Letters in Drug Design & Discovery - Volume 8, Issue 2, 2011

Multidrug resistant (MDR) bacteria constitute an ever-increasing threat to public health. The main mechanism of MDR is the active expulsion of drugs by bacterial pumps that expel unrelated compounds. The increase of knowledge, the understanding of efflux-mediated resistance and the translation of it into the development of original antimicrobials constitute important areas to report new results. The prevalence and the evolution of bacterial drug efflux mechanisms and its genetic regulation, the identification of the risk factors, and the synthesis and evaluation of molecules that obviate efflux-mediated resistance are subjects of interest at the moment, which may convincingly justify a special issue of Letters in Drug Design Discovery. Papers on molecular basis of drug efflux, structural genomics, molecular modelling, production of new molecules and their time stability, chemosensitizers or inhibitory agents are included in this issue. This is part of a multi- and inter-disciplinary approach to identify new targets and provide a generation of effective agents against efflux mechanisms in MDR bacterial pathogens. More general, infectious diseases cause more than 13 millions deaths each year. This high mortality impact will possibly increase, due to accelerated evolution of antibiotic resistance in important human pathogens and the scarcity of new anti-infective drug families. On the other hand, the development of antibiotic resistance is a natural evolutionary process which induces erosions of the effectiveness of the arsenal of antibiotics, leading to increased mortality in humans and even fauna. This is also related to global warming and the extreme conditions of natural and artificial origins in which humans are obliged to live and work nowadays. A significant number of papers, institutions and countries contribute to this issue; the articles are mainly review articles, but some of them are devoted to new results and the related methods obtained/ developed by the respective authors, who belong to prestigious research centres active in MDR studies. In total, 30 authors from 12 research institutions/universities located in 6 countries contribute to this special issue; most of them are participants at the European COST network named ATENS which includes among its members groups of outstanding scientists working to integrate in Europe the research efforts dedicated to fight against MDR . I hope this special volume of LDDD will provide the reader with an understanding of MDR research, its progress, and results obtained by some of the best scientific European communities dedicated to this subject.

Multi-drug resistance (MDR) of Gram-negative and Gram-positive bacteria is strongly associated with a modification of membrane permeability that alters the intracellular concentration of antimicrobial agents such as antibiotics, antiseptics and biocides. In order to combat this bacterial strategy, coordinated efforts from diverse and complementary scientific areas are essential - from clinical bacteriology, bacterial physiology, metabolic regulation, structural biology, biochemistry, biophysics, molecular modeling, chemical synthesis and systems biology. The COST (European Cooperation in the field of Scientific and Technical research) Action network BM0701 designated ATENS (Antibiotic Transport and Efflux: New Strategies to combat bacterial resistance) was initiated in 2008. The main aim of ATENS is to provide a framework, through collaboration, to increase knowledge and understanding of the efflux-mediated resistance in bacterial pathogens and to translate this knowledge into the development of novel antimicrobials. Its subobjectives are to determine prevalence and evolution of bacterial drug efflux mechanisms, identify risk factors, decipher genetic regulation of this mechanism, elucidate the functional and structural bases of efflux resistance, and to synthesize and evaluate molecules that obviate efflux-mediated resistance. More than 35 teams belonging to 20 member states constitute 4 Working Groups: Clinical and Veterinary Bacteriology; Molecular Basis of Drug Efflux; Structural Genomics, Bioinformatics and Molecular Modelling; they are also involved in the production of new molecules, chemosensitizers or inhibitory agents. This multi- and interdisciplinary approach should identify new targets and generate effective agents against efflux mechanisms in MDR bacterial pathogens. ATENS has already accomplished the following: produced more than 50 joint publications involving several BM0701 teams, including special issues focused on antibiotic resistance like the articles in this issue; organized five european COST meetings; and funded several students exchanges between partners to promote an efficient and fruitful transfer of knowledge. In addition, several COST members have contributed to International meetings in the field. We believe that our joint efforts will initiate new discoveries and strategies to overcome multidrug resistance.

A survey on MDR modulators containing heterocyclic moiety in the chemical structure for the past 20 years is provided. The discussion of the particular active compound is classified according to the heterocyclic rings involved. The most important biological results are also summarized.

Multidrug resistance (MDR) to antibiotics presents a serious therapeutic problem in the treatment of bacterial infections. The importance of this mechanism of resistance in clinical settings is reflected in the increasing number of reports of multidrug resistant isolates. In Salmonella enterica, the most common etiological agent of food borne salmonellosis worldwide, MDR is becoming a major concern. In Salmonella the main mechanisms of antibiotic resistance are mutations in target genes (such as DNA gyrase and topoisomerase IV) and the over-expression of efflux pumps. However, other mechanisms such as changes in the cell envelope; down regulation of membrane porins; increased lipopolysaccharide (LPS) component of the outer cell membrane; quorum sensing and biofilm formation can also contribute to the resistance seen in this microorganism. To overcome this problem new therapeutic approaches are urgently needed. In the case of efflux-mediated multidrug resistant isolates, one of the treatment options could be the use of efflux pump inhibitors (EPIs) in combination with the antibiotics to which the bacteria is resistant. By blocking the efflux pumps resistance is partly or wholly reversed, allowing antibiotics showing no activity against the MDR strains to be used to treat these infections. Compounds that show potential as an EPI are therefore of interest, as well as new strategies to target the efflux systems. Quorum sensing (QS) and biofilm formation are systems also known to be involved in antibiotic resistance. Consequently, compounds that can disrupt or inhibit these bacterial “communication systems” will be of use in the treatment of these infections.

The 3-[2-(dimethylamino)ethyl]-6-nitroquinazolin-4(3H)-one (BG1188) may modify the antibiotic susceptibility of human Gram-negative bacteria which present multidrug resistant phenotype. This application requires to know the time stability of BG1188 solutions. This paper reports for the first time about the time stability of the BG1188 solutions measured by optical absorption; the solvents were ultrapure de-ionized water and dimethyl sulfoxide (DMSO). In ultrapure water, BG1188 concentration varied between 2×10-3M (stock solution) and 10-6M, at pH = 6.7. In DMSO, the concentration was 10-3M (recommended by higher activity on bacteria). Solutions in ultrapure water were kept in dark at 4°C and measured at room temperature (22°C). The ultraviolet-visible (UV-Vis) absorption spectra have shown that only monomer forms were present in solutions. One of the obtained results is that the time evolution of the absorption spectra at 10-3M in ultrapure water shows a reproducibility within the measuring errors (±1.045%) for time intervals up to 1032 hours (more than 40 days) after preparation, showing a high stability of BG1188 solutions; generally, samples are stable within the experimental errors at concentrations higher than 10-5M, but the stability time interval may be variable: from 119 days at 10-4M to 34 days at 10-5M. As for solutions in the DMSO, the absorption spectra show that the samples exhibit modifications immediately after preparation, in terms of peak intensities and shapes, so that the BG1188 solutions in DMSO may be considered as unstable.

Pulmonary tuberculosis infections caused by multi-drug resistant Mycobacterium tuberculosis has progressed to extensively drug resistant status (XDR-TB). XDR-TB is very difficult to treat successfully and results in high mortality. Globally, XDR-TB is now a major threat, especially to India and countries that were once part of the Soviet Union. There is a potential alternative to current ineffective therapy that is solidly supported by in vitro, ex vivo and in vivo studies. It has reported successful therapies in 10 out of 12 non-responsive XDR-TB patients. That therapy is thioridazine, and it is the purpose of this mini-review to provide the rationale for thioridazine therapy, especially for compassionate reasons, when all other therapies have failed, depicting on extremely poor prognosis.

A quorum is the smallest number of people able to organize the decisions concerning functional activity. Similarly microbes use chemical signal molecules to make population size-dependent “decisions” by changing their gene regulations. The inhibition of quorum sensing (QS) by phenothiazines and structurally related molecules, e.g. amitriptyline, promethazine, acridine orange, imipramine, promazine, diethazine, desipramine, desertomycin and 5-fluorouracil as positive control was studied with Chromobacterium violaceum 026 as a sensor strain, which detects short carbon chain AHLs by the development of a purple pigment. The AHL was produced by Novospingobium Ezf 10-17, and the antibiotic-resistant clinical isolates, E. coli 31298. The QS was demonstrated as a signal transmission between the two bacterial strains. The most effective inhibitors of QS were amitriptyline, promethazine, acridine orange and desertomycin. Imipramine and diethazine were moderately active, while chlorprothixene was ineffective relative to 5-fluorouracil as positive control. The direct complex formation between AHL and QS inhibitors markedly reduced the QS in a chromogenic test. The AHL-neutralizing effect of the related compounds was shown by chromogenic method. The inhibition of QS signal transmission appears to be related to the quasi-planar structure and electron donor capacity of the conjugated π-electron system of the tricyclic framework. The results can be exploited in rational drug design as a new way to reduce the QS mediated processes eg. virulence of pathogens, to vary the formation of biofilms and to modify antibiotic resistance.

Background: Multidrug resistance modifiers were indentified against ABC transporters such as ABCB1, but there are other resistance mechanisms based upon such transporters as CTR1 or ATP7A/B membrane proteins. Cisplatin resistance due to the lower expression of CTR1 or to the over-expression of ATP7A/B is also responsible for therapeutic failures in many cancer types. Materials and Methods: 35 modified steroid derivatives were tested for their multidrug reversal and proliferation inhibitory activity comparing their effects in human ABCB1-transfected mouse T-cell lymphoma and cisplatin resistant human ovary carcinoma cell lines. The selected potent resistance reversal agents were tested in a checkerboard assay in the presence of the anticancer drug doxorubicin on mouse lymphoma or cisplatin on human ovary cancer cell lines. Results: Correlations between chemical structure of different steroidal compounds on their effects on drug resistance were investigated. A common characteristic feature of D-ring substituents with N or O atom in the position of 1,3 was found within the effective inhibitors of proliferation when comparing the effective compounds on the two cancer cell lines. Conclusion: We assume that the polar substituents forming a bidentate part may serve as a binding moiety on the polar protein-glycan surface of the cells and can result in an effect independent of the structure of the A ring.

This study aimed at analyzing the relationship between the expression of P-gp, MRP, LRP, GST-π and TopoIIα and acquired resistance to cisplatin in lung adenocarcinoma cell line A549. Drug resistance to cisplatin was determined using MTT assays. The expressions of P-gp, MRP, LRP, GST-π and TopoIIα were measured by immunofluorescence, Western blot and RT-PCR. The results showed that there was a significant elevation of IC50 of cisplatin in A549/DDP cells compared to A549 cells (73.95 ± 7.26 versus 7.28 ± 0.74). The expression of P-gp and MRP in A549/DDP cells was approximately 1.5-fold higher than that in A549 cells (61.99±2.63 versus 43.52±1.22 ; 34.11±2.17 versus 22.80±2.31 respectively), whereas TopoIIα expression was about 3-fold lower in A549/DDP cells than that in A549 cells (0.34±0.06 versus 0.92±0.03). In conclusion, acquired resistance to cisplatin was associated with the up-regulation of P-gp and MRP and the down-regulation of TopoIIα in human lung adenocarcinoma cell line A549.

Two series of gemfibrozil chiral analogues were synthesized and evaluated for their agonistic activity on PPARα receptor, belonging to the transcription factor family. A simple method to synthesize ureidic and thioureidic fibrates is described. Their ability to interact with the PPARα receptor was evaluated by a transactivation assay in comparison with gemfibrozil, with the aim of obtaining new hypolipidemic compounds. Compounds characterized by a methyl and an ethyl group in α-position to the carboxylic group of gemfibrozil showed a good increase of the transcriptional activity of the receptor, and their capability to activate the receptor seems to be effected by the stereochemistry and the size of the substituent on chiral center. The ureidic and thioureidic compounds did not induce PPARα activity.

A web-based G-protein coupled receptor (GPCR) ab initio modeling platform has been developed to support scientists involved in internal in silico drug discovery projects. Starting from a target GPCR sequence, the ab initio modeling workflow predicts an ensemble of 3-dimensional (3D) receptor structures which can subsequently be used for structure- based drug design activities. The workflow used to develop the web-based tools integrates MembStruk and Modeler application packages with significant improvements and enhancements made to the former. The workflow enabled through the web-based user interface encompasses the following steps: a) prediction of the transmembrane (TM) regions using the target GPCR sequence and other similar sequences; b) initial packing of the TM-helices; c) optimization of the TM-helical bundles by translation, rotation, and relaxation of the helices; d) addition of intra- and extra-cellular loops; e) final refinement of the 3D receptor structures including side chains and loops. A wizard-like workflow approach implemented in web-based GPCR modeling tools hides the complexity involved in various modeling steps and guides the user through the calculation one step at a time starting from a target sequence to a set of predicted 3D structures. The successful implementation of the workflow is presented by modeling the human adenosine A2A receptor (AA2AR) structure without the bound ligand and comparing the predicted structure of the TM-helices with the recently reported X-ray crystal structure.

Molecular topology and multilinear regression analysis have been applied to achieve QSAR models for the prediction of the in vitro antiprotozoal activity against T. b. rhodesiense, P. falciparum as well as the cytotoxicity of a group of compounds analogs to pentamidine. Once the models were validated, a molecular screening was carried out for the search of new compounds showing high activity on both parasites and low cytotoxicity.

Panax ginseng C. A. Meyer (PG), a well-known traditional Chinese medicine, has been widely used as a panacea in treatment of human diseases for a long time. In this study, we exerted systems biology and bioinformatics methods for the first time and analyzed the “giant components” of PG in the global component-target interaction network. Based upon this network, we further confirmed several key components and their related targets (e.g., NF-κB, COX-2, Bax, NOS, JNK, p21 and MMP3). Additionally, we demonstrated that ginsenosides and key targets were involved in several cancer-related pathways, which indicated that ginsenosides might play crucial roles in cancer treatments. Interestingly, we also found that the component-target network might provide new clues for further predicting new targets and elucidating the relationships amongst the network and side-effects. In conclusion, these inspiring findings demonstrate that the versatile effects of PG are mostly ascribed to the comprehensive interactions amongst ginsenosides and their pivotal targets.

The reaction of 2-(aryloxymethyl)benzoic acid hydrazide with carbon disulfide in the presence of potassium hydroxide followed by hydrazine hydrate afforded 4-amino-3{2-(aryloxymethyl)pheny}[1,2,4]triazole-5-thiol (5a-d). The cyclo-condensation of 5a-d with various aromatic carboxylic acids in the presence of phosphorous oxychloride and with phenacyl bromides afforded two series of fused heterocycles namely; 6-substituted-3-{2-(aryloxymethyl)phenyl}-1,2,4- triazolo[3,4-b][1,3,4]thiadizoles (6a-t) and 6-substituted-3-{2-(aryloxymethyl)phenyl)}[1,2,4]triazolo[3,4-b][1,3,4] thiadiazines (7a-p) respectively. The structures of these newly synthesized compounds were established on the basis of their IR, 1H-NMR 13C-NMR and Mass spectral data. All the title compounds were screened for their antimicrobial and antiinflammatory activities. Preliminary results indicated that some of them exhibit promising activities and they deserve more consideration as potential antimicrobial and anti-inflammatory agents.