Current Drug Discovery Technologies - Volume 15, Issue 2, 2018

Abstract: Background: In recent years human phospholipase D enzymes (PLD1 and PLD2 isozymes) have emerged as drug targets for various diseases such as cardiovascular disease, cancer, infectious diseases and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. The interest in PLD as a drug target is due to the fact that PLD enzymes belong to a superfamily of phospholipases that are essential to intracellular and extracellular signaling. Many bioactive lipid signaling molecules are generated by these enzymes including phosphatidic and lysophosphatidic acid, arachidonic acid, and diacylglycerol (DAG). More specifically PLDs are part of one pathway that generates phosphatidic acid which is a precursor to many lipids in the intracellular de novo pathway. The lipids produced from PA regulate many cellular events considered hallmarks of pathogenesis in cells; including proliferation, migration, invasion, angiogenesis, and vesicle transport. Hence, human PLD is a valid target for a variety of drug therapies. Methods: The focus of this review is phospholipase D inhibitory molecules. A survey of structure-based drug design studies for PLD enzymes was done by searching several literature databases. Studies that focused on the structural aspects of phospholipase D were compiled and analyzed for content. Particular attention was given to studies involving inhibitory molecules as the focus of this work. In addition, the protein data bank (PDB) was surveyed for three dimensional structures of PLD. Structural investigation via in silico docking utilizing the available three dimensional coordinates of PLD and recent potent PLD isozyme specific inhibitors was performed to gain insights into the mode of binding by drugs designed to inhibit PLDs. Results: Beginning with halopemide and derivatives such as FIPI (5-fluoro-2- indoyly des-chlorohalopemide) leading to PLD isozyme selective inhibitors such as novel triazaspirone-based series of PLD inhibitors, structures and IC50 values presented were found to be in the nanomolar range for either human PLD1 or PLD2. Selective oestrogen receptor modulators (SERMS), compounds used in the treatment of oestrogen-receptor-positive breast cancer, inhibited mammalian PLD enzymes in the low micromolar range. The first universal PLD inhibitor developed was devoid of the 6-OH moiety necessary for oestrogen receptor binding and anti-proliferation action. The universal PLD inhibitor contains a N,N-dimethylamino moiety which is known to reduce SERM activity and was found to inhibit several PLDs in the low micromolar range. The literature analyzed revealed a systematic approach to the biochemical evaluation of modes of binding of these inhibitors to the PLD enzymes. Finally, docking studies of several of the more potent PLD inhibitors correlates with biochemical studies with two modes of inhibitor binding to PLD: active site binding and allosteric binding. Conclusion: PLD inhibitors from diverse backgrounds continue to be developed as research progresses to the most potent and highly selective human PLD inhibitors with low or no off target activities. Docking studies strongly suggest both competitive (active site) and allosteric binding of these inhibitors to PLD. The three dimensional structure of PLD co-crystallized with potent inhibitors will be paramount to confirm the modes of binding for these molecules to PLD.

Background: The opioid system is considered a potential therapeutic target in a variety of neurological disorders. Delta opioid receptors (DORs) are broadly expressed in the brain, and their activation protects cells from hypoxic/ischemic insults by counteracting disruptions of ionic homeostasis and initiating neuroprotective pathways. The DOR agonist D-Ala2-D-Leu2-Enkephalin (DADLE) promotes neuronal survival, mitigates apoptotic pathways, and protects neurons and glial cells from ischemia-induced cell death, thus making DADLE a promising therapeutic option for stroke. The significant amount of research regarding DORs and DADLE in the last decades also suggests their potential in treating other neurological disorders. Methods: This review compiled relevant literature detailing the role of DORs and agonists in central nervous system function and neuropathologies. Results: Several studies demonstrate potential mechanisms implicating a key interaction between DORs and DADLE in conferring neuroprotective benefits. A better understanding of DOR function in disease-specific contexts is critical to transitioning DOR agonists into the clinic as a therapy for stroke and other neurological diseases. Conclusion: Evidence-based studies support the potential of the delta-opioid family of receptors and its ligands in developing novel therapeutic strategies for stroke and other brain disorders.

Background: Fluoroquinolones have been the centre of considerable scientific and clinical interest due to their broad spectrum pharmacological activities. Pefloxacin is an analogue of norfloxacin, which is a 3rd generation of fluoroquinolone antibiotic similar to ciprofloxacin. Pefloxacin is used to treat a variety of bacterial infections like respiratory tract, ear, nose and throat (ENT) infections, skin infections, and urinary tract infections. Hydrazone as a pharmacophore unit that attracts the medicinal chemists because of structure activity relationship (SAR) studies of fluoroquinolones especially the functionality at C-3 position. Consequently, recognition and development of potential ligands specifically for a protein target forms the primary goal in drug discovery process. Among the different theoretical approaches available, Gold and Glide are the molecular docking methods which find application protein ligand studies. In the current study, the DNA gyrase of Staphylococcus aureus has been used as the target protein to understand their possible interactions. Methods: The crystal structure of DNA gyrase (topoisomerase II) was downloaded from the Protein Data Bank (PDB ID: 2XCS, 3FOE) and molecular docking studies were performed using the docking programs like Gold 3.2 (Genetic Algorithm for Ligand Docking), Glide 5.0 (Grid Based Ligand Docking with Energetic). Melting points were uncorrected and determined in open capillary tubes in a melting point apparatus. TLC was performed on silica gel-G and spotting was done using iodine/ KMnO4 staining or UV-light. The following experimental procedures are representive of the general procedures used to synthesize all compounds. Results: The docking experiments of the title compounds with 2.1 Šcrystal structure of DNA gyrase 2XCS, 3FOE using Gold 3.2 and Glide 5.0 is carried out to understand the binding interactions of the novel ligands with the protein, contributing for antibacterial activity. The compounds in general exhibited good binding interactions like H-bonding interaction and π-π interactions which stabilize the protein-ligand complexes and responsible for good fitness scores in both the protocols. Conclusion: In summary, a new series of novel pefloxacin hydrazones 5a-5n were studied for their interactions with Staphylococcus aureus DNA gyrase protein by Glide 5.0 and Gold 3.2 molecular docking protocols [PDB IDS: 2XCS, 3FOE]. Among the tested molecules, compound 5g exhibited a good Glide score value of – 7.73 and Glide energy −51.24 with emodel value of −66.16. The nice docking scores of 5g, 5a, 8h, 5m and 5b revealed that these compounds are well accommodated on the active site residues of DNA gyrase enzyme. From the docking study, we have explored the probable binding mode and the binding pattern of compounds 5f, 5l, 5h, 5d, and 5n showed that they strongly interact with in the active site of Staphylococcus aureus of DNA gyrase enzyme. From screening results it is found that compounds having aromatic ring substituted with electron releasing groups are showing potent docking scores and exhibited better fitness than reference compounds CPF and CA4. An efficient combination of molecular modeling and biological activity provided an insight into QSAR guide lines that could aid in further development and optimization of the pefloxacin derivatives.

Background: Inhibition of Toll-like receptors (TLRs) signaling has been established as a new method for the development of anti-inflammatory drugs instead of NSAIDs (non-steroid anti-inflammatory drugs). Since the immunomodulatory role of G2013 (α-L-Guluronic acid) was reported in some recent experiments, we decided to assess the effects of G2013 on the protein expression of TLR2 and TLR4, their downstream signaling cascade, and the secretion of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). Methods: After blood sampling from 16 healthy donors, PBMCs were isolated and treated with/without lipopolysaccharide (LPS), lipopolyteichoic acid (LTA), and G2013. Flow cytometry was done for detecting the protein expression of TLR2 and TLR4. MyD88, IΚB, Tollip, and NF-ΚB mRNA expression were assessed by realtime PCR. ELISA was performed for assessing the concentration of IL-1β and IL-6. Results: G2013 at a concentration of 25 μg/mL (high dose) significantly downregulated NF-ΚB, IΚB and MyD88 mRNA expression and suppressed the secretion of IL-1β by PBMCs. The findings indicate that G2013 may exert its regulatory effect under normal condition via Tollip in a dose dependence pathway. Our results demonstrated that G2013 had no profound impact on the protein expression of TLR2 and TLR4. Conclusion: In conclusion, our findings point to the immunomodulatory effect of G2013 on the TLR2 and TLR4 signaling cascade and cytokine production by PBMCs. These findings could lead to an establishment of new safe anti-inflammatory drugs in the future.

Background: Lipopeptide synthetases play an important role in the production of lipopeptides. Lipopeptides are molecules made up of peptides and fatty acid moieties and have shown to have a broad range of antimicrobial activity. As infectious diseases have caused severe health problems mainly resulting from the development of antibiotic resistant strains of disease causing microorganisms there is a need of alternatives to antibiotics. The lipopeptide synthetase of the corresponding lipopeptides can be used as template to design these as drugs using computational techniques. Objective: The objective of this study was homology modeling and molecular docking of two lipopeptide synthetases, bacillomycin D synthetase and iturin A synthetase, with their ligands as a means of drug design. Method: Schrödinger software was used for homology modeling and molecular docking. Results: After the identification of ligands, molecular docking of these ligands with the lipopeptide (bacillomycin and iturin) synthetases was performed. The docking was tested on the parameters of docking score and glide energy. 5 out of 21 ligands were found to dock with bacillomycin D synthetase whereas 8 out of 20 ligands docked with the iturin A synthetase. Conclusion: The knowledge of the docking sites and docking characteristics of the lipopeptide synthetases mentioned in the paper with the ligands can provide advantages of high speed and reliability, reduced costs on chemicals and experiments and the ethical issues concerned with the use of animal models for screening of drug toxicity.

Background: Doxorubicin is an anthracycline antibiotic which inhibits DNA and RNA synthesis intercalating DNA double helix and inducing free radicals. Doxorubicin is used in the treatment of cancer diseases. The influence of Doxorubicin on human buccal cell was used as a model for the assessment of toxic effects in vitro. Objective: We studied the possibility of using the process of heterochromatinization in cell nuclei in toxicological investigations of drugs in vitro. Method: The exfoliated buccal epithelium cells of two donors (men) – Donor A (24 years) and Donor B (23 years) were used. Cells were subjected to Doxorubicin in concentration 0.2; 2; 20, and 200 μg/ml for 2 h. Results: A significant increase of chromatin condensation in a dose-dependent way was shown. Doxorubicin induced chromatin condensation for cells of donors A and B if ≥ 0.2 μg/ml, and ≥ 2 μg/ml, correspondingly. Cell viability assessed by combined staining with Hoechst 33342 and ethidium bromide revealed a significant increase of damaged cells if ≥ 2 μg/ml. Indigo carmine staining also revealed a significant increase in permeability of cell membranes if ≥ 20 μg/ml. In cells of donor A the intranuclear calcium concentration increased if Doxorubicin concentration was ≥ 0.2 μg/ml. In cells of donor B cytoplasmic and intranuclear calcium concentration decreased if Doxorubicin concentration ≥ 0.2 μg/ml. Conclusion: The comparison revealed high sensitivity of the method of chromatin changes registration in human buccal epithelium cells as a method of assessment of drug toxicity in vitro, and this method may be recommended for toxicological investigations.

Background: The development and introduction of combined therapy represent a challenge for analysis due to severe overlapping of their UV spectra in case of spectroscopy or the requirement of a long tedious and high cost separation technique in case of chromatography. Quality control laboratories have to develop and validate suitable analytical procedures in order to assay such multi component preparations. Methods: New spectrophotometric methods for the simultaneous determination of simvastatin (SIM) and nicotinic acid (NIA) in binary combinations were developed. These methods are based on chemometric treatment of data, the applied chemometric techniques are multivariate methods including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS). In these techniques, the concentration data matrix were prepared by using the synthetic mixtures containing SIM and NIA dissolved in ethanol. The absorbance data matrix corresponding to the concentration data matrix was obtained by measuring the absorbance at 12 wavelengths in the range 216 – 240 nm at 2 nm intervals in the zero-order. The spectrophotometric procedures do not require any separation step. The accuracy, precision and the linearity ranges of the methods have been determined and validated by analyzing synthetic mixtures containing the studied drugs. Conclusion: Chemometric spectrophotometric methods have been developed in the present study for the simultaneous determination of simvastatin and nicotinic acid in their synthetic binary mixtures and in their mixtures with possible excipients present in tablet dosage form. The validation was performed successfully. The developed methods have been shown to be accurate, linear, precise, and so simple. The developed methods can be used routinely for the determination dosage form.

Background: Therapy with inhaled nitric oxide (iNO) is effective in the management of pulmonary hypertension and severe hypoxemia. However, these benefits have not been demonstrated in preterm infants (<34 weeks). The objective of this report is to present the experience of eight cases of preterm neonates with respiratory distress syndrome (RDS) and refractory hypoxemia, with oligohydramnios history. Methods: We evaluated the clinical feature of 8 preterm neonates with severe hypoxemia who had maternal antecedents of oligoamnios, mainly due to premature rupture of membranes. They were treated with conventional management, with poor clinical response. Therefore, these neonates were treated with iNO, as a rescue strategy. iNO has been used with a dosage of 5 – 10 ppm. An echocardiogram was performed to determine the presence of structural malformations or persistent ductus arteriosus. Results: All the infants showed improvement in oxygenation. The neonates had signs of low flow pulmonary, confirmed by echocardiogram. Five preterm infants survived without complications associated with the therapy. Two died from pulmonary bleeding secondary to ductus arteriosus and another for pneumothorax. Conclusion: iNO therapy can be useful in a subgroup of preterm infants with a high risk of death secondary to hypoxemia. Although this report is based on a small number of cases, it follows the directions of other studies that suggest that iNO therapy can benefit preterm neonates, particularly those exposed to oligohydramnios.