Current Drug Targets - Volume 7, Issue 7, 2006

"In the field of science, the unknown has no foothold that is absolutely secure. As for those who first step" into this field, they can only hope that the mistake they may make would be a credit to them (A. Szent-Gyorgyi). Giuseppe Verdi, the great Italian master of dramatic compositions of operas, could not compose his famous La Traviata because the classic tuberculosis had eradicated. Recently, the incurable form of the disease appeared, that rapidly kills the person despite the treatments due to multidrug resistance of mycobacterium. Resistance develops regularly to a variety of drugs used in the treatment of bacterial, fungal, virus and protozoal infections and cancer. Do these diverse chemicals fail due to common resistance mechanisms? The answer is, yes, based on the 14 papers included in the part I and II of this theme issue. Resistance of bacteria, fungi, viruses, protozoa and cancer to medicinal compounds is now a common occurrence and the mechanism is similar.Accordingly, extrachromosomal DNA elements and membrane efflux proteins happen to be responsible for reduced effectivity of medicines in several systems. The theme issue will show how medicines designed to cure parasites can lose their effectivity and how their efficacy can be preserved by blocking special resistance mechanisms via resistance modifiers. During the last century, the development of medicinal compounds has been strongly influenced by the Magic Bullet concept of Paul Erhlich.. The theory was based on the concept that each drug would have a specific target and since each organism was distinctly different, a different drug was needed for each infection type. Accordingly, each drug approved by regulatory agencies is to be used for a single entity of disease. Hence, antibiotics are to be used for the management of bacterial or viral or fungal or parasitic infections, neuroleptics for the management of neuroses and psychoses, analgesics for the management of pain and cytotoxins for the management of cancer. The unraveling of the genetic code and subsequent characterization of whole genomes from microorganisms to Man, has shown that the genome of each cell class is nearly 95% similar. Therefore, it is not surprising that drugs that affect prokaryotic cells can also affect the cells of the eukaryote-the latter generally referred to as a "side effect". But what has been surprising is that drugs that affect the eukaryotic cell may also have effects on prokaryotic cells. Prolonged therapy with an inappropriate dose of a single antibiotic therapy that has been long known to provide the basis for the selection of antibiotic mono-resistant bacteria, fungi and parasites is now known to also promote the development of resistance to two or more antibiotics (multidrug resistance). The therapy of cancer has from the early onset involved the use of two or more cytotoxic drugs, since the cancer would quickly become refractory when only one drug would be used. Nevertheless, cancer cells that escape the toxicity of tri-drug therapy become resistant not only to these agents but also to a large gamut of other, non-related compounds. Multidrug resistant cancer is essentially incurable at the moment. (The development of multidrug resistance of bacteria, fungi, protozoa, parasites and cancer cells, given the fact that 95% of their genomes are similar, suggests that the mechanism or mechanisms by which multidrug resistance takes place may be either similar or identical. What are these mechanisms?) This theme issue (part I and II) will show how medicines designed to cure parasites and cancer can lose their effectivity and how their efficacy can be preserved by blocking special resistance mechanisms via resistance modifiers. There are examples of overlap of the drug resistance mechanisms in prokaryote and waseukaryote cells. Chemotherapy of infections and cancer shares similar histories and developed side by side for many years. The similarities of some DNA complexing agents that have antiplasmid and antiviral actions can be exploited for rational drug design. Resistance to chemotherapy may be intrinsic or may be induced by previous treatments. It can develop to a specific agent, a class of agents, or multiple compounds. The later, called multidrug resistance (MDR), frequently results from impaired retention of medicine caused by overexpression of particular transport proteins (the so called ABC transporters: MDR, MRP, LRP, BCRP), which function as energy dependent drug efflux pumps. To overcome this type of resistance, which is a major obstacle in chemotherapy of infections and cancer, various classes of reversal agents were developed. Unfortunately, their clinical efficacy was found to be weak, inspite of their remarkable effects in vitro. Approximately 6 years ago, a group of scientists formed a concerted action under the aegis of the European Commission to overcome drug resistance..........

Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.

Resistance of tumor cells to multiple structurally unrelated cytotoxic drugs, multidrug resistance (MDR), is the major limitation to the successful chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype is the result from decreased cellular drug accumulation mediated by the adenosine triphosphate binding cassette (ABC)-transporter MDR1/P-glycoprotein (MDR1/P-gp, ABCB1) encoded by the human MDR1 gene. Inhibition of the drug extrusion activity of MDR1/P-gp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in patients suffering on malignant diseases has been studied capaciously, but the clinical results have generally been disappointing. Thus, experimental therapeutic strategies to reverse MDR are under extensive investigation. These strategies included gene therapeutic approaches with antisense oligonucleotides (ODNs), ribozymes, or DNAzymes and, most recently, the application of the RNA interference (RNAi) technology. RNAi is a physiological double stranded RNA-triggered mechanism resulting in gene-silencing in a sequence-specific manner. Transient RNAi can be attained by application of small interferring RNAs (siRNAs), whereas a stable RNAi-mediated gene-silencing can be achieved by transfection of mammalian cells with short hairpin RNA (shRNA) encoding expression cassettes localized on plasmid or viral vectors. Transient and stable RNAi strategies were applied to overcome MDR1/P-gp-mediated MDR in different in vitro models derived from various neoplastic tissue and will be come up for discussion.

Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli , Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical π-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

Efflux pumps protect the bacterial cell by expelling toxic compounds before they reach intracellular targets. Because this mechanism actively contributes to the resistance of a given bacterium to more than one class of antibiotics, molecules that are able to block the relevant efflux pump are of potential significance to combat drug resistance caused by efflux pumps. Different quinoline derivatives including alkoxy, alkylamino, thioalkoxy and chloroquinolines have been previously reported to make Enterobacter aerogenes resistant isolates that over express the mechanism of efflux, noticeably more susceptible to structurally unrelated antibiotics. In addition, various quinoline derivatives significantly increase the intracellular concentration of chloramphenicol as reported with other inhibitors, thereby suggesting the inhibition of the drug transport by AcrAB-TolC pump, which is fully active in the clinicaly resistant isolates investigated. Here, we discuss the respective properties of this molecular family, taking into account the recent insights into the structural data of AcrB pump.

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

Multidrug resistance (MDR) is a major obstacle to the effective treatment of cancer. One of the underlying mechanisms of MDR is cellular overproduction of P-glycoprotein (P-gp) which acts as an efflux pump for various anticancer drugs. P-gp is encoded by the MDR1 gene and its overexpression in cancer cells has become a therapeutic target for circumventing multidrug resistance. A potential strategy is to co-administer efflux pump inhibitors, although such reversal agents might actually increase the side effects of chemotherapy by blocking physiological anticancer drug efflux from normal cells. Although many efforts to overcome MDR have been made using first and second generation reversal agents comprising drugs already in current clinical use for other indications (e.g. verapamil, cyclosporine A, quinidine) or analogues of the first-generation drugs (e.g. dexverapamil, valspodar, cinchonine), few significant advances have been made. Clinical trials with third generation modulators (e.g. biricodar, zosuquidar, and laniquidar) specifically developed for MDR reversal are ongoing. The results however are not encouraging and it may be that the perfect reverser does not exist. Other approaches to multidrug resistance reversal have also been considered: encapsulation of anthracyclines in liposomes or other carriers which deliver these drugs selectively to tumor tissues, the use of P-gp targeted antibodies such as UIC2 or the use of antisense strategies targeting the MDR1 messenger RNA. More recently, the development of transcriptional regulators appears promising. Also anticancer drugs that belong structurally to classes of drugs extruded from cells by Pgp but that are not substrates of this drug transporter may act as potent inhibitors of MDR tumors (e.g. epothilones, second generation taxanes). Taking advantage of MDR has also been studied. Bone marrow suppression, one of the major side effects of cancer chemotherapy, can compromise the potential of curative and palliative chemotherapy. It is conceivable that drug resistance gene transfer into bone marrow stem cells may be able to reduce or abolish chemotherapy-induced myelosuppression and facilitate the use of high dose chemotherapy. Clinical trials of retroviral vectors containing drug resistance genes have established that the approach is safe and are now being designed to address the therapeutically relevant issues.

Due to the high genetic variability of human immunodeficiency virus (HIV), treatment of AIDS (acquired immunodeficiency syndrome) patients with inhibitors of reverse trancriptase (RT) and drugs blocking the viral protease regularly results in the accumulation of drug resistant HIV variants and treatment failure. The sensitivity of clinically derived resistant HIV-1 strains to nucleotide RT inhibitors could be restored, however, in several laboratories by pharmacological depletion of the appropriate endogenous deoxynucleotide triphosphate (dNTP), and such a manipulation (induction of dCTP pool imbalance during reverse transcription in the presence of a non-nucleoside RT inhibitor) altered the mutation spectrum of the HIV-1 genome, resulting in a lower level of HIV resistance to certain drugs. The cytoplasmic singlestranded DNA cytidine deaminases APOBEC3G and APOBEC3F block HIV replication by introducing premature stop codons into the viral genome. We suggest that the resulting crippled, defective HIV (dHIV) variants could interfere with replication of "wild type" viruses and curbe disese progression in long term non-progressor individuals. Vif, an accessory protein encoded by HIV, counteracts APOBEC3G/F action. We speculate that small molecule inhibitors of Vif could permit lethal or sublethal mutagenesis of HIV genomes. We suggest that an artificial dHIV construct carrying a mutated vif gene (coding for a Vif protein unable to block APOBEC3G/F) could have a therapeutic effect as well in HIV infected individuals and AIDS patients.

The antibiotic resistance is now common place throughout the globe. Two highly problematic antibiotic resistant infections are those produced by multi-drug resistant Mycobacterium tuberculosis (MDRTB) and methicillin resistant Staphylococcus aureus (MRSA). Although vancomycin is useful for therapy of MRSA, there is now evidence that resistance to this antibiotic is taking place. Intracellular infections of MRSA are very difficult to manage and are recurrent especially when invasive prosthetic devices are employed. This mini-review provides cogent evidence that both intracellular MDRTB and intracellular MRSA can be killed by concentrations of the non-antibiotic phenothiazine, Thioridazine, at concentrations in the medium that are below those present in the plasma of patients treated with this agent. Although thioridazine has been claimed to cause arrhythmias and even sudden death, the frequencies of these episodes are rare and when present, they are related to the patients underlying cardiac status as opposed to the direct effect of the agent itself. The authors do not suggest that thioridazine be used indiscriminately for MDRTB or intracellular infections produced by MRSA. However, there are circumstances where there are no alternative forms of therapy and the patient faces an unfavourable prognosis. For these highly selective and controlled situations, the use of thioridazine in the manner employed for the therapy of psychosis is recommended (compassionate therapy).

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is the consequence of the over-expression of a variety of proteins that extrude the chemotherapic from the cell, lowering its concentration below the effective one. The ABC (ATP Binding Cassette) is a ubiquitous and important family of such transporter proteins. Members of this super family are present in mammals as well as in prokaryotic organisms and use ATP as the energy source to activate the extrusion process. P-glycoprotein (Pgp) and Multidrug Resistance Proteins (MRP1 and sister proteins) are the most important and widely studied members of ABC super family. Our knowledge about the structures and functions of transporter proteins has definitely improved in recent years, following the resolution of the structure of bacterial pumps which opened the way to the building of homology models for the more complex Pgp and MRP. It can be anticipated that these results will have a strong impact on the design of more potent and safer MDR reverters. A huge number of small molecules, many of natural origin, are able to reverse multidrug resistance by inhibiting the functions of Pgp, MRP1 and sister proteins and their action has been considered a possible way to reverse MDR. However, while a few compounds have reached clinical trials, none of them has, so far, been cleared for therapeutic use. Two main reasons are at the base of this difficulty: i) MDR is a complex phenomenon that may arise from several different biochemical mechanisms, with the consequence that inhibition of transporter proteins may be insufficient to reverse it; ii) the physiological role of Pgp and sister proteins requires more potent modulators with proper selectivity and pharmacokinetic in order to avoid unwanted side effects. This paper first reviews the most recent discoveries on the structures and functions of the ABC super family, in particular Pgp and MRP. Then, the medicinal chemistry of MDR reverters, in light of these findings, is discussed and the molecules that are presently in development are reviewed.