Identification of 2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-N-phenylpropanamides as a novel class of potent DprE1 inhibitors

The identification of a novel series of DprE1 inhibitors based on a 2-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)amino)-N-phenylpropanamide scaffold is described herein. SAR exploration around the HTS hit 1 led to the identification of multiple analogues with potent DprE1 inhibition and good whole-cell antimycobacterial activity.     

Design,synthesis, and evaluation of a novel macrocyclic anti-EV71 agent

We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a potent agent targeting enterovirus A71 (EV71). The compound has a 15-membered macrocyclic ring in a defined conformation. Yamaguchi esterification reaction was used to close the 15-membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction. The crystallographic characterization of the complex between this compound and its target, 3C protease from EV71, validated the design and paved the way for the generation of a new series of anti-EV71 agents.     

Rational design,synthesis and biological profiling of new KDM4C inhibitors

The human histone demethylases of the KDM4 family have been related to diseases such as prostate and breast cancer. Majority of currently known inhibitors suffer from the low permeability and low selectivity between the enzyme isoforms. In this study, toxoflavin motif was used to design and synthesize new KDM4C inhibitors with improved biological activity and in vitro ADME properties. Inhibitors displayed good passive cellular permeability and metabolic stability. However, diminishing of redox liability and consequently non-specific influence on cell viability still remains a challenge.     

A bicyclic pentapeptide-based highly potent and selective pan-SIRT1/2/3 inhibitor harboring Nε-thioacetyl-lysine

Past few years have seen an active pursuit of the inhibitors for the deacylation catalyzed by the seven human sirtuins (i.e. SIRT1-7) as valuable chemical biological/pharmacological probes of this enzymatic deacylation and lead compounds for developing novel therapeutics for human diseases. In the current study, we prepared eight monocyclic and one bicyclic analogs of a linear pentapeptide-based potent (sub-μM IC₅₀’s) pan-SIRT1/2/3 inhibitor Zheng laboratory discovered recently that harbors the catalytic mechanism-based SIRT1/2/3 inhibitory warhead N^ε-thioacetyl-lysine at its central position. We found that the bicyclic analog exhibited largely comparable SIRT1/2/3 inhibitory potencies to those of the parent linear pentapeptide, however, the former is proteolytically much more stable than the latter. Moreover, the bicyclic analog displayed very weak inhibition against SIRT5/6/7, was cell permeable, and exhibited an anti-proliferative effect on the human SK-MEL-2 melanoma cells. This bicyclic analog could be a lead for the future development of more potent and still selective pan-SIRT1/2/3 inhibitors whose use in studies on human sirtuin biology, pharmacology, and medicinal chemistry could complement with the use of the potent inhibitors selective for a single human sirtuin.     

Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II,a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.     

Proteinaceous Inhibitor Versus Fructose as Modulators of Pteris deflexa Invertase Activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer M r 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had β -fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with K m of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

Why minimal is not optimal: Driving the mammalian cell cycle—and drug discovery—with a physiologic CDK control network

Progression through the eukaryotic cell division cycle is governed by the activity of cyclin-dependent kinases (CDKs). For a CDK to become active it must (1) bind a positive regulatory subunit (cyclin) and (2) be phosphorylated on its activation (T) loop. In metazoans, multiple CDK catalytic subunits, each with a distinct set of preferred cyclin partners, regulate the cell cycle, but it has been difficult to assign functions to individual CDKs in vivo. Biochemical analyses and experiments with dominant-negative alleles suggested that specific CDK/cyclin complexes regulate different events, but genetic loss of interphase CDKs (Cdk2, -4 and -6), alone or in combination, did not block proliferation of cells in culture. These knockout and knockdown studies suggested redundancy or plasticity built into the CDK network but did not address whether there was true redundancy in normal cells with a full complement of CDKs. Here, we discuss recent work that took a chemical-genetic approach to reveal that the activity of a genetically non-essential CDK, Cdk2, is required for cell proliferation when normal cyclin pairing is maintained. These results have implications for the systems-level organization of the cell cycle, for regulation of the restriction point and G?/S transition and for efforts to target Cdk2 therapeutically in human cancers.     

HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

Metallo-β-lactamase: Inhibitors and reporter substrates

Metallo-β-lactamases represent an emerging clinical threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biological imaging. Despite progress in molecular probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design.