Stevastelins,a novel group of immunosuppressants,inhibit dual-specificity protein phosphatases

Background: Since the molecular target of the immunosuppressive reagents FK506 and cyclosporin A was revealed to be protein phosphatase PP2B (calcineurin), many researchers have been screening the protein phosphatase inhibitors from microbial metabolites to develop new immunosuppressive reagents. We isolated stevastelin B, which is composed of valine, threonine, serine and 3,5-dihydroxy-2,4-dimethyl stearic acid, and stevastelin A, which is a sulphonylated derivative of stevastelin B. To understand the action mechanism of stevastelins A and B, we synthesized a series of stevastelin derivatives and investigated their structure-activity relationships.Results: A series of stevastelin derivatives have been systematically synthesized. Stevastelin B inhibited gene expression that is dependent on interleukin-2 (IL-2) or IL-6 promoters in situ, but it had no inhibitory activity against any protein phosphatases in vitro. In contrast, stevastelin A, which is a sulphonylated derivative of stevastelin B, inhibited the phosphatase activity of a dual-specificity phosphatase, VH1-related human protein (VHR), in vitro, but it had no inhibitory activity against gene expression or cell-cycle progression in situ.Conclusions: Stevastelin B is a novel immunosuppressant. It inhibited IL-2 or IL-6 dependent gene expression but did not inhibit the phosphatase activity of calcineurin. The structure-activity relationships show that the acidic functional group on the threonine residue and the stearic acid moiety in the stevastelin molecule are important for inhibitory effects on the dephosphorylation activity of VHR in vitro. Stevastelin B might be sulphonylated or phosphorylated after incorporation into the target cell, and then it interacts with protein tyrosine phosphatases and regulates cell-cycle progression.     

A dendritic cell-like biomimetic nanoparticle enhances T cell activation for breast cancer immunotherapy

Cancer immunotherapy has remarkably improved the therapeutic effect of melanoma and non-small cell lung cancer in the clinic. Nevertheless, it showed disappointing clinical outcomes for treating immunosuppressive tumors, wherein aggressive T cells are rather limited in tumor sites. Therefore, regulating the behavior of T cells in tumor sites to increase their attack ability for suppressing the immunosuppressive tumor is highly desirable. Inspiringly, we designed a dendritic cell-like biomimetic nanoparticle (DMSNs³@HA) to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors. In this work, anti-CD3 and anti-CD28 were responsible for mimicking dendritic cells to activate T cells, and anti-PD-1 for blocking the pathway of PD-1/PD-L1 to break the immune “brake”, which synergistically regulated the behavior of T cells to attack cancer cells. Experimental results indicated that DMSNs³@HA can effectively activate T cells and improve their immune response to significantly inhibit the growth of breast cancer. Moreover, it also proved that T cell activation combining immune checkpoint blocking induced the “1 + 1 >2” immunotherapy effect against immunosuppressive tumors. We expect that this strategy will provide new insights into tumor immunotherapy by modulating T cell behavior.

A dendritic cell-like biomimetic nanoparticle has been designed to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors.     

Synthesis and Immunosuppressive Activity of New Amino Alcohol Derivatives(Ⅰ)

A series of new amino alcohol derivatives was synthesized and evaluated for their immunosuppressive activity on mouse peripheral blood lymphocytes.The structures were confirmed by means of 1H NMR,13C NMR,IR and MS.Most of the compounds display moderate to potent inhibitory activity.Compound 9d shows the most activity among them that are expected as a powerful candidate for safer immunosuppressant for organ transplantations and the treatment of autoimmune diseases.     

Diketoacetonylphenalenone,Derived from Hawaiian Volcanic Soil-Associated Fungus Penicillium herquei FT729, Regulates T Cell Activation via Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathway

In immunological responses, controlling excessive T cell activity is critical for immunological homeostasis maintenance. Diketoacetonylphenalenone, derived from Hawaiian volcanic soil-associated fungus Penicillium herquei FT729, possesses moderate anti-inflammatory activity in RAW 264.7 cells but its immunosuppressive effect on T cell activation is unknown. In the present study, diketoacetonylphenalenone (up to 40 μM) did not show cytotoxicity in T cells. Western blot analysis showed treatment with diketoacetonylphenalenone did not alter the expression of anti-apoptotic proteins. Pretreatment with diketoacetonylphenalenone suppressed the interleukin-2 production in activated T cells induced by T cell receptor-mediated stimulation and PMA/A23187. The CFSE-proliferation assay revealed the inhibitory effect of diketoacetonylphenalenone on the proliferation of T cells. The expression of surface molecules on activated T cells was also reduced. We discovered the suppression of the TAK1-IKKα-NF-κB pathway by pretreatment with diketoacetonylphenalenone abrogated mitogen-activated protein kinase (MAPK) signaling in activated T cells. These results suggest that diketoacetonylphenalenone effectively downregulates T cell activity via the MAPK pathway and provides insight into the therapeutic potential of immunosuppressive reagents.     

通过调控肿瘤代谢增强T细胞抗肿瘤活性

肿瘤免疫治疗是一种新型的癌症治疗方法。 然而,现阶段肿瘤免疫疗法的临床响应率低等问题严重制约了其进一步应用。 其根本原因在于肿瘤组织免疫抑制微环境限制了T细胞的抗肿瘤活性,而肿瘤组织代谢改变在肿瘤免疫抑制微环境形成过程中起关键作用。 本文重点总结了通过调控肿瘤的有氧糖酵解、氨基酸代谢和脂肪酸代谢增强T细胞的抗肿瘤活性。 最后,提出当前肿瘤代谢调控研究方面仍然存在的问题,并对纳米载体在肿瘤代谢领域的发展前景进行了展望。     

Molecular dynamics simulation reveals structural and thermodynamic features of kinase activation by cancer mutations within the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a major target for drugs in treating lung carcinoma as it promotes cell growth and tumor progression. Structural studies have demonstrated that EGFR exists in an equilibrium between catalytically active and inactive forms, and dramatic conformational transitions occur during its activation. It is known that EGFR mutations promote such conformational changes that affect its activation and drug efficacy. The most common point mutation in lung cancer patients is a leucine to arginine substitution at amino acid 834 (L834R). In a recent article, we have studied changes in drug binding affinities due to cancer mutations of EGFR using ensemble molecular dynamics (MD) simulations. Here, we address an enhanced activation mechanism thought to be associated with this mutation. Using extended timescale MD simulations, the structural and energetic properties are studied for both active and inactive conformations of EGFR. The thermodynamic stabilities of these two conformations are characterized by free energy landscapes estimated from molecular mechanics/Poisson-Boltzmann solvent area calculations. Our study reveals that the L834R mutation introduces conformational changes in both states, adjusting the relative stabilities of active and inactive conformations and hence the activation of the EGFR kinase.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells.

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine prefferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

SHP2 is a target of the immunosuppressant tautomycetin

SHP2 phosphatase is a positive transducer of growth factor and cytokine signaling. SHP2 is also a bona fide oncogene; gain-of-function SHP2 mutations leading to increased phosphatase activity cause Noonan syndrome, as well as multiple forms of leukemia and solid tumors. We report that tautomycetin (TTN), an immunosuppressor in organ transplantation, and its engineered analog TTN D-1 are potent SHP2 inhibitors. TTN and TTN D-1 block T?cell receptor-mediated tyrosine phosphorylation and ERK activation and gain-of-function mutant SHP2-induced hematopoietic progenitor hyperproliferation and monocytic differentiation. Crystal structure of the SHP2?TTN D-1 complex reveals that TTN D-1 occupies the SHP2 active site in a manner similar to that of a peptide substrate. Collectively, the data support the notion that SHP2 is a cellular target for TTN and provide a potential mechanism for the immunosuppressive activity of TTN. Moreover, the structure furnishes molecular insights upon which therapeutics targeting SHP2 can be developed on the basis of the TTN scaffold.     

Enhanced antitumor chemo-immunotherapy by local co-delivery of chemotherapeutics,immune checkpoint blocking antibody and IDO inhibitor using an injectable polypeptide hydrogel

The efficiency of antitumor immunotherapy is usually limited by the immunosuppressive tumor microenvironment (TME). In this study, we developed a chemo-immunotherapy strategy that is able to improve the immunosuppressive TME for enhancing the antitumor efficacy. The chemo-immunotherapy was achieved by the topical co-delivery of a chemotherapeutic drug, Doxorubicin (DOX), an immune checkpoint blocking antibody targeting programmed cell death protein 1 (aPD-1), and an indoleamine-2,3-dioxygenase (IDO) inhibitor, 1-methyl-d -tryptophan (d -1MT) by using a thermosensitive polypeptide hydrogel. It was revealed that the sustained DOX release from the hydrogel caused the immunogenic cell death (ICD) of B16F10 cells in vitro, and the tumor cell lysates subsequently promoted the activation of dendritic cells (DCs). After intratumoral injection into B16F10 melanoma-bearing mice, the DOX/aPD-1/D-1MT co-loaded hydrogel exhibited enhanced tumor inhibition efficacy and prolonged animal survival time, compared to the DOX/aPD-1/D-1MT mixed solution, DOX-loaded hydrogel or DOX/aPD-1 co-loaded hydrogel. The improvement of immunosuppressive TME and enhancement of antitumor immune response after the local chemo-immunotherapy were demonstrated by the augmented activation of DCs and increased infiltration of CD8⁺ and CD4⁺ T cells, as well as enhanced secretion of pro-inflammatory cytokines. Therefore, the hydrogel-based local chemo-immunotherapy system holds great potential for effective antitumor treatment.     

A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1.

Heme oxygenase (HO) catalyzes the oxygen-dependent degradation of heme to biliverdinIXalpha, CO, and free iron ion via three sequential monooxygenase reactions. Although the distinct active-site structure of HO from cytochrome P450 families suggests unique distal protein machinery to activate molecular oxygen, the mechanism and the key amino acid for the oxygen activation have not been clear. To investigate the functionality of highly conserved polar amino acids in the distal helix of HO-1, we have prepared alanine mutants: T135A, R136A, D140A, and S142A, and found drastic changes in the heme degradation reactions of D140A. In this paper, we report the first evidence that D140 is involved in the oxygen activation mechanism in HO-1. The heme complexes of HO mutants examined in this study fold and bind heme normally. The pK(a) values of the iron-bound water and autoxidation rates of the oxy-form are increased with R136A, D140A, and S142A mutations, but are not changed with T135A mutation. As the wild-type, T135A, R136A, and S142A degrade heme to verdohemeIXalpha with H(2)O(2) and to biliverdinIXalpha with the NADPH reductase system. On the other hand, D140A heme complex forms compound II with H(2)O(2), and no heme degradation occurs. For the NADPH reductase system, the oxy-form of D140A heme complex is accumulated in the reaction, and only 50% of heme is degraded. The stopped flow experiments suggest that D140A cannot activate iron-bound dioxygen and hydroperoxide properly. To investigate the carboxylate functionality of D140, we further replaced D140 with glutamic acid (D140E), phenylalanine (D140F), and asparagine (D140N). D140E degrades heme normally, but D140N shows reactivity similar to that of D140A. D140F loses heme degradation activity completely. All of these results indicate that the carboxylate at position 140 is essential to activate the iron-bound dioxygen and hydroperoxide. On the basis of the present findings, we propose an oxygen activation mechanism involving the hydrogen-bonding network through the bridging water and D140 side chain.     

WSS2220, a novel cyclic tetrapeptide with a new sulfonoamino acid, exhibits potent and selective inhibitory activity against GlyT1

In the course of our screening program for novel glycine transporter inhibitors, we obtained a novel cyclic tetrapeptide (WSS2220) from the culture broth of Nonomuraea sp. The structure of WSS2220 was elucidated based on physicochemical data. WSS2220 contained a novel amino acid (4-oxo-3′-sulfoisoleucine), the absolute stereochemistry of which was determined by chemical modifications and the modified Mosher’s method. WSS2220 selectively inhibited glycine transporter type1 with an IC₅₀ of 20 nM.     

镧对MC3T3细胞钙激活钾电流及其动力学的影响

利用全细胞膜片钳技术,研究了稀土镧离子对非兴奋性小鼠成骨细胞(MC3T3)钙激活外向钾电流及其激活和失活动力学的影响.结果表明:MC3T3细胞钙激活外向钾电流随着电极内液中游离Ca2+浓度的增加而增加,且具有电压和胞内游离Ca2+依赖性特征.细胞外液中的稀土镧可浓度依赖性地抑制MC3T3细胞钙激活外向钾电流,其半数抑制浓度(EC50)为8.23±1.45μmol/L.50μmol/L氯化镧可使钾电流的激活曲线向正电位方向移动,而使其失活曲线向负电位方向移动,但对激活曲线和失活曲线的斜率因子k值影响都不大.研究表明:抑制钾通道电流,可使细胞膜去极化,细胞的兴奋性增加,从而增加胞外Ca2+向胞内流动,引起胞内Ca2+浓度的增加,由此而诱发一系列的生理和分子生物学事件.这一过程可能是稀土镧影响MC3T3成骨细胞生长和功能的分子作用机制之一.     

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.     

β1 integrins show specific association with CD98 protein in low density membranes

Background  

The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear.     

A New Synthesis of 4, 4-Diaryl/Diheteroaryl-3-butenyl Derivatives of Nipecotic Acids as GABA Transporter Inhibitors

A new method for the synthesis of 4, 4-diaryl/diheteroaryl-3-butenyl derivatives of nipecotic acid as GABA transporter inhibitors is described. The key intermediates 4-tosyl-1, 1-diaryl/diheteroaryl-1-butene 10a-d were synthesized by Wittig reaction, and followed by alkylation with （R）-3-piperidinecarboxylate. The resulting N-cycloalkylated amino acid esters 11a-d were saponified and then acidified to get the target compounds 1a-d. The preliminary bioassays showed that la-d exhibited excellent inhibition of [3H]-GABA uptake in vitro of culture cells.     

A Cisplatin‐Loaded Immunochemotherapeutic Nanohybrid Bearing Immune Checkpoint Inhibitors for Enhanced Cervical Cancer Therapy

The efficacy of conventional chemotherapy is hindered by cancer cell escape from the immune system. A multifunctional nanohybrid system is reported for effective immunochemotherapy against cervical cancer. This nanohybrid contains both immune checkpoint inhibitor and cisplatin anticancer prodrug, showing improved cellular accumulation and increased binding of Pt to DNA and resulting in elevated apoptosis than using cisplatin alone when tested in cervical cancer cells. The immune checkpoint inhibitor enables the inhibition of indoleamine‐2,3‐dioxygenase and reverses immunosuppressive T cells to recognize cancer cells, leading to T cell proliferation and activation, cancer cell cycle arrest, and ultimately increased cancer cell death. The nanohybrid is also active in vivo against the growth of human cervical tumors. Overall, a strategy is provided using a multifunctional nanohybrid system to boost the antitumor activity of cisplatin.     

Total synthesis of cyclic tetrapeptide FR235222, a potent immunosuppressant that inhibits mammalian histone deacetylases

[structure: see text] The total synthesis of FR235222, a potent immunosuppressant with in vivo activities, has been achieved. The key steps include assembling its (2S,9R)-2-amino-9-hydroxy-8-oxodecanoic acid residue via an olefin cross-metathesis of a methyl (R)-lactate-derived homoallyl ketone with protected allyl amino acid and constructing its trans-(2R,4S)-4-methylproline unit from protected (R)-pyroglutamic acid in seven steps.     

Flowers from Kalanchoe pinnata are a rich source of T cell-suppressive flavonoids

The chemical composition and immunosuppressive potential of the flowers from Kalanchoe pinnata (Crassulaceae) were investigated. We found that the aqueous flower extract was more active than the leaf extract in inhibiting murine T cell mitogenesis in vitro. Flavonoids isolated from the flower extract were identified and quantitated based on NMR and HPLC-DAD-MS analysis, respectively. Along with quercetin, four quercetin glycosyl conjugates were obtained, including quercetin 3-O-beta-D-glucuronopyranoside and quercetin 3-O-beta-D-glucopyranoside, which are described for the first time in K. pinnata. All flavonoids inhibited murine T cell mitogenesis and IL-2 and IL-4 production without cell toxicity. This is the first report on the pharmacological activity of flowers of a Kalanchoe species, which are not used for curative purposes. Our findings show that K. pinnata flowers are a rich source of T-suppressive flavonoids that may be therapeutically useful against inflammatory diseases.     

靶向iNOS的雷公藤甲素氨基酸前药的合成

雷公藤甲素(TL)是从卫矛科雷公藤中提取的环氧二萜内酯,具有显著的抗炎和免疫抑制活性,但TL本身毒性较大、水溶性差、治疗窗窄,极大地限制其在临床上的应用.以炎症部位过度表达的诱导型一氧化氮合酶(iNOS)为靶点,在TL结构中引入iNOS底物及其类似物设计合成系列雷公藤甲素氨基酸前药,提高其水溶性和靶向性.雷公藤甲素氨基...     

采用隔离池的毛细管电泳-间接紫外吸收法测定茶叶中的氨基酸

改进的毛细管电泳-间接紫外吸收法采用了自制隔离池,以对氨基苯甲酸(PAB)为背景电解质,对茶叶中的氨基酸进行了测定。PAB能够提高分离效率,降低检出限。隔离池的使用避免了PAB的电极反应,降低了基线噪声,维持了两缓冲液池间的电流导通。研究了背景电解质的浓度、pH值以及电渗流改性剂的种类和浓度对氨基酸分离的影响。在优化的实验条件下,16种氨基酸在14 min内达到了基线分离,峰面积的相对标准偏差小于5%(n=5),检出限为1.7~4.5 μmol/L,回收率为83.0%~106%。该法快速、便捷和灵敏,已成功应用于茶叶中11种游离氨基酸的检测。