Surface-enhanced Raman spectroscopy study of the interaction of the antitumoral drug emodin with human serum albumin

Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.     

Variability in Response to Drugs

Variability in the response to drugs is due to three principal components—the disease, the responsiveness of tissues, and the concentration of the drug at its site of action (as reflected by its plasma concentration). The relative contributions of these components will differ not only for different drugs but also for different effects of the same drug. Rational drug therapy depends on knowledge of all three factors.     

纳米靶向给药系统载体材料的研究进展

纳米靶向给药系统可以增强药物在病变部位的浓度和疗效,同时也可以最大限度地降低药物的毒副作用,因此已成为现代药剂学研 究的重要内容,其中对纳米载体材料的研究也越来越多。对2013年度国内学者在纳米靶向给药制剂中载体材料的研究与开发进展进行综述。     

DMSO (dimetyl sulfoxide), morphine and analgesia

DMSO was compared to morphine in rats to determine its relative analgesic effects. DMSO produces analgesia that is comparable in magnitude to morphine although its duration (6–7 hrs) is longer than that of morphine (≤ 2 hrs). DMSO apparently produced analgesia both by an action at the site at which it was administered as well as at a site that was remote to the site of administration. The mechanism of action of DMSO is apparently different from that of morphine because naloxone, a specific narcotic antagonist, does not block the analgesic effect of DMSO. However, DMSO has toxic effects such as hematuria (bloody urine). Therefore, the toxicity observed may restrict the clinical usefulness of DMSO as an analgesic drug.     

Photochemical affinity labeling of the macrolide binding site on the 70S E. coli ribosome

Photoactivation of the alpha, beta-unsaturated ketone-epoxide system of [3H] dihydrorosaramycin at a wavelength above 300 nm allows the covalent attachment of the antibiotic to its receptor site. The radioactivity is mainly associated to proteins L1, L5, L6, S1; as a consequence, the binding site of this type of drug could be located at the peptidyltransferase center and in between both subunits.     

3ircular dichroism simulation shows a site-II-to-site-I displacement of human serum albumin-bound diclofenac by ibuprofen

Purpose: The purpose of this study was to confirm the hypothesis that a site-II-to-site-I displacement takes place when some nonsteroidal anti-inflammatory drugs are displaced by another drug from their high-affinity binding site to a site of lower affinity on human serum albumin (HSA).Methods: Diclofenac, sodium salt, was used as a representative example because of its prominent reversal of the Cotton effect. Effects of site-specific drugs on the free fraction of diclofenac were determined by equilibrium dialysis, and effects on induced circular dichroism (CD) of diclifenac bound to HSA were studied by CD and CD simulation techniques.Results: Ibuprofen, a site-II-specific drug, altered the CD spectrum of the diclofenac-HSA complex at a molar ratio of 0.5∶1 to that obtained at a higher ratio (5∶1) without ibuprofen. The induced CD spectrum obtained in the presence of ibuprofen was very similar to one that assumed that all diclofenac displaced from its high-affinity binding site (site II) became rebound to a lower-affinity site (site I). The rebinding could be influenced by a free energy linkage between the two sites which would make site I (or parts thereof) more suitable for diclofenac binding.Conclusion: We have confirmed the existence of a site II-to-site displacement, which is very striking and pharmacologically important, because the concentration of unbound drug being displaced is much lower than expected for a competitive mechanism.     

A Remote Controlled Valve in Liposomes for Triggered Liposomal Release

In order to reduce the toxicity and increase the efficacy of drugs, there is a need for smart drug delivery systems. Liposomes are one of the promising tools for this purpose. An ideal liposomal delivery system should be stable, long-circulating, accumulate at the target site and release its drug in a controlled manner. Even though there have been many developments to this end, the dilemma of having a stable liposome during circulation but converting it into a leaky structure at the target site is still a major challenge. So far, most attempts have focused on destabilizing the liposome in response to a specific stimulus at a target site, but with limited success. Our approach is to keep the stable liposome but build in a remote-controlled valve as a new release mechanism, instead. The valve is a pore-forming bacterial membrane protein. It has been engineered such that, after being reconstituted into the liposomes, its opening and closing can be controlled on command by the ambient pH, light or a combination of both. In addition, a much higher degree of flexibility for fine-tuning of the liposome's response to its environment is achieved.     

Reversible human serum albumin binding of lipocrine: a circular dichroism study

Lipocrine has been selected as an effective candidate for in vivo investigation because of its multiple biological properties, namely inhibition of AChE and BChE activities, inhibition of AChE-induced Aβ aggregation, and ability to protect cells against reactive oxygen species. To evaluate the possibility for lipocrine to become a lead and to be developed as a multipotent drug for the treatment of Alzheimer's disease, ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters need to be determined. Among ADMET parameters, distribution plays a key role in determining the lead drugability, and the drug binding to plasma proteins greatly influences the drug distribution. Here, the human serum albumin (HSA) binding of lipocrine has been studied by circular dichroism (CD) spectroscopy. The reversible binding of lipocrine is stereoselective as shown by the well-defined induced CD spectrum in its binding to HSA. The intensity of the CD signal changes upon changing the [drug]/[HSA] molar ratio, showing a different behavior for a [drug]/[HSA] up to 2/1 or over this molar ratio, suggesting a binding to multiple sites. Competition experiments show that lipocrine interacts significantly with all the main binding sites on the serum carrier. A direct competition has been monitored for site II and bilirubin-binding site, whereas a noncooperative binding should better describe the displacement observed at site I. Rac-lipocrine and its enantiomers are characterized by two different binding modes. Almost the same induced CD spectra were obtained for both (R)- and (S)-lipocrine complexed to HSA, suggesting a similar stereochemistry for the bound enantiomers.     

A remote controlled valve in liposomes for triggered liposomal release

In order to reduce the toxicity and increase the efficacy of drugs, there is a need for smart drug delivery systems. Liposomes are one of the promising tools for this purpose. An ideal liposomal delivery system should be stable, long-circulating, accumulate at the target site and release its drug in a controlled manner. Even though there have been many developments to this end, the dilemma of having a stable liposome during circulation but converting it into a leaky structure at the target site is still a major challenge. So far, most attempts have focused on destabilizing the liposome in response to a specific stimulus at a target site, but with limited success. Our approach is to keep the stable liposome but build in a remote-controlled valve as a new release mechanism, instead. The valve is a pore-forming bacterial membrane protein. It has been engineered such that, after being reconstituted into the liposomes, its opening and closing can be controlled on command by the ambient pH, light or a combination of both. In addition, a much higher degree of flexibility for fine-tuning of the liposome's response to its environment is achieved.     

Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution

Enzymes of glycolysis in Trypanosoma brucei have been identified as potential drug targets for African sleeping sickness because glycolysis is the only source of ATP for the bloodstream form of this parasite. Several inhibitors were previously reported to bind preferentially to trypanosomal phosphoglucose isomerase (PGI, the second enzyme in glycolysis) than to mammalian PGIs, which suggests that PGI might make a good target for species-specific drug design. Herein, we report recombinant expression, purification, crystallization and X-ray crystal structure determination of T. brucei PGI. One structure solved at 1.6 A resolution contains a substrate, D-glucose-6-phosphate, in an extended conformation in the active site. A second structure solved at 1.9 A resolution contains a citrate molecule in the active site. The structures are compared with the crystal structures of PGI from humans and from Leishmania mexicana. The availability of recombinant tPGI and its first high-resolution crystal structures are initial steps in considering this enzyme as a potential drug target.     

Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

Magnetic drug targeting through a realistic model of human tracheobronchial airways using computational fluid and particle dynamics

Biomechanics and Modeling in Mechanobiology - Magnetic drug targeting (MDT) is a local drug delivery system which aims to concentrate a pharmacological agent at its site of action in order to...     

Inhibition of tubulin assembly by antileprosy drug dapsone

The effect of dapsone on assembly-disassembly process of bovine brain tubulin was examined. The drug was found to readily bind tubulin dimer and that in its presence colchicine binding to tubulin was enhanced. Although dapsone associated with tubulin at a site other than the colchicine binding site, distinct inhibition of microtubule assembly was detected.     

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.     

Interaction of a spin-labeled adenine-acridine conjugate with a DNA duplex containing an abasic site model

The abasic site is a common lesion in DNA that is also formed as an intermediate in the base excision repair of damaged bases. We have previously reported the adenine-acridine conjugate 1 that was designed to bind to the abasic site and interfere with the repair process. High-field NMR had shown that 1 forms specific complexes with a DNA duplex containing an apurinic abasic site model. We report here the dynamics of the interaction of the nitroxide-labeled analogue 3 of the conjugate 1 with the same apurinic oligonucleotide and with the parent unmodified duplex. Identical study of the labeled acridine subunit 5 used as a reference is also reported. In the presence of the apurinic duplex and depending on the concentrations and drug ratios, three species are observed: the radical "free in solution", the "intercalation" complex characterized by its similarity to that observed in the presence of the parent unmodified duplex, and the "abasic-site-specific" complex which is the sole species visible at low drug ratios. The experimental data reinforced by molecular modeling of the complex and theoretical calculation of correlation times suggest (i) the most immobilized form corresponds to that observed by NMR and (ii) complexation of the drug is little or not modified by the spin-label. We also show that the abasic site constitutes a binding site for the propylaminoacridine intercalator 5.     

淋病奈瑟菌临床菌株porB基因分型及其突变与耐药相关性研究

了解淋病奈瑟菌临床菌株porB基因型及其产物120和121位氨基酸突变与耐药的相关性。采用多重PCR(mPCR)检测淋病奈瑟菌临床菌株porB基因型,扩增产物测序后分析其编码蛋白的120和121位氨基酸突变情况,二倍平皿稀释法检测临床菌株对青霉素和四环素的耐药性。184株淋病奈瑟菌临床菌株中,99.5%(183/184)检出porB基因,其中61株(33.3%)为porB1A基因型,122株(66.7%)为porB1B基因型。122株porB1B基因型菌株中,117株(95.9%)porB基因120和/或121位氨基酸发生突变,5株(4.1%)porB1B及所有porB1A基因型菌株porB基因120和/或121位氨基酸未突变。117株porB基因120和/或121位氨基酸突变的porB1B基因型菌株中,97.4%(114/117)和95.7%(112/117)分别对青霉素和四环素耐药,2.6%菌株(3/117)对青霉素和四环素敏感。61株porB1A基因型菌株中,仅有2株(3.3%)对青霉素和四环素耐药。研究中采用的mPCR能快速、准确地对淋病奈瑟菌临床菌株porB基因进行分型,这些菌株主要携带porB1B基因且该基因型菌株对青霉素和四环素耐药率显著高于porB1A基因型(P<0.01),该耐药性与porB1B基因120和/或121位氨基酸突变密切相关。     

EVALUATING THE EFFICACY OF AMIKACIN IN LOW-CLEARANCE UNILAMELLAR LIPOSOMES IN A S. AUREUS LOCAL INFECTION MODEL

Traditional therapies for Staphylococcal infections such as osteomyelitis or localized abscesses have a difficult time penetrating into tissue sites. To effectively ameliorate these infections, prolonged therapy and/or high doses of antibiotics are frequently required. Aminoglycosides, such as amikacin, are not routinely utilized for treating local infections due to poor efficacy associated with ineffective tissue penetration, toxicity, and poor penetration in an acid millieu. We postulated that a formulation of amikacin in small unilamellar liposomes might readily be engulfed by inflammatory macrophages facilitating drug delivery to the site of infection. This increased drug load to the site of bacterial infection may result in enhanced bactericidal action compared to conventional aminoglycosides. Tissue drug concentrations were determined for liposomal amikacin (L-AN) and conventional amikacin (AN). Plasma amikacin levels were determined for L-AN. The L-AN was very effective at concentrating at the site of infection compared to AN. Following confirmation of adequate tissue drug levels, a rodent subcutaneous abscess infection using S. aureus as the bacterial challenge agent was evaluated. Sprague-Dawley rats were intravenously administered L-AN every other day due to its prolonged half-life, while the comparator agent, AN, was administered daily. Abscess size, weights, severity, histology, and tissue colony counts were examined. In efficacy studies, L-AN was superior to AN in reducing colony counts.     

Evidence of an adriamycin binding site in the secretory granules of the mast cell

Adriamycin induced significant non-cytotoxic histamine release from rat peritoneal mast cells to which the drug showed a very high affinity. The relationship between adriamycin-induced exocytosis and its uptake by purified rat peritoneal mast cells was studied. Adriamycin induced histamine release and was highly concentrated in mast cells at 37 degrees C but not at 0 degrees C. However, if exocytosis was provoked by other secretagogues like compound 48/80, protamine, concanavalin A, and ionophore A23187, and cells were then treated with adriamycin at 0 degrees C, the concentrations of the antineoplastic drug significantly increased. Adriamycin binding to purified granular material was similar to that of intact cells treated at 37 degrees C, but was not modified by metabolic inhibitors, extremes of temperature (0 or 45 degrees C) or by the carboxylic ionophore monensin. On the contrary, sodium cromoglycate limited adriamycin binding to granular materials as well. In addition, sodium cromoglycate, but not monensin, displaced the antineoplastic drug from mast cells, even when added after adriamycin. We conclude that the high affinity of adriamycin for mast cells is ascribable to the externalization of a granular binding site, as a consequence of the exocytotic process. The experiments with sodium cromoglycate suggest that this binding site could be in common with the antiallergic drug.     

Novel Epigenetic Target Therapy for Prostate Cancer: A Preclinical Study

Epigenetic events are critical contributors to the pathogenesis of cancer, and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy. Classic demethylating agents, such as 5-Aza-2′-deoxycytidine (Decitabine), hold the potential for reprograming somatic cancer cells demonstrating high therapeutic efficacy in haematological malignancies. On the other hand, epigenetic treatment of solid tumours often gives rise to undesired cytotoxic side effects. Appropriate delivery systems able to enrich Decitabine at the site of action and improve its bioavailability would reduce the incidence of toxicity on healthy tissues. In this work we provide preclinical evidences of a safe, versatile and efficient targeted epigenetic therapy to treat hormone sensitive (LNCap) and hormone refractory (DU145) prostate cancers. A novel Decitabine formulation, based on the use of engineered erythrocyte (Erythro-Magneto-Hemagglutinin Virosomes, EMHVs) drug delivery system (DDS) carrying this drug, has been refined. Inside the EMHVs, the drug was shielded from the environment and phosphorylated in its active form. The novel magnetic EMHV DDS, endowed with fusogenic protein, improved the stability of the carried drug and exhibited a high efficiency in confining its delivery at the site of action in vivo by applying an external static magnetic field. Here we show that Decitabine loaded into EMHVs induces a significant tumour mass reduction in prostate cancer xenograft models at a concentration, which is seven hundred times lower than the therapeutic dose, suggesting an improved pharmacokinetics/pharmacodynamics of drug. These results are relevant for and discussed in light of developing personalised autologous therapies and innovative clinical approach for the treatment of solid tumours.     

Structure-based in-silico rational design of a selective peptide inhibitor for thymidine monophosphate kinase of mycobacterium tuberculosis

Tuberculosis still remains one of the most deadly infectious diseases. The emergence of drug resistant strains has fuelled the quest for novel drugs and drug targets for its successful treatment. Thymidine monophosphate kinase (TMPK) lies at the point where the salvage and de novo synthetic pathways meet in nucleotide synthesis. TMPK in M.tb has emerged as an attractive drug target since blocking it will affect both the pathways involved in the thymidine triphosphate synthesis. Moreover, the unique differences at the active site of TMPK enzyme in M.tb and humans can be exploited for the development of ideal drug candidates. Based on a detailed evaluation of known inhibitors and available three-dimensional structures of TMPK, several peptidic inhibitors were designed. In silico docking and selectivity analysis of these inhibitors with TMPK from M.tb and human was carried out to examine their differential binding at the active site. The designed tripeptide, Trp-Pro-Asp, was found to be most selective for M.tb. The ADMET analysis of this peptide indicated that it is likely to be a drug candidate. The tripeptide so designed is a suitable lead molecule for the development of novel TMPK inhibitors as anti-tubercular drugs.