Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1)

Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells

Chemical structures of Largazole, synthetic analogs, and Trichostatin A.

<p>Largazole (L) includes a substituted 4-methythiazoline linearly fused to a thiazole, a 3-hydroxy-7-mercaptohept-4-enoic acid, a thioester moiety, and a hydrocarbon tail. Analogs include a substituted ketone (K) and ester (E) in place of the thioester moiety, a macrocycle lacking the thioester moiety and hydrocarbon tail (M), an analog containing a macrocycle broken at carbon-3 of the enoic acid (S), and a thiol analog lacking the thioester moiety (T). Trichostatin A (TSA) contains a hydroxamic acid functional group.</p

Investigation into the selectivity of Largazole ketone.

<p>(A) Largazole ketone (K) fails to inhibit the ligation of ubiquitin onto Uba1p, a homologue of UBA1 from <i>S. pombe</i>. Formation of Uba1p-ubiquitin adducts was determined by thioester assay utilizing fluorescein-ubiquitin. Uba1p (1.03 µM) was incubated with either DMSO or various concentrations of K serially diluted from 1000 µM to 31 µM. (B) K inhibits ligation of SUMO-1 onto human SUMO E1 in a concentration-dependent fashion. Reduction of E1-SUMO adducts was determined by thioester assay utilizing fluorescein-SUMO-1. hSUMO E1 (500 nM) was incubated with either DMSO or various concentrations of K serially diluted from 1000 µM to 31 µM.</p

Largazole (L) and largazole ester (E) inhibit ubiquitin E1 in a dose dependent manner <i>in vitro</i>.

<p>(A,C) L and E inhibit transfer of ubiquitin onto E1 in a concentration-dependent manner. Thioester assay of E1 activity using fluorescein ubiquitin (Ub-F). Thioester bond formation between E1 and Ub-F is ATP-dependent (lane 2 vs. lane 1). In addition, DMSO has no effect on the formation of the thioester linkage as seen in lane 2 of both gels. 50 nM E1 was incubated with decreasing concentrations of L (A) or E (C) for 15 minutes at room temperature followed by addition of a cocktail containing ATP and Ub-F. After 5 minutes of incubation, the reactions were resolved by SDS-PAGE under non-reducing conditions. Ub-F was used to show equal loading. (B,D) Thioester assay of the ubiquitin transfer from E1 to E2 (Cdc34). Largazole or Largazole ester, when preincubated with 50 nM E1 for 15 minutes, inhibit the transfer of ubiquitin from E1 to Cdc34 in a concentration-dependent manner. (E) Largazole selectively inhibits the activity of E1 not E2. 50 nM E1 was pre-charged with ATP and then added to Cdc34 that was previously incubated with decreasing concentrations (1 mM–16 µM) of L in thioester reaction mixture. (F) Largazole ester inhibits E2 at high concentrations. Pre-charged E1 was added to reactions that contained Cdc34 pre-incubated with E ranging from 1 mM to 16 µM and resolved by SDS-PAGE under non-reducing conditions. Complete inhibition of ubiquitin transfer to E2 was observed at 1 mM of E, with only modest inhibition at 500 µM. (G) Largazole thiol (T) has no effect on transfer of ubiquitin onto E1. The reaction was carried out as described in A,C.</p

Largazole stabilizes p27 expression in Kip16 cells and inhibits p27 ubiquitination <i>in vitro.</i>

<p>(A) Fluorescent and corresponding bright-field images of Kip16 cells treated with varying concentrations of Largazole (L). L treatment induces the expression of GFP-p27 in a dose-dependent fashion. Addition of MG132 (1 µM) prevents the degradation of GFP-p27 via the ubiquitination and subsequent proteasomal degradation pathway. The vehicle control, DMSO, has no effect on the reporter protein stabilization. (B) L fails to inhibit the phosphorylation of p27 by the Cdk2/CyclinE complex compared to the positive control. L (250 µM, lane 3, and 125 µM, lane 4) was incubated with the Cdk2/CyclinE complex prior to the autophosphorylation of Cdk2/CyclinE step. Phosphorylated-p27 was identified by protein standard. (C) L, K, and E reduce polyubiquitinated forms of p27 while M and S have no inhibitory effects. Ubiquitin-activating enzyme E1 (100 nM), UBA1, was incubated with 100 µM of each compound prior to the reaction. (D) E reduces polyubiquitinated forms of Trf1 in a dose-dependent fashion. UBA1 (100 nM) was incubated with either DMSO or various concentrations of E ranging from 250 µM to 1 µM prior to the reaction.</p

A Practical Synthesis of Indoles via a Pd-Catalyzed C–N Ring Formation

A method for the synthesis of <i>N</i>-functionalized C2-/C3-substituted indoles via Pd-catalyzed C–N bond coupling of halo-aryl enamines is described. The general strategy utilizes a variety of amines and β-keto esters which are elaborated into halo-aryl enamines as latent precursors to indoles. The preferred conditions comprising the RuPhos precatalyst and RuPhos in the presence of NaOMe in 1,4-dioxane tolerate a variety of substituents and are scalable for the construction of indoles in multigram quantities

Discovery and Optimization of Tetramethylpiperidinyl Benzamides as Inhibitors of EZH2

The identification and development of a novel series of small molecule Enhancer of Zeste Homologue 2 (EZH2) inhibitors is described. A concise and modular synthesis enabled the rapid development of structure–activity relationships, which led to the identification of <b>44</b> as a potent, SAM-competitive inhibitor of EZH2 that dose-dependently decreased global H3K27me3 in KARPAS-422 lymphoma cells

Discovery of Benzotriazolo[4,3‑<i>d</i>][1,4]diazepines as Orally Active Inhibitors of BET Bromodomains

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 <i>in vivo</i>, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo­[<i>b</i>]­isoxazolo­[4,5-<i>d</i>]­azepines and benzotriazolo­[4,3-<i>d</i>]­[1,4]­diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice