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HDAC11-IN-1 (Compound 14-NC6OH) TFA 是一种选择性 HDAC11 的大环抑制剂，Ki 为 40 nM。HDAC11-IN-1 TFA 具有良好的细胞穿透性且能抑制 YAP1 和 SOX2 的表达。

组蛋白去乙酰化酶（HDAC）是一类表观遗传调控因子，通过去除整个蛋白质组中赖氨酸侧链上的酰基来影响多种蛋白质功能。最新发现的人类同工酶HDAC11在底物偏好性和组织表达谱方面与其他HDAC存在差异。目前关于该酶生物学功能的阐释甚少，迄今为止仅开发出少数几种有助于推进相关研究的化学探针。本研究通过氯烷渗透实验发现，大环抑制剂对HDAC11具有选择性，并能穿透培养细胞的细胞质膜。我们的工作证实，将全新大环合成与N-烷基化羟肟酸基团相结合，是靶向HDAC11的有效策略。此外，这项研究展示了应用大环肽库合成直接制备高亲和力、细胞渗透性配体的潜力。所发现的抑制剂为研究HDAC11的生物学功能提供了工具化合物。

组蛋白去乙酰化酶11（HDAC11）是唯一一种依赖Zn2+的HDAC酶，能高效去除赖氨酸残基侧链上的长链酰基修饰ε-N-肉豆蔻酰赖氨酸（Kmyr）[对Kmyr的选择性超过ε-N-乙酰赖氨酸（Kac）10,000倍以上]。该同工酶是人类HDAC家族第四类中的唯一成员，主要由一个催化结构域和较短的N端、C端组成。4HDAC11在物种间高度保守，但与大多数其他HDAC不同，它并非在所有组织中普遍表达，主要存在于大脑、睾丸和骨骼肌中。HDAC11在免疫反应中发挥关键作用， 作为关键抗炎细胞因子IL-10的转录抑制因子发挥作用。此外，HDAC11基因敲除小鼠在高脂饮食下能抵抗体重增加，并表现出更好的整体代谢健康，表明HDAC11可能成为代谢性疾病的潜在治疗靶点。8 最近研究表明，抑制HDAC11是巨噬细胞活化的一种策略，凸显了其在炎症治疗和适应性细胞疗法中的治疗潜力。

HDAC11的底物结合口袋中含有一个可被不同锌结合基团靶向的催化性Zn2+离子，这构成了多种HDAC抑制剂的设计基础。天然存在的大环肽类（如阿匹西丁A、特拉毒素A和B）通常含有锌结合基团，是I类HDAC的高效抑制剂。研究显示，这些环肽的特拉毒素及其含羟肟酸类似物（如阿匹西丁AAsuha）也能强效抑制HDAC11，但仍保持对I类HDAC的选择性1。为实现对HDAC11的选择性抑制，我们设想在锌结合部分引入烷基将更易被HDAC11活性位点容纳——该位点能高效催化Kmyr的水解。类似策略曾报道用于开发基于N-烷基化酰肼（如SIS17）的HDAC11选择性小分子抑制剂，最近还应用于修饰特拉毒素A的环氧酮部分以提升对HDAC11的选择性（TD034）。然而羟肟酸的N-烷基化修饰尚未被用于HDAC11选择性研究，尽管已有报道表明该修饰会降低对I类HDACs的亲和力。

研究采用两种不同策略开发靶向细胞内HDAC11的新型工具化合物，以探究该酶的生物学功能。我们首先尝试以天然产物衍生的优势肽大环化合物为起点，通过引入N-烷基化羟肟酸基团作为锌结合基团，虽获得了高效力但HDAC11选择性有限。随后我们将这些锌结合基团与全新高通量大环化合物合成及HDAC11筛选相结合，最终获得了强效、选择性高且具细胞渗透性的酶抑制剂。实验数据表明，N-烷基化羟肟酸基序可作为通用HDAC11靶向基序，但更重要的是发现大环结构对结合亲和力的贡献决定了选择性程度。筛选获得的先导大环化合物（经最小修饰的类似物14-NCAOH）在CAPA实验中显示出细胞渗透性。此外研究发现，非烷基化羟肟酸类似物（14-NHOH）能增加组蛋白乙酰化水平，而14-NC6OH则能提升培养细胞中HDAC11调控的YAP1和SOX2表达——这些发现与重组酶筛选结果及小鼠脑裂解液中酶活性记录的选择性特征相符。

参考文献

Danková D, et al. Discovery of De Novo Macrocycle Inhibitors of Histone Deacetylase 11. JACS Au. 2025 Feb 16;5(3):1299-1307.  [Content Brief]

定义
酶是用于生化反应的非常有效的催化剂。它们通过提供较低活化能的替代反应途径来加快反应速度。酶作用于底物并产生产物。一些物质降低或什至停止酶的催化活性被称为抑制剂。
发现
1965年，Umezawa H分析了微生物产生的酶抑制剂，并分离出了抑制亮肽素和抗痛药的胰蛋白酶和木瓜蛋白酶，乳糜蛋白酶抑制的胰凝乳蛋白酶，胃蛋白酶抑制素抑制胃蛋白酶，泛磷酰胺抑制唾液酸酶，乌藤酮抑制酪氨酸羟化酶，多巴汀抑制多巴胺3-羟硫基嘧啶和多巴胺3-羟色胺酶酪氨酸羟化酶和多巴胺J3-羟化酶。最近，一种替代方法已应用于预测新的抑制剂：合理的药物设计使用酶活性位点的三维结构来预测哪些分子可能是抑制剂1。已经开发了用于识别酶抑制剂的基于计算机的方法，例如分子力学和分子对接。
结构特征
已经确定了许多抑制剂的晶体结构。已经确定了三种与凝血酶复合的高效且选择性的低分子量刚性肽醛醛抑制剂的晶体结构。这三种抑制剂全部在P3位置具有一个新的内酰胺部分，而对胰蛋白酶选择性最高的两种抑制剂在P1位置具有一个与S1特异性位点结合的胍基哌啶基。凝血酶的抑制动力学从慢到快变化，而对于胰蛋白酶，抑制的动力学在所有情况下都快。根据两步机理2中稳定过渡态络合物的缓慢形成来检验动力学。
埃米尔•菲舍尔（Emil Fischer）在1894年提出，酶和底物都具有特定的互补几何形状，彼此恰好契合。这称为“锁和钥匙”模型3。丹尼尔·科什兰（Daniel Koshland）提出了诱导拟合模型，其中底物和酶是相当灵活的结构，当底物与酶4相互作用时，活性位点通过与底物的相互作用不断重塑。
在众多生物活性肽的成熟过程中，需要由其谷氨酰胺（或谷氨酰胺）前体形成N末端焦谷氨酸（pGlu）。游离形式并与底物和三种咪唑衍生抑制剂结合的人QC的结构揭示了类似于两个锌外肽酶的α/β支架，但有多个插入和缺失，特别是在活性位点区域。几种活性位点突变酶的结构分析为针对QC相关疾病5的抑制剂的合理设计提供了结构基础。
作用方式
酶是催化化学反应的蛋白质。酶与底物相互作用并将其转化为产物。抑制剂的结合可以阻止底物进入酶的活性位点和/或阻止酶催化其反应。抑制剂的种类繁多，包括：非特异性，不可逆，可逆-竞争性和非竞争性。可逆抑制剂 以非共价相互作用（例如疏水相互作用，氢键和离子键）与酶结合。非特异性抑制方法包括最终使酶的蛋白质部分变性并因此不可逆的任何物理或化学变化。特定抑制剂 对单一酶发挥作用。大多数毒药通过特异性抑制酶发挥作用。竞争性抑制剂是任何与底物的化学结构和分子几何结构非常相似的化合物。抑制剂可以在活性位点与酶相互作用，但是没有反应发生。非竞争性抑制剂是与酶相互作用但通常不在活性位点相互作用的物质。非竞争性抑制剂的净作用是改变酶的形状，从而改变活性位点，从而使底物不再能与酶相互作用而产生反应。非竞争性抑制剂通常是可逆的。不可逆抑制剂与酶形成牢固的共价键。这些抑制剂可以在活性位点附近或附近起作用。
功能
工业应用中， 酶在商业上被广泛使用，例如在洗涤剂，食品和酿造工业中。蛋白酶用于“生物”洗衣粉中，以加速蛋白质在诸如血液和鸡蛋等污渍中的分解。商业上使用酶的问题包括：它们是水溶性的，这使得它们难以回收，并且一些产物可以抑制酶的活性（反馈抑制）。
药物分子，许多药物分子都是酶抑制剂，药用酶抑制剂通常以其特异性和效力为特征。高度的特异性和效力表明该药物具有较少的副作用和较低的毒性。酶抑制剂在自然界中发现，并且也作为药理学和生物化学的一部分进行设计和生产6。
天然毒物 通常是酶抑制剂，已进化为保护植物或动物免受天敌的侵害。这些天然毒素包括一些已知最剧毒的化合物。
神经气体（ 例如二异丙基氟磷酸酯（DFP））通过与丝氨酸的羟基反应生成酯，从而抑制了乙酰胆碱酯酶的活性位点。
参考
1、Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.
2、Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.
3、Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.
4、Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.
5、Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.
6、Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

Definition
Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction pathway of lower activation energy. Enzyme acts on substrate and gives rise to a product. Some substances reduce or even stop the catalytic activities of enzymes are called inhibitors.

Discovery
In 1965, Umezawa H analysed enzyme inhibitors produced by microorganisms and isolated leupeptin and antipain inhibiting trypsin and papain, chymostatin inhibiting chymotrypsin, pepstatin inhibiting pepsin, panosialin inhibiting sialidases, oudenone inhibiting tyrosine hydroxylase, dopastin inhibiting dopamine 3-hydroxylase, aquayamycin and chrothiomycin inhibiting tyrosine hydroxylase and dopamine J3-hydroxylase . Recently, an alternative approach has been applied to predict new inhibitors: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors 1. Computer-based methods for identifying inhibitor for an enzyme have been developed, such as molecular mechanics and molecular docking.

Structural Characteristics
The crystal structures of many inhibitors have been determined. The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined. All the three inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism 2.

Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model 3. Daniel Koshland suggested induced fit model where substrate and enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 4.

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The structural analyses of several active-site-mutant enzymes provide a structural basis for the rational design of inhibitors against QC-associated disorders 5.

Mode of Action
Enzymes are proteins that catalyze chemical reactions. Enzymes interact with substrate and convert them into products. Inhibitor binding can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. Reversible inhibitors bind to enzymes with non-covalent interactions like hydrophobic interactions, hydrogen bonds, and ionic bonds. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible. Specific Inhibitors exert their effects upon a single enzyme. Most poisons work by specific inhibition of enzymes. A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site.  The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible. Irreversible Inhibitors form strong covalent bonds with an enzyme.  These inhibitors may act at, near, or remote from the active site .

Functions
Industrial application, enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in 'biological' washing powders to speed up the breakdown of proteins in stains like blood and egg. Problems using enzymes commercially include: they are water soluble which makes them hard to recover and some products can inhibit the enzyme activity (feedback inhibition) .

Drug molecules, many drug molecules are enzyme inhibitors and a medicinal enzyme inhibitor is usually characterized by its specificity and its potency. A high specificity and potency suggests that a drug will have fewer side effects and less toxic. Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry 6.

Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known.

Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.

References

Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.

Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.

Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.

Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.

Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.