Major Biological Targets Research Articles

Network Pharmacology-Based Strategy Integrated with Molecular Docking and In Vitro Experimental Validation to Explore the Underlying Mechanism of Fangji Huangqi Decoction in Treating Rheumatoid Arthritis.

Fangji Huangqi decoction (FHD), as a classic traditional Chinese medicine formula, has been clinically proven effective against rheumatoid arthritis (RA), yet its therapeutic mechanism remains unclear. This study employed network pharmacology and molecular docking methods to explore the major active components, biological targets, and signaling pathways of FHD. Subsequently, lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used as the in vitro model to validate the modulating effects of FHD on molecules/inflammatory mediators using various biomedical techniques/kits such as MTT assay, Griess reagents, flow cytometry, RT-qPCR, and immunoblotting. Network pharmacology analyses indicated a total of 20 major active components and 30 core biological targets of FHD against RA. Pathway enrichment analyses demonstrated the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the efficacy of the formula. Furthermore, experimental evidence demonstrated that FHD dose-dependently and significantly inhibited the productions of nitric oxide (NO) and reactive oxygen species; lowered the mRNA expression levels of proinflammatory mediators including iNOS, COX-2, TNF-α, ΙL-1β, and IL-6; decreased protein levels of the phosphorylated forms of p38, ERK, JNK, and NF-κB p65. Additionally, the results of molecular docking showed that tetrandrine, licochalcone A, oxonantenine, isorhamnetin, and kaempferol in FHD exerted the potent capability of binding to target molecules in the focused signaling pathway, probably being the potential effective substances for FHD. Our network pharmacology study integrated with cellular validation has elucidated that FHD exerts downregulating effects of the MAPK and NF-κB signaling pathway, ultimately leading to inhibitory effects on the productions of proinflammatory mediators in LPS-stimulated RAW264.7 cells. This work comprehensively demonstrated the effective substances, key targets, and signaling pathways involved in the anti-RA effects of the formula, and these findings provide a further understanding of the underlying mechanism of FHD in managing RA.

MD Simulation Studies for Selective Phytochemicals as Potential Inhibitors against Major Biological Targets of Diabetic Nephropathy.

Diabetes is emerging as an epidemic and is becoming a public health concern worldwide. Diabetic nephropathy is one of the serious complications of diabetes, and about 40% of individuals with diabetes develop diabetic nephropathy. The consistent feature of diabetes and its associated nephropathy is hyperglycemia, and in some cases, hyperamylinemia. Currently, the treatment includes the use of medication for blood pressure control, sugar control, and cholesterol control, and in the later stage requires dialysis and kidney transplantation, making the management of this complication very difficult. Bioactive compounds, herbal medicines, and extracts are extensively used in the treatment and prevention of several diseases, and some are reported to be efficacious in diabetes too. Therefore, in this study, we tried to identify the therapeutic potential of phytochemicals used in in silico docking and molecular dynamic simulation studies using a library of 5284 phytochemicals against the two potential targets of type 2 diabetes-associated nephropathy. We identified two phytochemicals (i.e., gentisic acid and michelalbine) that target human amylin peptide and dipeptidyl peptidase-4, respectively, with good binding affinity. These phytochemicals can be further evaluated using in vitro and in vivo studies for their anti-hyperglycemia and anti-hyperamylinemia effects.

In Silico Computations of Selective Phytochemicals as Potential Inhibitors Against Major Biological Targets of Diabetes Mellitus.

In the past few years, several developments have been made to understand and control the complications and harmful side-effects associated with the disorder diabetes mellitus (DM). Many new steps have been taken in a better understanding of the pathophysiology of the disease. With the advancement in the field of medical sciences, various novel therapies have been developed to efficiently control the pathological effects of diabetes mellitus. Recently, phytochemicals possessing various medicinal properties have opened up a new vast range of opportunities to design novel therapeutic drugs against diabetes mellitus. The present study aims to identify and screen phytochemicals as potent and novel inhibitors against diabetes mellitus. Three major biological targets of diabetes mellitus named Cytochrome P450, glycogen synthase kinase and PPARγ are targeted using phytochemicals by performing pharmacological properties prediction, molecular docking and density functional theory studies. Out of 108 phytochemicals, 20, 12 and 3 phytochemicals showed higher binding affinity values as compared to chemically synthesized drugs against cytochrome P450, glycogen synthase kinase and PPARγ, respectively. The screened phytochemicals have strong inhibitory potential against diabetes mellitus and in future, these compounds, holding immense potential, can be considered as candidate drugs for treating diabetes mellitus.

Imaging the Cardiac Extracellular Matrix.

Cardiovascular disease is the global leading cause of death. One route to address this problem is using biomedical imaging to measure the molecules and structures that surround cardiac cells. This cellular microenvironment, known as the cardiac extracellular matrix, changes in composition and organization during most cardiac diseases and in response to many cardiac treatments. Measuring these changes with biomedical imaging can aid in understanding, diagnosing, and treating heart disease. This chapter supports those efforts by reviewing representative methods for imaging the cardiac extracellular matrix. It first describes the major biological targets of ECM imaging, including the primary imaging target of fibrillar collagen. Then it discusses the imaging methods, describing their current capabilities and limitations. It categorizes the imaging methods into two main categories: organ-scale noninvasive methods and cellular-scale invasive methods. Noninvasive methods can be used on patients, but only a few are clinically available, and others require further development to be used in the clinic. Invasive methods are the most established and can measure a variety of properties, but they cannot be used on live patients. Finally, the chapter concludes with a perspective on future directions and applications of biomedical imaging technologies.

18 - Characterisation of the Novel Products of Protein Oxidation by the Inflammatory Oxidant Hypochlorous Acid (HOCl) Using Raman Spectroscopy

Background The heme enzyme myeloperoxidase that is released by activated leukocytes at sites of inflammation, uses H2O2 and chloride ions to form the bactericidal agent hypochlorous acid (HOCl). Inappropriate or excessive production of this powerful oxidant can however cause host tissue damage, with this being implicated in multiple human inflammatory diseases. Proteins are major biological targets for HOCl, with sulfur- and amine-containing residues particularly susceptible to damage. Reaction of HOCl with amines (e.g. the α-amino groups of oxidised glutathione, GSSG, or the amine group of Lys side-chains) generates chloramines (RNHCl) that undergo subsequent decomposition to aldehydes (R’CHO) and nitriles (R’C≡N). Hypothesis: Nitriles may be characteristic biomarkers of HOCl and chloramine generation, which can be readily detected by Raman spectroscopy via their characteristic absorbance bands. Methods: Sulfur- and amine-containing model peptides were incubated for 24 h with up to 20-fold molar excess HOCl, before drying and analysing by Raman spectroscopy Results Analysis of the HOCl-treated model peptides by Raman spectroscopy shows dose-dependent changes in the wavelength and intensity of characteristic Raman bands assigned to disulfides (500–550 cm-1), and the formation of a new Raman absorption at ~2255 cm-1 assigned to stretches of nitrile groups. The latter peak was detected in a dose-dependent manner on HOCl treatment of Lys-containing protein and peptide models, N-acetyl-histidine and oxidised glutathione (GSSG). Conclusions HOCl-mediated oxidation of proteins and peptides modifies protein and peptide side-chains in a manner that can be readily detected by Raman spectroscopy. The absorption band attributed to C≡N stretching at ~2255 cm-1 is well removed from other features in the Raman spectra, making it a potential marker of HOCl-mediated protein oxidation, chloramine formation and decomposition.

Synthesis, Molecular Modelling and Biological Evaluation of Novel Heterodimeric, Multiple Ligands Targeting Cholinesterases and Amyloid Beta.

Cholinesterases and amyloid beta are one of the major biological targets in the search for a new and efficacious treatment of Alzheimer’s disease. The study describes synthesis and pharmacological evaluation of new compounds designed as dual binding site acetylcholinesterase inhibitors. Among the synthesized compounds, two deserve special attention—compounds 42 and 13. The former is a saccharin derivative and the most potent and selective acetylcholinesterase inhibitor (EeAChE IC50 = 70 nM). Isoindoline-1,3-dione derivative 13 displays balanced inhibitory potency against acetyl- and butyrylcholinesterase (BuChE) (EeAChE IC50 = 0.76 μM, EqBuChE IC50 = 0.618 μM), and it inhibits amyloid beta aggregation (35.8% at 10 μM). Kinetic studies show that the developed compounds act as mixed or non-competitive acetylcholinesterase inhibitors. According to molecular modelling studies, they are able to interact with both catalytic and peripheral active sites of the acetylcholinesterase. Their ability to cross the blood-brain barrier (BBB) was confirmed in vitro in the parallel artificial membrane permeability BBB assay. These compounds can be used as a solid starting point for further development of novel multifunctional ligands as potential anti-Alzheimer’s agents.

Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal–lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH 4, which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥ 1 μM for oxidized amino acids and peptides and ≥ 10 μM for oxidized proteins, compared with ca. 100 μM for H 2O 2, indicating that H 2O 2 is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.

Electron ionization mass spectrometry of the ryanodine receptor‐based Ca2+‐channel stabilizer S‐107 and its implementation into routine doping control

New insights into the biochemistry of cardiac arrhythmia and skeletal muscle fatigue have yielded new drug candidates to counteract these phenomena. Major biological targets have become ryanodine receptor (RyR)-based Ca(2+)-release channels, which tend to 'leak' under various circumstances including strenuous exercise and, thus, cause aberrant calcium sparks that entail impaired muscle function. Therapeutics, which are referred to as rycals, are currently being developed to treat cardiac arrhythmia via enhancement of calstabin-ryanodine affinities that causes a stabilization of the RyR. These therapeutics possess potential for misuse in sports, and an early implementation of target analytes such as the benzothiazepine derivatives S-107 and JTV-519 or putative metabolites into doping control screening procedures is recommended. Reference compounds, deuterated analogues, and a putative metabolic product were synthesized, and electron ionization mass spectra of these products were studied and dissociation pathways elucidated by means of tandem mass spectrometry (MS/MS) and accurate mass measurements. The characterized analytes were incorporated into existing sports drug testing assays based on liquid-liquid extraction and subsequent gas chromatography/mass spectrometry (GC/MS) analysis, and specificity, lower limit of detection (4-6 ng/mL), intraday and interday precision (1.5-17.2%), as well as recovery (63-66%) were determined. The established procedure proved suitable for routine doping control analysis to detect a potential misuse of the drug candidate S-107 in elite sport.

Singlet-oxygen-mediated amino acid and protein oxidation: Formation of tryptophan peroxides and decomposition products

Proteins are major biological targets for oxidative damage within cells owing to their high abundance and rapid rates of reaction with radicals and excited-state species, including singlet oxygen. Reaction of Tyr, Trp, and His residues, both free and on proteins, with singlet oxygen generates peroxides in high yield. Peroxides have also been detected on proteins within intact cells on exposure to visible light in the presence of a photosensitizer. The structures of some of these materials have been elucidated for free amino acids, but less is known about peptide- and protein-bound species. In this study we have characterized Trp-derived peroxides, radicals, and breakdown products generated on free Trp and Trp residues in peptides and proteins, using LC/MS/MS. With free Trp, seven major photoproducts were characterized, including two isomeric hydroperoxides, two alcohols, two diols, and N-formylkynurenine, consistent with singlet oxygen-mediated reactions. The hydroperoxides decompose rapidly at elevated temperatures and in the presence of reductants to the corresponding alcohols. Some of these materials were detected on proteins after complete enzymatic (Pronase) hydrolysis and LC/MS/MS quantification, providing direct evidence for peroxide formation on proteins. This approach may allow the quantification of protein modification in intact cells arising from singlet oxygen formation.

Protective Effect of Tetrahydrobiopterin Against Reactive Oxygen Species- and Nitric Oxide-induced Vascular Endothelial Cell Injury

Abstract Vascular endothelial cells are one of the major biological targets of reactive oxygen species (ROS) in ischemiareperfusion, diabetes, hypercholesterolemia and inflammation. Accumulated evidence has shown that tetrahydrobiopterin (BH4), which is an essential cefactor for nitric oxide synthase (NOS) activity, protected the cells against ROS and nitric oxide (NO) toxicities. Although NOS releases NO, which regulates vascular tone and immune surveillance under normal conditions, NOS also produces superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of ROS under pathological conditions. Since BH4 is easily oxidized by ROS, oxidative stress may induce ROS release from NOS by the decomposition of BH4. BH4 also has an antioxidative activity and scavenging activity for ROS. In this review, the r o l e of BH4 in the function of' NOS, and the mechanisms underlying the protective effect of BH4 against ROS- and/or NO-induced endothelial cell injury will be discussed.

Loss of a prolyl oligopeptidase confers resistance to lithium by elevation of inositol (1,4,5) trisphosphate.

The therapeutic properties of lithium ions (Li+) are well known; however, the mechanism of their action remains unclear. To investigate this problem, we have isolated Li+-resistant mutants from Dictyostelium. Here, we describe the analysis of one of these mutants. This mutant lacks the Dictyostelium prolyl oligopeptidase gene (dpoA). We have examined the relationship between dpoA and the two major biological targets of lithium: glycogen synthase kinase 3 (GSK-3) and signal transduction via inositol (1,4,5) trisphosphate (IP3). We find no evidence for an interaction with GSK-3, but instead find that loss of dpoA causes an increased concentration of IP3. The same increase in IP3 is induced in wild-type cells by a prolyl oligopeptidase (POase) inhibitor. IP3 concentrations increase via an unconventional mechanism that involves enhanced dephosphorylation of inositol (1,3,4,5,6) pentakisphosphate. Loss of DpoA activity therefore counteracts the reduction in IP3 concentration caused by Li+ treatment. Abnormal POase activity is associated with both unipolar and bipolar depression; however, the function of POase in these conditions is unclear. Our results offer a novel mechanism that links POase activity to IP3 signalling and provides further clues for the action of Li+ in the treatment of depression.

Photophysical and photobiological processes in the photodynamic therapy of tumours

Photodynamic therapy (PDT) is an innovative and attractive modality for the treatment of small and superficial tumours. PDT, as a multi-modality treatment procedure, requires both a selective photosensitizer and a powerful light source which matches the absorption spectrum of the photosensitizer. Quadra Logic's Photofrin ®, a purified haematoporphyrin derivative, is so far the only sensitizer approved for phase III and IV clinical trials. The major drawbacks of this product are the lack of chemical homogeneity and stability, skin phototoxicity, unfavourable physicochemical properties and low selectivity with regard to uptake and retention by tumour vs. normal cells. Second-generation photosensitizers, including the phthalocyanines, show an increased photodynamic efficiency in the treatment of animal tumours and reduced phototoxic side effects. At the time of writing of this article, there were more than half a dozen new sensitizers in or about to start clinical trials. Most available data suggest a common mechanism of action. Following excitation of photosensitizers to long-lived excited singlet and/or triplet states, the tumour is destroyed either by reactive ringlet oxygen species (type II mechanism) and/or radical products (type I mechanism) generated in an energy transfer reaction. The major biological targets of the radicals produced and of singlet oxygen are well known today. Nucleic acids, enzymes and cellular membranes are rapidly attacked and cause the release of a wide variety of pathophysiologically highly reactive products, such as prostaglandins, thromboxanes and leukotrienes. Activation of the complement system and infiltration of immunologically active blood cells into the tumorous region enhance the damaging effect of these aggressive intermediates and ultimately initiate tumour necrosis. The purpose of this review article is to summarize the up-to-date knowledge on the mechanisms responsible for the induction of tumour necrotic reactions.

Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells

One of the major biological targets of free radical oxidations, prone, for anatomical reasons, to oxidative challenges, is the cardiovascular system. In the present paper the effect of hydrogen peroxide on intracellular ionized calcium ([Ca 2+] i) homeostasis in smooth muscle cells (SMC) is studied, the major aim of the study being a better understanding of the protective effect of antioxidants and Ca 2+ channel blockers. The exposure of SMC to 300 μ m H 2O 2 induced a rapid increase of [Ca 2+] i, followed by a decrease to a new constant level, higher than the basal before the oxidative challenge. When incubation medium was Ca 2+ free, the pattern of [Ca 2+] i change was different. The rapid increase was still observed, but it was followed by a rapid decrease to a level only slightly above the basal before the oxidative challenge. The involvement of intracellular Ca 2+ stores was tested by using vasopressin, a hormone able to induce discharge of inositol 1,4,5-triphosphate-sensitive Ca 2+ stores. When H 2O 2 was added after vasopressin no [Ca 2+] i increase was observed. Treatment of cells, in which the stable increase of [Ca 2+] i was induced by H 2O 2, with disulfide reducing compounds, induced a progressive decrease of [Ca 2+] i toward the level observed before the oxidative challenge. Calcium channel blockers and antioxidants, on the other hand, effectively prevented the stabilization of [Ca 2+] i at the high steady-state, after the internal Ca 2+ release phase. Dihydropyridine Ca 2+ channel blockers were by far more active than verapamil and among those the most active was lacidipine. Also the antioxidants trolox and N,N′-diphenyl-1,4-phenylenediamine both prevented the [Ca 2+] i unbalance. These results suggest that Ca + channel blockers and antioxidants, although inactive on oxidative stress-induced Ca 2+ release from intracellular stores, prevent the increased influx apparently related to a membrane thiol oxidation.