The prospect of FKBP51 as a drug target

The FK506 binding protein 51 (FKBP51) is best known as an Hsp90-associated co-chaperone that regulates the responsiveness of steroid hormone receptors. In human genetic association studies, FKBP51 has repeatedly been associated with emotion processing and numerous stress-related affective disorders. It has also been implicated in contributing to the glucocorticoid hyposensitivity observed in New World primates. More recently, several research groups have consistently shown a protective effect of FKBP51 knockout or knockdown on stress endocrinology and stress-coping behavior in animal models of depression and anxiety. The principal druggability of FKBP51 is exemplified by the prototypic FKBP ligands FK506 and rapamycin. Moreover, FKBP51 is highly suited for X-ray co-crystallography, which should facilitate the rational drug design of improved FKBP51 ligands. In summary, FKBP51 has emerged as a promising new drug target for stress-related disorders that should be amenable to drug discovery.     

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

A series of DNA-linked RNases H, in which the 15-mer DNA iscross-linked to the Thermus thermophilus RNase HI (TRNH) variantsat positions 135, 136, 137 and 138, were constructed and analyzedfor their abilities to cleave the complementary 15-mer RNA.Of these, that with the DNA adduct at position 135 most efficientlycleaved the RNA substrate, indicating that position 135 is themost appropriate cross-linking site among those examined. Toexamine whether DNA-linked RNase H also site-specifically cleavesa highly structured natural RNA, DNA-linked TRNHs with a seriesof DNA adducts varying in size at position 135 were constructedand analyzed for their abilities to cleave MS2 RNA. These DNAadducts were designed such that DNA-linked enzymes cleave MS2RNA at a loop around residue 2790. Of the four DNA-linked TRNHswith the 8-, 12-, 16- and 20-mer DNA adducts, only that withthe 16-mer DNA adduct efficiently and site-specifically cleavedMS2 RNA. Primer extension revealed that this DNA-linked TRNHcleaved MS2 RNA within the target sequence.     

Inhibitors of FabI, an enzyme drug target in the bacterial fatty acid biosynthesis pathway

The modern age of drug discovery, which had been slowly gathering momentum during the early part of the twentieth century, exploded into life in the 1940s with the isolation of penicillin and streptomycin. The immense success of these early drug discovery efforts prompted the general view that many infectious diseases would now be effectively controlled and even eradicated. However this initial optimism was misplaced, and pathogens such as multidrug-resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus present a major current threat to human health. Drug resistance arises through the unrelenting pressure of natural selection, and there is thus a continuing need to identify novel drug targets and develop chemotherapeutics that circumvent existing drug resistance mechanisms. In this Account, we summarize current progress in developing inhibitors of FabI, the NADH-dependent enoyl reductase from the type II bacterial fatty acid biosynthesis pathway (FAS-II), a validated but currently underexploited target for drug discovery. The FabI inhibitors have been divided into two groups, based on whether they form a covalent adduct with the NAD (+) cofactor. Inhibitors that form a covalent adduct include the diazaborines, as well as the front-line tuberculosis drug isoniazid. The NAD adducts formed with these compounds are formally bisubstrate enzyme inhibitors, and we summarize progress in developing novel leads based on these pharmacophores. Inhibitors that do not form covalent adducts form a much larger group, although generally these compounds also require the cofactor to be bound to the enzyme. Using structure-based approaches, we have developed a series of alkyl diphenyl ethers that are nanomolar inhibitors of InhA, the FabI from M. tuberculosis, and that are active against INH-resistant strains of M. tuberculosis. This rational approach to inhibitor development is based on the proposal that high-affinity inhibition of the FabI enzymes is coupled to the ordering of a loop of amino acids close to the active site. Compounds that promote loop ordering are slow onset FabI inhibitors with increased residence time on the enzyme. The diphenyl ether skeleton has also been used as a framework by us and others to develop potent inhibitors of the FabI enzymes from other pathogens such as Escherichia coli, S. aureus, and Plasmodium falciparum. Meanwhile chemical optimization of compounds identified in high-throughput screening programs has resulted in the identification of several classes of heteroaromatic FabI inhibitors with potent activity both in vitro and in vivo. Finally, screening of natural product libraries may provide useful chemical entities for the development of novel agents with low toxicity. While the discovery that not all pathogens contain FabI homologues has led to reduced industrial interest in FabI as a broad spectrum target, there is substantial optimism that FabI inhibitors can be developed for disease-specific applications. In addition, the availability of genome sequencing data, improved methods for target identification and validation, and the development of novel approaches for determining the mode of action of current drugs will all play critical roles in the road ahead and in exploiting other components of the FAS-II pathway.     

Functionalized bacterial cellulose nanowhiskers as long‐lasting drug nanocarrier for antibiotics and anticancer drugs

A nano drug carrier based on sustainable and biocompatible nanocellulose was developed for use in prolonged drug releases. The grafting of β‐cyclodextrin (βCD) on bacterial cellulose nanowhiskers (BCNC) using citric acid (CA) as a green linker was performed. This led to the formation of functionalized BCNC‐grafted‐βCD (BCNC‐g‐βCD). Broad‐spectrum antibiotic Ciprofloxacin (CIP) and anticancer drugs Doxorubicin (DOX) and Paclitaxel (PTX) were used as model drugs. These model drugs were conjugated to BCNC‐g‐βCD to form the drug‐nanocarrier systems (BCNC‐g‐βCD‐drug). The change in the nanowhiskers’ surface chemistry, morphology, and crystallinity was characterized by FTIR, solid‐state ¹³C NMR, scanning electron microscopy (SEM), atomic force microscopy (AFM), and x‐ray diffraction (XRD). The functionalized nanowhiskers showed a significant increase in the drug payloads, which ranged from 495 ±4–810 ±7 μg/mg, along with a radical improvement in the drug release profiles. For all of the developed drug‐conjugated nanocarriers, the initial burst releases were reduced effectively. The observed drug releases showed a sustained and controlled manner, with cumulative releases of 75–90 % over 5–5.5 days. Nevertheless, an improved drug release performance was observed in the acidic pH of 6.4 that mimicked extracellular tumor cells. In vitro drug release data were fitted zero‐order kinetic model with drug release constants (K₀) of 0.68, 0.74, and 0.79 μg drug/h (at pH 6.4 and 37 °C) for BCNC‐g‐βCD‐CIP, BCNC‐g‐βCD‐DOX, and BCNC‐g‐βCD‐PTX nanosystems, respectively. The observed higher payloads along with the slow releases of drugs from the developed nanocarrier suggests its promising potential for reducing the frequent daily dosing and minimizing systemic toxicity of loaded drugs.     

Targeting Intra-Pulmonary P53-Dependent Long Non-Coding RNA Expression as a Therapeutic Intervention for Systemic Lupus Erythematosus-Associated Diffuse Alveolar Hemorrhage

Diffuse alveolar hemorrhage (DAH) in systemic lupus erythematosus (SLE) is associated with significant mortality, requiring a thorough understanding of its complex mechanisms to develop novel therapeutics for disease control. Activated p53-dependent apoptosis with dysregulated long non-coding RNA (lncRNA) expression is involved in the SLE pathogenesis and correlated with clinical activity. We examined the expression of apoptosis-related p53-dependent lncRNA, including H19, HOTAIR and lincRNA-p21 in SLE-associated DAH patients. Increased lincRNA-p21 levels were detected in circulating mononuclear cells, mainly in CD4+ and CD14+ cells. Higher expression of p53, lincRNA-p21 and cell apoptosis was identified in lung tissues. Lentivirus-based short hairpin RNA (shRNA)-transduced stable transfectants were created for examining the targeting efficacy in lncRNA. Under pristane stimulation, alveolar epithelial cells had increased p53, lincRNA-p21 and downstream Bax levels with elevated apoptotic ratios. After pristane injection, C57/BL6 mice developed DAH with increased pulmonary expression of p53, lincRNA-p21 and cell apoptosis. Intra-pulmonary delivery of shRNA targeting lincRNA-p21 reduced hemorrhage frequencies and improved anemia status through decreasing Bax expression and cell apoptosis. Our findings demonstrate increased p53-dependent lncRNA expression with accelerated cell apoptosis in the lungs of SLE-associated DAH patients, and show the therapeutic potential of targeting intra-pulmonary lncRNA expression in a pristane-induced model of DAH.     

Evaluation of alginate–chitosan bioadhesive beads as a drug delivery system for the controlled release of theophylline

This study describes the preparation of mucoadhesive alginate–chitosan beads containing theophylline intended for colon‐specific delivery. The calcium alginate beads were coated with chitosan by the ionotropic hydrogelation method with a polyelectrolyte complex reaction between two oppositely charged polyions. The release profiles of theophylline from the beads were determined by ultraviolet–visible absorption measurement at 272 nm. Scanning electron microscopy was used for morphology observation. The in vitro mucoadhesive tests for particles were carried out with the freshly excised jejunum of Sprague‐Dawley rats. The bead particles, which ranged in size from 200 to 400 μm, exhibited excellent mucoadhesive properties. The results showed that the formulated coated beads succeeded in controlling the release of theophylline over a 24‐h period. In conclusion, the release of theophylline was found to be dependent on the composition of the beads, the component polymer and its possible interactions, and the bioadhesiveness. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Stimuli-responsive hybrid microgels for controlled drug delivery: Sorafenib as a model drug

Microgels (MGs) are synthetic colloidal hydrogel particles made of three dimensional polymer networks. Their chemical composition is crucial for their use as intelligent drug release systems operated by temperature control. Herein, several MGs using N-isopropylacrylamide (Nipam)/N-isopropylmethacrylamide (Nipmam), chitosan and acrylic/methacrylic acid have been synthesized by free radical polymerization reactions (NC MGs) and the effects of surfactants and different reaction times on size and swelling properties have been investigated. MGs have been identified and characterized by dynamic light scattering and atomic force microscopy, and finally used to optimize the encapsulation protocol of the hydrophobic drug sorafenib. The drug delivery system here described has encapsulation efficiency of 40% and releases 10% of the entrapped drug over about 16 h after the temperature is raised above the volume phase transition temperature. Data suggest that MGs with optimized composition may act as properly instructed entities able to trap and release biomolecules following external stimuli.     

In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction

After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.     

Evaluation of an oleic acid water-in-oil-in-water-type multiple emulsion as potential drug carriervia the enteral route

A water-in-oil-in-water (W/O/W) emulsion composed of oleic acid was used as a carrier of carboxyfluorescein (CF)via the enteral route, as a model for future drug transport. The absorption of CF in the small intestine of rats given the emulsion (W/O/W group) was compared with the absorption in a group administered CF alone (CF group), and a surface-active agent in CF solution (MM group). Higher amounts of CF were absorbed in the W/O/W and MM groups than in the CF group. At 120 min, the amount of CF remaining in the intestinal tract was smaller in the MM group than in the W/O/W group. In the early period, CF excretion into bile was higher in the MM group than in the W/O/W group was higher than in the MM group (non-specific). The blood CF level was significantly higher at 240 and 360 min in the W/O/W group than in the other two groups. The highest concentration in lymph was found in the W/O/W group. The W/O/W emulsion was considered superior to the micelles because it maintained a higher blood level of CF over long periods and transferred it to the lymph. This suggests that the W/O/W emulsion is applicable as a drug carriervia the enteral route.     

Novel metronidazole-loaded hydrogel as a gastroretentive drug delivery system

A metronidazole-loaded hydrogel was synthesized by free radical polymerization using dimethylaminoethyl methacrylate (DMAEMA) monomer and triethyleneglycol dimethacrylate (TEGDA) and methylene bisacrylamide (MBA) as cross-linkers. The DMAEMA hydrogels were cross-linked with 5 and 10% MBA or with 0.1, 0.5, 1 and 4% TEGDA as cross-linking agents. Ammonium persulfate and tetramethyl ethylene diamine were used as initiator and catalyst, respectively. The prepared hydrogels were characterized, and the effect of cross-linking agent content on the swelling behavior and in vitro drug release of hydrogels was investigated. The results of X-ray diffractometry, differential scanning calorimetry and Fourier transform infrared spectroscopy studies indicated that the prepared hydrogels possessed an amorphous morphology and there was not any interaction between the hydrogel polymers and metronidazole as drug, which resulted in the dependence of drug release on the physicochemical characteristics of hydrogel such as swelling, polymer erosion, and surface morphology. According to the results, the hydrogel containing 0.5% TEGDA which was prepared by freeze-drying method exhibited a porous structure with a high swelling ratio and displayed a sustained and complete drug release. It could be concluded that the hydrogel developed by this facile method is a good candidate with a potential for use in gastroretentive drug delivery systems.     

Application of molecularly imprinted polymer as a drug retaining matrix

Copolymerization by gamma irradiation of 2-hydroxyethyl methacrylate with ethylene glycol dimethacrylate in the presence of hydrocortisone as model drug gave a molecular imprinted polymer (MIP). The hydrocortisone was removed from the matrix with chloroform and incorporated again from methanol solution. Release data show that 96% of the incorporated drug is retained after 30 days storage in a methanol/water (3 : 1 v/v) mixture. In comparison with hydrocortisone, only a small quantity of testosterone is absorbed by the matrix.     

The intestinal mucosa as a target for dietary polyunsaturated fatty acids

Several studies have reported beneficial effects of dietary polyunsaturated fatty acids (PUFA) on various aspects of both human and animal health, and particular reference has been made to their effects on systemic immune responses. Both immune stimulation and immune suppression have been reported, with the outcome dependent on the type of PUFA, the target cell, as well as the immune competence of the cells before exposure. The systemic and the mucosal immune systems are discrete entities, which have evolved specific approaches in the defense of the host. The latter comprises several interconnected tissues, which communicate with one another through the action of soluble mediators and the trafficking of cellular components. After the oral mucosa, the intestinal epithelium and its associated gutassociated lymphoid tissue are the primary targets of dietary components. Absorption of dietary PUFA and its incorporation into intestinal tissues has been well studied, but the consequences of these events in relation to local immune responses have received little attention. This article describes some of the immune mechanisms operating at this barrier and, where possible, pinpoints areas for which a modulatory role for PUFA has already been demonstrated. Although not an exhaustive treatise of the subject, it is hoped that this review will foster research into the specific interaction between dietary PUFA and cell populations comprising the intestinal barrier.