Progress in small-molecule inhibitors targeting PD-L1.

PD-L1 is a transmembrane protein overexpressed by tumor cells. It binds to PD-1 on the surface of T-cells, suppresses T-cell activity and hinders the immune response against cancer. Clinically, several monoclonal antibodies targeting PD-1/PD-L1 have achieved significant success in cancer immunotherapy. Nevertheless, their disadvantages, such as unchecked immune responses, high cost and long half-life, stimulated pharmacologists to develop small-molecule inhibitors targeting PD-1/PD-L1. After a batch of excellent inhibitors with a biphenyl core structure were firstly reported by BMS, more and more researchers focused on small-molecule inhibitors targeting PD-L1 rather than PD-1. Numerous small-molecule inhibitors were extensively designed and synthesized in the past few years. In this paper, the structural characteristics of PD-L1 and complexes of PD-L1 with its inhibitors are elaborated and small molecule inhibitors developed in the last decade are summarized as well. This paper aims to provide insights into further designing and synthesis of small molecule inhibitors targeting PD-L1.

Study on the chemical reactivity difference of primary hydroxyl groups in iridoid glycosides.

Iridoid glycoside, which belongs to the polyhydroxy compound, is a kind of active ingredient of traditional Chinese medicine with a wide range of sources, and has many pharmacological effects such as anti-cancer, anti-inflammatory, anti-virus, hypoglycemic and so on. Its structure contains many hydroxyl groups, including two primary hydroxyl groups. The chemical reactivity of primary hydroxyl groups has very little difference, so it is very important to control the selectivity of hydroxyl groups under certain conditions. In this paper, the difference between the two primary hydroxyl groups in iridoid glycoside was calculated based on computer simulation and verified this result through designed experiments. This study will provide an important way for site-directed modification of hydroxyl in iridoid glycoside in the future.

Design, Synthesis and Biological Evaluation of Novel Catalpol Derivatives as Potential Pancreatic Cancer Inhibitors.

A series of C10-position imidazole-modified catalpol derivatives are specifically designed and synthesized for serving as potential pancreatic cancer inhibitors, which are characterized by 1 H NMR, 13 C NMR and high-resolution mass spectrometry (HRMS). They were evaluated by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) test on two human pancreatic cancer cells PANC-1, BxPC-3 and normal pancreatic cell HPDE6-C7, which showed the significant inhibitory effected on the growth of human pancreatic cancer cells of PANC-1 and BxPC-3, especially 91.6% efficacy on BxPC-3, and 73.1% on PANC-1. Simulation studies like molecular docking supported strong binding of vascular endothelial growth factor receptor 2 (VEGFR-2) protein tyrosine kinase (PDB ID: 4AGD), a target of pancreatic cancer. A novel imidazol-modified catalpol compound 3i with strong inhibitory effect on pancreatic cancer cells, which could potentially develop into anti-pancreatic cancer drug candidates in the future.

Advances in the Study of Hexokinase 2 (HK2) Inhibitors.

PURPOSE: The Warburg effect is an important metabolic feature of tumours, and hexokinase is the first ratelimiting enzyme of the glycolytic pathway during tumour metabolism. Among hexokinase subtypes, hexokinase 2 (HK2) is increasingly proving to be a key target for cancer treatment. This study presents the challenges and potential strategies for developing HK2 inhibitors by systematically summarising the characteristics of HK2 inhibitors reported in the literature and patents. METHODS: In this study, we analysed the HK2 active site using molecular docking and evaluated the structure, biochemical and physiological function, activity, and action mechanism of reported HK2 inhibitors using databases (Science, SCI Finder, CNKI, and WANFANG DATA). RESULTS: In total, 6 natural inhibitors of HK2, 9 synthetic inhibitors of HK2, and 3 compounds with patent-pending HK2 inhibitory effects were obtained by searching 87 articles. These inhibitors have poor efficacy and specificity when used alone and have numerous side effects; therefore, there is an urgent need to develop HK2 inhibitors with improved activity and high selectivity. CONCLUSION: HK2 has received much attention in anticancer drug development, but most previous studies have focused on elucidating the action mechanism of HK2 in carcinogenesis, whereas the development of its small-molecule inhibitors has rarely been reported. In this study, we analysed and illustrated the eutectic structure of small molecules with the catalytic structural domain of HK2 to develop highly selective and low-toxicity HK2 inhibitors.

Non-Structural Carbohydrate Storage Strategy Explains the Spatial Distribution of Treeline Species.

Environmental factors that drive carbon storage are often used as an explanation for alpine treeline formation. However, different tree species respond differently to environmental changes, which challenges our understanding of treeline formation and shifts. Therefore, we selected Picea jezoensis and Betula ermanii, the two treeline species naturally occurring in Changbai Mountain in China, and measured the concentration of non-structural carbohydrates (NSC), soluble sugars and starch in one-year-old leaves, shoots, stems and fine roots at different elevations. We found that compared with P. jezoensis, the NSC and soluble sugars concentrations of leaves and shoots of B. ermanii were higher than those of P. jezoensis, while the starch concentration of all the tissues were lower. Moreover, the concentration of NSC, soluble sugars and starch in the leaves of B. ermanii decreased with elevation. In addition, the starch concentration of B. ermanii shoots, stems and fine roots remained at a high level regardless of whether the soluble sugars concentration decreased. Whereas the concentrations of soluble sugars and starch in one-year-old leaves, shoots and stems of P. jezoensis responded similarly changes with elevation. These findings demonstrate that compared with P. jezoensis, B. ermanii has a higher soluble sugars/starch ratio, and its shoots, stems and fine roots actively store NSC to adapt to the harsh environment, which is one of the reasons that B. ermanii can be distributed at higher altitudes.

Astragaloside IV Prevents Cardiac Remodeling in the Apolipoprotein E-Deficient Mice by Regulating Cardiac Homeostasis and Oxidative Stress.

BACKGROUND: Hypercholesterolemia is a risk factor for the development of cardiac hypertrophy. Astragaloside IV (AST-IV) possesses cardiovascular protective properties. We hypothesize that AST-IV prevents cardiac remodeling with hypercholesterolemia via modulating tissue homeostasis and alleviating oxidative stress. METHODS: The ApoE-/- mice were treated with AST-IV at 1 or 10 mg/kg for 8 weeks. The blood lipids tests, echocardiography, and TUNEL were performed. The mRNA expression profile was detected by real-time PCR. The myocytes size and number, and the expressions of proliferation (ki67), senescence (p16INK4a), oxidant (NADPH oxidase 4, NOX4) and antioxidant (superoxide dismutase, SOD) were observed by immunofluorescence staining. RESULTS: Neither 1 mg/kg nor 10 mg/kg AST-IV treatment could decrease blood lipids in ApoE-/- mice. However, the decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS) in ApoE-/- mice were significantly improved after AST-IV treatment. The cardiac collagen volume fraction declined nearly in half after AST-IV treatment. The enlarged myocyte size was suppressed, and myocyte number was recovered, and the alterations of genes expressions linked to cell cycle, proliferation, senescence, p53-apoptosis pathway and oxidant-antioxidants in the hearts of ApoE-/- mice were reversed after AST-IV treatment. The decreased ki67 and increased p16INK4a in the hearts of ApoE-/- mice were recovered after AST-IV treatment. The percentages of apoptotic myocytes and NOX4-positive cells in AST-IV treated mice were decreased, which were consistent with the gene expressions. CONCLUSION: AST-IV treatment could prevent cardiac remodeling and recover the impaired ventricular function induced by hypercholesterolemia. The beneficial effect of AST-IV might partly be through regulating cardiac homeostasis and anti-oxidative stress.

In Vitro Effects of Hollow Gold Nanoshells on Human Aortic Endothelial Cells.

Gold nanoparticles are emerging as promising biomedical tools due to their unique nanoscale characteristics. Our purpose was to synthesize a hollow-shaped gold nanoparticle and to investigate its effect on human aortic endothelial cells (HAECs) in vitro. Hollow gold nanoshells with average 35-nm diameters and 10-nm shell thickness were obtained by galvanic replacement using quasi-spherical nanosilver as sacrifice-template. Our results showed that hollow gold nanoshells in the culture medium could be internalized into the cytoplasm of HAECs. No cytotoxicity effect of hollow gold nanoshells on HAECs was observed within the test concentrations (0-0.8 µg/mL) and test exposure period (0-72 h) by tetrazolium dye assay. Meanwhile, the release of cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without hollow gold nanoshells). The concentrations of vasodilators, nitric oxide, and prostacyclin I-2 were not changed, but the vasoconstrictor endothelin-1 was decreased by hollow gold nanoshells treatment in HAECs. HAECs exposed to hollow gold nanoshells resulted in suppressing expressions of genes involved in apoptosis and activating expressions of genes of adhesion molecules. Moreover, we demonstrated by in vitro endothelial tube formation that hollow gold nanoshells (0.8 µg/mL) could not inhibit angiogenesis by the HAECs. Altogether, these results indicate that the structure and major function of HAECs would not be disrupted by hollow gold nanoshell treatment.

Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

The effects of different initiation time of exercise training on left ventricular remodeling and cardiopulmonary rehabilitation in patients with left ventricular dysfunction after myocardial infarction.

PURPOSE: The purpose of this study was to determine whether different initiation of exercise training (ET) produces different effect sizes for left ventricular (LV) remodeling and cardiopulmonary rehabilitation in patients with LV dysfunction after myocardial infarction (MI). METHOD: Trials evaluating ET outcomes identified by searches in OVID MEDLINE, EMBASE, PubMed and WEB OF SCIENCE were used. Meta-analysis was conducted with the use of the software STATA 11.0. The results were expressed as the standardized mean difference (SMD), with corresponding 95% CI and p value. RESULTS: The largest changes in LV remodeling and cardiopulmonary capacity rehabilitation were obtained when programs began the acute phase after MI. With the healing of MI, the beneficial effects of ET on LV ejection fraction (LVEF), LV end-systolic diameter (LVDs) and peak VO2 were gradually weakened even worse. The incidence of major adverse cardiac events was not significantly increased in acute phase post-MI. Sensitivity analyses show that ET still had significant effect in reducing LVDs and increasing peak VO2, while ET no longer had statistical effect in increasing LVEF but showed favorable trends when the same research institution's works were excluded. CONCLUSIONS: ET has favorable effects on LV remodeling and cardiopulmonary rehabilitation in LV dysfunction post-MI patients. The greatest benefits are obtained when ET starts at the acute phase following MI. IMPLICATIONS FOR REHABILITATION: Early exercise training is safe and feasible in acute and healing phase after myocardial infarction. Early exercise training could attenuate LV remodeling and improve cardiopulmonary capacity in patients with myocardial infarction after hospital discharge (around one week post-MI). Exercise training has favorable effects on LV remodeling and cardiopulmonary capacity rehabilitation. Exercise training should be treated to have the same roles with drugs in secondary prevention of myocardial infarction.

Beneficial effects of astragaloside IV against angiotensin II-induced mitochondrial dysfunction in rat vascular smooth muscle cells.

Angiotensin II (Ang II)-induced mitochondrial dysfunction is a prominent characteristic of the majority of cardiovascular diseases. Astragaloside IV (As-IV), the major active ingredient of Astragalus membranaceus (Fisch.) Bge. (a traditional Chinese herbal medicine), possesses antioxidant properties. The present study was carried out to examine whether As-IV can reverse Ang II-induced mitochondrial dysfunction in vascular smooth muscle cells (VSMCs) and to elucidate the underlying molecular mechanisms. Cultured rat aortic VSMCs treated with Ang II (1 µM) for 24 h exhibited mitochondrial dysfunction, including a decrease in mitochondrial oxygen consumption rates (OCRs), adenosine triphosphate (ATP) production and mitochondrial DNA (mtDNA) levels, as well as the disruption of mitochondrial structural integrity. Following treatment with Ang II, As-IV (50 µg/ml) was added to the culture medium followed by incubation for a further 24 h. The administration of As-IV significantly increased the mitochondrial OCRs, ATP production and the mtDNA levels, and reversed the mitochondrial morphological changes which occurred in the VSMCs. Treatment with As-IV also reversed the Ang II-induced increase in the production of reactive oxygen species (ROS), the increase in NADPH oxidase and xanthine oxidase activity, as well as the decrease in mitochondrial membrane potential (ΔΨm) and manganese superoxide dismutase (Mn-SOD) activity. Furthermore, treatment with As-IV led to an increase in the mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and mitochondrial transcription factor A (Tfam), and in the protein expression of PGC-1α, parkin and dynamin 1-like protein 1 (Drp1) in the VSMCs. These results indicate that As-IV exerts beneficial effects on Ang II-induced mitochondrial dysfunction in rat VSMCs and that these effects are mediated through the inhibition of ROS overproduction, as well as the promotion of mitochondrial autophagy and mitochondrial biogenesis. These data demonstrate the antioxidant properties of As-IV.

Cardiac troponin I is abnormally expressed in non-small cell lung cancer tissues and human cancer cells.

Cardiac troponin I (cTnI) is the only sarcomeric protein identified to date that is expressed exclusively in cardiac muscle. Its expression in cancer tissues has not been reported. Herein, we examined cTnI expression in non-small cell lung cancer (NSCLC) tissues, human adenocarcinoma cells SPCA-1 (lung) and BGC 823 (gastric) by immunohistochemistry, western blot analysis and real-time PCR. Immunopositivity for cTnI was demonstrated in 69.4% (34/49) NSCLC tissues evaluated, and was strong intensity in 35.3% (6/17) lung squamous cell carcinoma cases. The non-cancer-bearing lung tissues except tuberculosis (9/9, 100%) showed negative staining for cTnI. Seven monoclonal antibodies (mAbs) against human cTnI were applied in immunofluorescence. The result showed that the staining pattern within SPCA-1 and BGC 823 was dependent on the epitope of the cTnI mAbs. The membrane and nucleus of cancer cells were stained by mAbs against N-terminal peptides of cTnI, and cytoplasm was stained by mAbs against the middle and C-terminal peptides of cTnI. A ~25 kD band was identified by anti-cTnI mAb in SPCA-1 and BGC 823 extracts by western blot, as well as in cardiomyocyte extracts. The cTnI mRNA expressions in SPCA-1 and BGC 823 cells were about ten thousand times less than that in cardiomyocytes. Our study shows for the first time that cTnI protein and mRNA were abnormally expressed in NSCLC tissues, SPCA-1 and BGC 823 cells. These findings challenge the conventional view of cTnI as a cardiac-specific protein, enabling the potential use of cTnI as a diagnostic marker or targeted therapy for cancer.

The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells.

One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and the concentrations no more than 0.02 mg/ml had little toxic effect which were revealed by tetrazolium dye assay. Meanwhile, the cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without DMSA-Fe2O3). However, the endocrine function for endothelin-1 and prostacyclin I-2, as well as the urea transporter function, was altered even without obvious evidence of cell injury in this context. We also showed by real-time PCR analysis that DMSA-Fe2O3 exposure resulted in differential effects on the expressions of pro- and anti-apoptosis genes of HAECs. Meanwhile, it was noted that DMSA-Fe2O3 exposure could activate the expression of genes related to oxidative stress and adhesion molecules, which suggested that inflammatory response might be evoked. Moreover, we demonstrated by in vitro endothelial tube formation that even a small amount of DMSA-Fe2O3 (0.01 and 0.02 mg/ml) could inhibit angiogenesis by the HAECs. Altogether, these results indicate that DMSA-Fe2O3 have some cytotoxicity that may cause side effects on normal endothelial cells.

Astragaloside IV alleviates hypoxia/reoxygenation-induced neonatal rat cardiomyocyte injury via the protein kinase a pathway.

BACKGROUND: Astragaloside IV (As-IV) exerts beneficial effects on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury, possibly through normalization of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) function. The exact mechanisms remain unknown. This study was designed to investigate the role of protein kinase A (PKA) in the protective effect of As-IV on SERCA2a function. METHODS: Cultured cardiomyocytes from neonatal rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation (H/R) with or without As-IV treatment. Myocyte injury was determined by the creatine kinase (CK)-MB fraction in supernatant. Myocardial SERCA2a activity and PKA kinase activity were assessed. PKA subunit mRNA expression and Ser(16) phosphorylated phospholamban (Ser(16)-PLN) protein expression were detected by real-time PCR and Western blot, respectively. RESULTS: The administration of As-IV significantly decreased CK-MB release and restored SERCA2a activity in H/R cardiomyocytes. The mRNAs of PKA subunits, PKA-RIα, PKA-RIIα, PKA-RIIß, PKA-Cα and PKA-Cß, were downregulated in H/R cardiomyocytes. However, PKA-Cα mRNA expression was significantly increased after As-IV treatment. Meanwhile, there was a tendency to recovery of the H/R-induced PKA kinase activity decrease after As-IV treatment. The expression of Ser(16)-PLN protein, which is specifically phosphorylated by PKA, was upregulated in As-IV-treated H/R cardiomyocytes. CONCLUSIONS: These results suggest that the cardioprotection of As-IV may be through the upregulation of PKA and Ser(16)-PLN, thereby restoring SERCA2a function in H/R injury.

Astragaloside IV improved intracellular calcium handling in hypoxia-reoxygenated cardiomyocytes via the sarcoplasmic reticulum Ca-ATPase.

Although astragaloside IV, a saponin isolated from Astragalus membranaceus, has been shown to protect the myocardium against ischemia/reperfusion injury, its effect on the status of sarcoplasmic reticulum (SR) Ca2+ transport in the injured myocardium remains largely unknown. In this study, we investigated whether in cultured cardiomyocytes subjected to hypoxia and reoxygenation (H/R) administration of astragaloside IV during H/R attenuates the myocardial cell injury and prevents changes in Ca2+ handling activities and gene expression of SR Ca2+ pump. Cultured cardiomyocytes from neonatal rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation. Myocyte injury was determined by the release of cardiac troponin I in supernatant. Astragaloside IV significantly inhibited cardiac troponin I release after H/R in a dose-dependent manner. The diastolic [Ca2+]i measured with Fura-2/AM was significantly increased after reoxygenation. Astragaloside IV prevented the rise of diastolic [Ca2+]i and the depression of caffeine-induced Ca2+ transients caused by H/R. Furthermore, the observed depressions in SR Ca2+-ATPase activity as well as the mRNA and protein expression of SR Ca2+-ATPase in hypoxic-reoxygenated cardiomyocytes were attenuated by astragaloside IV treatment. These results suggest that the beneficial effect of astragaloside IV in H/R-induced injury may be related to normalization of SR Ca2+ pump expression and, thus, may prevent the depression in SR Ca2+ handling.

Cardiac protective effect of Astragalus on viral myocarditis mice: comparison with Perindopril.

In clinical practice, Astragali Radix (Astragalus), the root of Astragalus membranaceus Bunge, has been widely applied to treat patients with viral diseases, including viral myocarditis in China. The present study was designed to evaluate the protective effects of Astragalus on the function of sarcoplasmic reticulum calcium ATPase (SERCA2) activity and endothelin system at acute and chronic periods of myocarditis mice induced by CVB(3) infection. Astragalus feeding (2.2 mg/kg/day) could significantly increase the survival rate, alleviate pathological alterations and serum cardiac troponin I (cTnI), as well as restore impaired SERCA activity at the acute stage. Low affinity and capacity of ETR were reversed with Astragalus after the first CVB(3) inoculation up to 7 days and after the second virus inoculation up to 150 days. In the meantime, the contents of cardiac ET-1 and ANP were reduced. Comparison the myocarditis mice treated with Perindopril (0.44 mg/kg/day), an ACE inhibitor, shows that Astragalus achieved a similar effect on survival rate, SERCA2 and ET system. These results indicated that the beneficial effects of Astragalus and Perindopril for treating viral myocarditis might be partly mediated by preserving the functions of SERCA 2 activity and ET system.

[Role of calcineurin in the progression of cardiac hypertrophy in renovascular hypertensive rats].

The present study was to investigate the mRNA, protein expression and the activity of calcineurin in the hypertrophic heart, and to determine the effect of calcineurin inhibitor--cyclosporine A (CsA) on the regression of cardiac hypertrophy in renovascular hypertensive rats. Renovascular hypertension was induced by two kidney-one clip methods. Two months after the operation, cardiac hypertrophy was determined by histological analysis performed in some rats (2K1C-2M), then the rats were subdivided into 2 groups: (1) 3-month old two kidney-one clip group (2K1C-3M) with rats receiving 0.9% NaCl per day for one month, and (2) CsA-treated group with rats treated with CsA for one month. Sham-operated rats were used as control. The ratio of the left ventricular weight to tibial length (LVW/TL), the area of cardiac myocyte, mRNA and protein expression and the activity of calcineurin were determined. Both the LVW/TL and the cardiomyocyte area were significantly larger in 2K1C-2M and 2K1C-3M rats than in age-matched sham-operated rats. Treatment with CsA significantly attenuated the increase in the LVW/TL as well as the cardiomyocyte area. The mRNA, protein expression and the activity of calcineurin were significantly higher in 2K1C-2M and 2K1C-3M rats than those in the age-matched sham-operated rats, while the elevation of mRNA, protein expression and activity of calcineurin were significantly suppressed in the CsA-treated rats. In conclusion, calcineurin plays a role in the progression of cardiac hypertrophy in renovascular hypertensive rats. The inhibition of calcineurin can reverse cardiac hypertrophy.