Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity

Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivatives of fumagillin have shown promise in clinical studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivatives has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin’s molecular scaffold but have improved pharmacological profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochemical enzymatic assays against three MetAP isoforms. Inhibitors with the same relative and absolute stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallographic analysis revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochemically active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clinically investigated fumagillin derivative beloranib

Additional file 3: Figure S2. of Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma

ALDH1A2 expression in HNSCC cell lines and morphological phenotype upon inhibition of ALDH1A2-RAR signaling. Western blot analysis with whole cell lysate demonstrates protein expression of ALDH1A2 and key regulators of RA signaling in UMSCC-17B, Detroit562, FaDu, Cal27 and SCC25 cells (A), and ALDH1A2 protein levels in newly established HNSCC cell lines from Lausanne (B). Detection of β-Actin served as control for quantity and quality of protein lysates. Relative RA levels were determined by HPLC analysis with whole cell lysate of untreated (C), and DMSO-treated control or WIN18.446-treated Cal27 and FaDu cells (D). Bars represent mean values ¹ SD of three independent replicates. Graphs indicate relative survival fraction of Cal27 and FaDu cells, which were treated with the indicated concentration of Adapalene (E) or Fenretinide (F). Data represent mean values ¹ SD of three independent replicates. (TIF 301 kb

Additional file 1: Figure S1. of Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma

Association between subgroups with high or low protein expression of CRABP2 (A), FABP5 (B), RARα (C), RARβ (D) or PPARα/δ (E) and overall survival of OPSCC patients was assessed by univariate Kaplan-Meier analysis. (F) Kaplan-Meier analysis demonstrates overall survival probability for subgroups with indicated staining patterns for ALDH1A2, CRABP2 and FABP5. Number at risk indicates the total amount of patients per subgroup, which were alive and not censored at the indicated time points and were considered to calculate the overall survival probability. P values were calculated by log-rank tests. (TIF 1257 kb

Additional file 5: Figure S4. of Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma

(A) Representative phase contrast pictures of FaDu, Detroit562 and UMSCC-17B cells, which were treated with DMSO, 3 μM WIN18.446 or 3 μM BMS493 for four days. (B) Representative fluorescent pictures of FaDu and Detroit562 cells, which were treated as described in (A), and were stained with Phalloidin-Alexa488 (green signal). Nuclear staining was done with H33342 (blue signal). Migration of Detroit562 (C) and FaDu cells (D), which were treated with DMSO (white bars), 3 μM WIN18.446 (grey bars) or 3 μM BMS493 (black bars), in a scratch wounding assay was determined by the relative gap closure at the indicated time points. * p value ≤ 0.05. (TIF 4129 kb

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

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

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

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

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

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