Simplified adenine base editors improve adenine base editing efficiency in rice

Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE‐P1S (Adenine Base Editor‐Plant version 1 Simplified) containing the ecTadA*7.10‐nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE‐P1 consisting of the ecTadA‐ecTadA*7.10‐nSpCas9 (D10A) fusion. We found that the protein expression level of ABE‐P1S is higher than that of ABE‐P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE‐P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10‐nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH‐Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.     

Alpha-mercaptoketone based histone deacetylase inhibitors

In an effort to discover novel non-hydroxamic acid histone deacetylase (HDAC) inhibitors, a novel alpha-mercaptoketone was identified in a high-throughput screen. Lead optimization of the screening hit, led to a number of potent HDAC inhibitors. In particular, alpha-mercaptoketone 19y (KD5150) exhibited nanomolar in vitro activity and inhibition of tumor growth in vivo.     

Design of novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.     

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5–10 mM) and nicotinamide (1–5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants.     

Sulfamides as novel histone deacetylase inhibitors

The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted in potent HDAC1 and HDAC6 inhibitors.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.     

Fluoroalkene modification of mercaptoacetamide-based histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.     

Discovery of histone deacetylase 8 selective inhibitors

We have developed an efficient method for synthesizing candidate histone deacetylase (HDAC) inhibitors in 96-well plates, which are used directly in high-throughput screening. We selected building blocks having hydrazide, aldehyde and hydroxamic acid functionalities. The hydrazides were coupled with different aldehydes in DMSO. The resulting products have the previously identified ‘cap/linker/biasing element’ structure known to favor inhibition of HDACs. These compounds were assayed without further purification. HDAC8-selective inhibitors were discovered from this novel collection of compounds.     

Trifluoromethyl ketones as inhibitors of histone deacetylase

Trifluoromethyl ketones were found to be inhibitors of histone deacetylases (HDACs). Optimization of this series led to the identification of submicromolar inhibitors such as 20 that demonstrated antiproliferative effects against the HT1080 and MDA 435 cell lines.     

Alpha-keto amides as inhibitors of histone deacetylase

Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.     

Zinc binding groups for histone deacetylase inhibitors

Zinc binding groups (ZBGs) play a crucial role in targeting histone deacetylase inhibitors (HDACIs) to the active site of histone deacetylases (HDACs), thus determining the potency of HDACIs. Due to the high affinity to the zinc ion, hydroxamic acid is the most commonly used ZBG in the structure of HDACs. An alternative ZBG is benzamide group, which features excellent inhibitory selectivity for class I HDACs. Various ZBGs have been designed and tested to improve the activity and selectivity of HDACIs, and to overcome the pharmacokinetic limitations of current HDACIs. Herein, different kinds of ZBGs are reviewed and their features have been discussed for further design of HDACIs.     

Amino acid derivatives as histone deacetylase inhibitors

Ongoing clinical studies indicate that inhibitors of Class I and Class II histone deacetylase (HDAC) enzymes show great promise for the treatment of cancer. Zolinza (SAHA, Zolinza) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma. As a part of an ongoing effort to identify novel small molecules to target these important enzymes, we have prepared several classes of amino acid-derived HDAC1 inhibitors. The design rationale and in vitro activity against the HDAC1 enzyme and HCT116 cell line are described in this letter.     

Chlamydocin-hydroxamic acid analogues as histone deacetylase inhibitors

Chlamydocin-hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloalkanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin-hydroxamic acid analogue and compared it with the solution structure of chlamydocin.     

Mercaptoamide-based non-hydroxamic acid type histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. A mercaptoamide functionality was designed as a bidentate zinc chelator and incorporated into the hydroxamic acid based SAHA (1) scaffold in order to identify non-hydroxamate compounds as potential inhibitors of histone deacetylases. Two sets of mercaptoamides 2 and 3 with varying spacer length were synthesized and their HDAC inhibitory activity was evaluated. Low micromolar inhibition was observed for mercaptoamides 2e, 3b, and 3d.     

组蛋白去乙酰化酶抑制剂(HDAIs)对异染色质的作用

组蛋白乙酰化和去乙酰化可调节染色体的多种功能,例如基因表达和染色体分离等。研究发现,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors,HDACIs)可诱导分化、生长阻断和肿瘤细胞凋亡,目前HDACIs正作为抗肿瘤药物进行临床试验,在肿瘤治疗中显示出具有较好的应用前景。然而,人们对于HDACIs在生物体内是如何发挥作用以及不同类型细胞为何会有不同的应答途径却关注甚少。本综述通过讨论HDACIs对周期和非周期细胞中组蛋白去乙酰化酶的抑制结果,来阐明组蛋白乙酰化模式的动力学特征,特别是对基因组异染色质的作用。     

Design and synthesis of phthalimide-type histone deacetylase inhibitors

Several hydroxamic acid derivatives with a substituted phthalimide group as a linker and/or cap structure, prepared during structural development studies based on thalidomide, were found to have histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that nature of the substituent introduced at the phthalimide nitrogen atom, introduction of a hydroxamic acid structure, and distance between the N-hydroxyl group and the cap structure are important for HDAC-inhibitory activity.