potent HDAC Inhibitors via Late-Stage Modification.

New Thailandepsin B pseudo-natural products have been prepared. Our synthetic strategy offers the possibility to introduce varying warheads via late stage modification. Additionally, it gives access to the asymmetric branched allylic ester moiety of the natural product in a highly diastereoselective manner applying rhodium-catalyzed hydrooxycarbonylation. The newly developed pseudo-natural products are extremely potent and selective HDAC inhibitors. The non-proteinogenic amino acid d-norleucine was obtained enantioselectively by a recently developed method of rhodium-catalyzed hydroamination.

Metabolite Patterns in Human Myeloid Hematopoiesis Result from Lineage-Dependent Active Metabolic Pathways.

Assessment of hematotoxicity from environmental or xenobiotic compounds is of notable interest and is frequently assessed via the colony forming unit (CFU) assay. Identification of the mode of action of single compounds is of further interest, as this often enables transfer of results across different tissues and compounds. Metabolomics displays one promising approach for such identification, nevertheless, suitability with current protocols is restricted. Here, we combined a hematopoietic stem and progenitor cell (HSPC) expansion approach with distinct lineage differentiations, resulting in formation of erythrocytes, dendritic cells and neutrophils. We examined the unique combination of pathway activity in glycolysis, glutaminolysis, polyamine synthesis, fatty acid oxidation and synthesis, as well as glycerophospholipid and sphingolipid metabolism. We further assessed their interconnections and essentialness for each lineage formation. By this, we provide further insights into active metabolic pathways during the differentiation of HSPC into different lineages, enabling profound understanding of possible metabolic changes in each lineage caused by exogenous compounds.

Avoiding chemotherapy prescribing errors: Analysis and innovative strategies.

BACKGROUND: At Freiburg University Medical Center, chemotherapy prescriptions are processed via a computerized physician order entry (CPOE) tool and clinically checked by a designated chemotherapy surveillance team. Any error detected is reported instantly, corrected, and prospectively recorded. The objective of the current study was to gain insight into the causes, potential consequences, and future preventability of chemotherapy prescribing errors. METHODS: A detailed analysis of 18,823 consecutive antineoplastic orders placed in 2013 through 2014 was performed. In cooperation with information technology (IT) specialists, the intercepted errors were analyzed for effective future prevention using IT measures. Potential error consequences were determined by case discussions between pharmacists and physicians. RESULTS: Within 24 months, a total of 406 chemotherapy prescribing errors were intercepted that affected 375 (2%) of the total orders. Errors were classified as clinically relevant in 279 of the chemotherapy orders (1.5%). In these cases, reduced therapeutic efficacy (0.44%), the need for increased monitoring (0.48%), prolonged hospital stay (0.55%), and fatality (0.02%) were avoided as potential consequences. The most efficient conventional measures for error prevention comprised checking the order history and patient's medical record, and a detailed knowledge of chemotherapy protocols. Of all the errors analyzed, 61% would be avoided through further software development. The improvements identified are implemented through a validated next-generation CPOE tool. CONCLUSIONS: The upgraded CPOE tool can be shared across other hospitals to raise safety standards and spread potential benefits across a wider patient population. The current analysis also highlighted that approximately 30% to 40% of errors cannot be avoided electronically. Therefore, pharmacovigilance initiatives remain indispensable.

Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: towards the design of photoswitchable sirtuin inhibitors.

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end-to-end distance. The resulting affinity shifts are intended to enable real-time photomodulation of sirtuins in vitro.