Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Histone deacetylase-10 liberates spermidine to support polyamine homeostasis and tumor cell growth.

Cytosolic histone deacetylase-10 (HDAC10) specifically deacetylates the modified polyamine N8-acetylspermidine (N8-AcSpd). Although intracellular concentrations of N8-AcSpd are low, extracellular sources can be abundant, particularly in the colonic lumen. Extracellular polyamines, including those from the diet and microbiota, can support tumor growth both locally and at distant sites. However, the contribution of N8-AcSpd in this context is unknown. We hypothesized that HDAC10, by converting N8- AcSpd to spermidine, may provide a source of this growth-supporting polyamine in circumstances of reduced polyamine biosynthesis, such as in polyamine-targeting anticancer therapies. Inhibitors of polyamine biosynthesis, including α-difluoromethylornithine (DFMO), inhibit tumor growth, but compensatory uptake of extracellular polyamines has limited their clinical success. Combining DFMO with inhibitors of polyamine uptake have improved the antitumor response. However, acetylated polyamines may use different transport machinery than the parent molecules. Here, we use CRISPR/Cas9-mediated HDAC10-knockout cell lines and HDAC10-specific inhibitors to investigate the contribution of HDAC10 in maintaining tumor cell proliferation. We demonstrate inhibition of cell growth by DFMO-associated polyamine depletion is successfully rescued by exogenous N8-AcSpd (at physiological concentrations), which is converted to spermidine and spermine, only in cell lines with HDAC10 activity. Furthermore, we show loss of HDAC10 prevents both restoration of polyamine levels and growth rescue, implicating HDAC10 in supporting polyamine-associated tumor growth. These data suggest the utility of HDAC10-specific inhibitors as an antitumor strategy that may have value in improving the response to polyamine-blocking therapies. Additionally, the cell-based assay developed in this study provides an inexpensive, high-throughput method of screening potentially selective HDAC10 inhibitors.

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout.

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

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.

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R-S-S-Alk) rapidly convert into the corresponding thioethers (R-S-Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N-t-butyl-iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R-S-S-Alk) to tautomerize into the thiosulfoxide (R-(S=S)-Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

A Validated "Pool and Split" Approach to Screening and Optimization of Copper-Catalyzed C-N Cross-Coupling Reactions.

A general method for the quick identification of effective catalytic systems for copper-catalyzed C-N cross-couplings is described. This is based on evaluating mixtures of copper sources, ancillary ligands, and bases in different solvents followed by two deconvolution procedures, which aim at identifying the most proper reagent combination in only three distinct steps. Despite being a high-throughput approach in nature, the proposed method utilizes only frugal technological platforms such as 24-well microplates while offering a screening efficiency-the number of executed experiments vs the total number of possible experiments-higher than 95%. To facilitate visualization and mining of the high-throughput experimentation (HTE) data, Visual Basic scripts have been developed, which allow streamlining the extraction of raw HPLC data into TIBCO Spotfire for the graphical display in the form of pie charts. The unique capabilities of this "pool and split" approach have been demonstrated by applying it to literature known cross-coupling reactions. In every case, the described experimental setup was validated by retrieving the original literature conditions in addition to exposing several additional solutions with a minimum number of parallel experiments. Finally, examples are provided for the successful application of this HTE screening workflow to internal projects.

Direct Imaging of Protein-Specific Methylation in Mammalian Cells.

Abundant post-translational modification through methylation alters the function, stability, and/or localization of a protein. Malfunctions in post-translational modification are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now, their usage was limited to cell lysates. Herein, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and FRET detected by means of fluorescence lifetime imaging microscopy. Through this approach, methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines has been successfully imaged. To further demonstrate its potential, the localization-dependent methylation state of Foxo1 in the cellular context has been visualized.

Multimechanism biological profiling of tetrahydro-ß-carboline analogues as selective HDAC6 inhibitors for the treatment of Alzheimer's disease.

With the intensive research on the pathogenesis of Alzheimer's disease (AD), inhibition of HDAC6 appears to be a potential therapeutic approach for AD. In this paper, a series of tetrahydro-ß-carboline derivatives with hydroxamic acid group were fast synthesized. Among all, the most potent 15 selectively inhibited HDAC6 with IC50 of 15.2 nM and markedly increased acetylated alpha-tubulin levels. In cellular assay, 15 showed excellent neurotrophic effect by increasing the expression of GAP43 and Beta-3 tubulin markers. Besides, 15 showed neuroprotective effects in PC12 or SH-SY5Y cells against H2O2 and 6-OHDA injury through activation of Nrf2, catalase and Prx II, and significantly reduced H2O2-induced reactive oxygen species (ROS) production. In vivo, 15 significantly attenuated zebrafish anxiety-like behaviour and memory deficits in a SCOP-induced zebrafish model of AD. To sum up, multifunctional 15 might be a good lead to develop novel tetrahydrocarboline-based agents for the treatment of AD.

Decrypting lysine deacetylase inhibitor action and protein modifications by dose-resolved proteomics.

Lysine deacetylase inhibitors (KDACis) are approved drugs for cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and multiple myeloma, but many aspects of their cellular mechanism of action (MoA) and substantial toxicity are not well understood. To shed more light on how KDACis elicit cellular responses, we systematically measured dose-dependent changes in acetylation, phosphorylation, and protein expression in response to 21 clinical and pre-clinical KDACis. The resulting 862,000 dose-response curves revealed, for instance, limited cellular specificity of histone deacetylase (HDAC) 1, 2, 3, and 6 inhibitors; strong cross-talk between acetylation and phosphorylation pathways; localization of most drug-responsive acetylation sites to intrinsically disordered regions (IDRs); an underappreciated role of acetylation in protein structure; and a shift in EP300 protein abundance between the cytoplasm and the nucleus. This comprehensive dataset serves as a resource for the investigation of the molecular mechanisms underlying KDACi action in cells and can be interactively explored online in ProteomicsDB.

Tetrahydro-ß-carboline derivatives as potent histone deacetylase 6 inhibitors with broad-spectrum antiproliferative activity.

A series of tetrahydro-ß-carboline (THßC)-based hydroxamic acids were rationally designed and synthesized as novel selective HDAC6 inhibitors (sHDAC6is) by the application of scaffold hopping strategy. Several THßC analogues were highly potent (IC50 < 5 nM) and selective against HDAC6 enzyme and exhibited good antiproliferative activity against human multiple myeloma (MM) cell. Molecular docking interpreted the structure activity relationship (SAR). Target engagement of HDAC6 was confirmed in RPMI-8226 cells using the WB assay. In vitro, (1S, 3R)-1-(4-chlorophenyl)-N-(4-(hydroxycarbamoyl)benzyl)-2,3,4,9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxamide (14g) showed potent broad antiproliferative activity against various tumors including leukemia, colon cancer, melanoma, and breast cancer cell lines, better than ACY-1215. Moreover, 14g also showed good pharmacokinetics properties in mice via oral administration.

Chemoproteomic target deconvolution reveals Histone Deacetylases as targets of (R)-lipoic acid.

Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic (R/S)-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, (R/S)-lipoic acid is an approved drug for the treatment of diabetic neuropathy. However, its mechanism of action remains elusive. Here, we performed chemoproteomics-aided target deconvolution of lipoic acid and its active close analog lipoamide. We find that histone deacetylases HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC10 are molecular targets of the reduced form of lipoic acid and lipoamide. Importantly, only the naturally occurring (R)-enantiomer inhibits HDACs at physiologically relevant concentrations and leads to hyperacetylation of HDAC substrates. The inhibition of HDACs by (R)-lipoic acid and lipoamide explain why both compounds prevent stress granule formation in cells and may also provide a molecular rationale for many other phenotypic effects elicited by lipoic acid.

Structural Basis for the Selective Inhibition of HDAC10, the Cytosolic Polyamine Deacetylase.

The cytosolic class IIb histone deacetylase HDAC10 is an emerging target for drug design. As an inducer of autophagy, its selective inhibition suppresses the autophagic response that otherwise attenuates the efficacy of cytotoxic cancer chemotherapy drugs. HDAC10 is a zinc-dependent polyamine deacetylase exhibiting maximal catalytic activity against N8-acetylspermidine. As revealed in the structure of Danio rerio (zebrafish) HDAC10, two conserved structural motifs direct this narrow substrate specificity: a 310 helix containing the P(E,A)CE motif that sterically constricts the active site and an electrostatic "gatekeeper," E274, that confers selectivity for cationic polyamine substrates. To accelerate drug design efforts targeting human HDAC10, we now report the preparation of "humanized" zebrafish HDAC10 in which two amino acid substitutions, A24E and D94A, yield an active site contour more similar to that of human HDAC10. X-ray crystal structures of this HDAC10 variant complexed with Tubastatin A and indole analogues bearing pendant tertiary amines reveal that inhibitors capable of hydrogen bonding with gatekeeper E274 exhibit high affinity and selectivity for HDAC10 over HDAC6 (the other class IIb isozyme). Moreover, these structures reveal that the P(E,A)CE motif helix can shift by up to 2 Å to accommodate the binding of bulky inhibitors. Thus, slender polyamine-like inhibitor structures are not exclusively required for selective, high affinity binding to HDAC10. Indeed, the flexibility of the P(E,A)CE motif helix could conceivably enable the binding of certain protein substrates.

Novel 2, 5-diketopiperazine derivatives as potent selective histone deacetylase 6 inhibitors: Rational design, synthesis and antiproliferative activity.

Histone deacetylase 6 (HDAC6) has gained popular attention for its wide participation in various pathological process recently. In this paper, a series of novel derivatives containing 2, 5-diketopiperazine (DKP) skeleton were developed as potent selective HDAC6 inhibitors (sHDAC6is). Most of these compounds exhibited low nanomolar IC50 values toward HDAC6, and the best compound was 21b (IC50 = 0.73 nM) which had 144-10941-fold selectivity over other HDAC isoforms. Western blot assay further validated these compounds to be sHDAC6is. Molecular simulation of 21b was conducted to rationalize the high binding affinity for HDAC6. In the cytotoxicity experiment, 18a, 18b and 18d gave superior or comparable influence on the growth of two multiple myeloma cells U266 and RPMI-8226 compared to ACY-1215. Moreover, the combination of 18a and adriamycin showed synergistic effect against non-small cell lung cancer cell A549. 18a and 18b also demonstrated appropriate drug metabolism in human liver microsome (HLM).

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors.

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC50 value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.

Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated.

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.