姜花属植物二萜成分研究进展

本文对姜花属植物二萜类成分及其药理活性的研究进行了综述。     

Phenanthridin-6-one derivatives as the first class of non-steroidal pharmacological chaperones for Niemann-Pick disease type C1 protein

Niemann-Pick disease type C is a fatal, progressive neurodegenerative disease mostly caused by mutations in Nieamnn-Pick type C1 (NPC1), a late endosomal membrane protein that is essential for intracellular cholesterol transport. The most prevalent mutation, I1061T (Ile to Thr), interferes with the protein folding process. Consequently, mutated but intrinsically functional NPC1 proteins are prematurely degraded via proteasome, leading to loss of NPC1 function. Previously, we reported sterol derivatives as pharmacological chaperones for NPC1, and showed that these derivatives can normalize folding-defective phenotypes of I1061T NPC1 mutant by directly binding to, and stabilizing, the protein. Here, we report a series of compounds containing a phenanthridin-6-one scaffold as the first class of non-steroidal pharmacological chaperones for NPC1. We also examined their structure-activity relationships.     

The discovery of benzoxazine sulfonamide inhibitors of NaV1.7: Tools that bridge efficacy and target engagement

The voltage-gated sodium channel Na_V1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of Na_V1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat Na_V1.7 versus human Na_V1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse Na_V1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.     

Oxidative transformation of tunichromes – Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains.     

Identification of cross‐reactive B‐cell epitopes between Bos d 9.0101(Bos Taurus) and Gly m 5.0101 (Glycine max) by epitope mapping MALDI‐TOF MS

Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross‐allergenicity described between soy and milk proteins. We have previously identified several cross‐reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1‐casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α‐casein‐specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross‐reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI‐TOF MS analysis. On a second approach, the peptide mixture was resolved by RP‐HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI‐TOF MS. This novel MS based approach led us to identify and characterize four peptides on α‐casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross‐reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross‐reactivity, to further develop new and more effective vaccines for food allergy.     

Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased k_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^?1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.     

Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.     

Sibutramine Treatment in Obesity: Predictors of Weight Loss Including Rorschach Personality Data

Objective: To study personality and clinical factors in weight loss by sibutramine (Meridia and Reductil), an anti‐obesity drug enhancing satiety. Research Methods and Procedures: The subjects were 30 obese patients [43 ± 12 years (mean ± SD), BMI 40 ± 4 kg/m²]. The treatment comprised 15 mg of sibutramine administered daily and monthly dietary advice. Weight loss after 6 months of treatment was evaluated. For psychological assessment, the Rorschach method (Comprehensive System) and the Beck Depression Inventory were used. Results: A multiple linear regression model including the Rorschach predictors’ physical demand states (animal movement, designated as FM) being intrusive or difficult to hold and a dependency orientation (food contents) could explain 47% of 6 months of weight loss. A model including initial weight loss in addition to the Rorschach predictors explained 58% of the 6‐month weight loss. Discussion: The personality factors predicted greater weight loss. In particular, patients with difficulties concerning physical demand states, which would include hunger, could have reduced their eating behavior with enhanced satiety, resulting in greater weight loss. Enhanced satiety could also have helped patients with a dependent need for food to limit food intake. Being enrolled in a treatment program could also have provided essential support for patients with dependency needs. Furthermore, initial weight loss was a predictor of greater weight loss in sibutramine treatment, in accordance with prior research.     

Field‐based comparison of ligand and coactivator binding sites of nuclear receptors

A structure‐based comparison of the ligand‐binding domains of 35 nuclear receptors from five different subfamilies is presented. Their ligand and coactivator binding sites are characterized using knowledge‐based contact preference fields for hydrophobic and hydrophilic interactions implemented in the MOE modeling environment. Additionally, for polar knowledge‐based field points the preference for negative or positive electrostatic interactions is estimated using the Poisson‐Boltzmann equation. These molecular‐interaction fields are used to cluster the nuclear receptor family based on similarities of their binding sites. By analyzing the similarities and differences of hydrophobic and polar fields in binding pockets of related receptors it is possible to identify conserved interactions in ligand and coactivator binding pockets, which support e.g. design of specific ligands during lead optimization or virtual screening as docking filter. Examples of remarkable similarities between ligand binding sites of members from phylogenetically different nuclear receptor families (RXR, RAR, HNF4, NR5) and differences between closely related subtypes (LXR, RAR, TR) are discussed in more detail. Significant similarities and differences of coactivator binding sites are shown for NR3Cs, LXRs and PPARs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 884–894, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com