Current Drug Targets - Volume 3, Issue 4, 2002

Hepatitis C virus (HCV), a member of the Flaviviridae family, has been recognised to be responsible for both parenterally transmitted and sporadic non-A and non-B hepatitis affecting 1-3% of the world population. HCV is a positive stranded RNA virus encoding a single polyprotein which contains at least ten unique structural and non-structural proteins. Amongst these the structural protein E2 has been of special interest for vaccine development and the serine protease NS3, which is responsible for cleavage of the polyprotein, for the development of small molecule inhibitors. We will focus on the contribution of computational techniques and the use of structural information for the design and discovery of novel therapeutic agents for these targets. Both drug discovery and vaccine design efforts will be discussed taking into account also the problem of emerging resistance.

Thymidylate synthase (TS) is an essential enzyme in de novo synthesis of thymidylate, and is required for DNA synthesis and cell proliferation in the absence of exogenous thymidine. As a consequence, TS is a target for anticancer chemotherapy by several drugs, including 5-fluorouracil (5-FU) and raltitrexed (Tomudex), in treatment of colorectal and other tumors. TS overexpression due to increased gene transcription and mRNA translation can mediate drug resistance. Decreased cellular uptake and polyglutamylation of TS-targeting drugs (raltitrexed, for example), increased drug efflux, altered metabolism of cytotoxic drugs (for example, 5-FU), and other events can decrease the effectiveness of TS-targeting drugs. Recent preclinical and clinical studies have addressed the resistance problem by using combinations of different drugs that target TS,or by combining TS-targeting and non-TS-targeting drugs. Our approach has been to circumvent and / or prevent TS overexpression-mediated drug resistance by employing antisense oligodeoxynucleotides (ODNs) to downregulate TS mRNA and protein levels. These studies have revealed that targeting the 3' end of human TS mRNA downregulates TS mRNA and protein, inhibits cell proliferation, and sensitizes HeLa cells to raltitrexed, 5-FU, and 5-fluorodeoxyuridine (5-FUdR) in vitro (Ferguson et al., Br. J. Pharmacol. 127, 1777-1786, 1999). In addition, growth of human HT29 colon carcinoma cell explants in immunocompromised mice is inhibited by antisense downregulation of TS (Berg et al., J.Pharmacol. Exp. Therap. 298, 477-484, 2001). TS-overexpressing, 5-FUdR-resistant HeLa cells have been established in order to examine resistance mechanisms and cross-resistance to 5-FU and raltitrexed. Treatment of 5-FUdR-resistant HeLa cells with TS antisense ODN effectively reduces TS mRNA and protein levels, and decreases the IC50 of 5-FUdR by up to 80% (Ferguson et al., Br. J. Pharmacol., 134, 1437-1446, 2001). These results indicate that antisense ODN treatment improves the efficacy of anti-TS chemotherapeutic drugs in vitro and in vivo , and is effective in overcoming tumor cell resistance to these drugs. However, cellular responses to antisense targeting of different TS mRNA domains are complex. In fact, targeting the translation start site (but not other TS mRNA regions) stimulates TS gene transcription (DeMoor et al., Exp. Cell Res., 243, 11-21, 1998). Distinctive cellular responses to targeting of specific TS mRNA regions provide exciting therapeutic opportunities. Antisense ODN treatment to modulate TS activity, in combination with TS-targeting chemotherapeutic drugs, has the potential to be an effective anti-tumor therapy.

Protozoan parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the second cause of death, mainly due to the emergence of parasite resistance to conventional drugs. P-glycoprotein (Pgp)-like transporters overexpression is a very efficient mechanism to reduce the intracellular accumulation of many drugs in cancer cells and parasitic protozoans including Plasmodium and Leishmania, thus conferring a multidrug resistance (MDR) phenotype. Therefore, there is a great clinical interest in developing inhibitors of these transporters to overcome such a resistance. Pgps are active pumps belonging to the ATPbinding cassette (ABC) superfamily of proteins, and consist of two homologous halves, each containing a transmembrane domain (TMD) involved in drug efflux, and a cytosolic nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis. Most conventional cancer MDR modulators interact with the drug-binding sites on the TMDs of Pgps, but they are also usually transported and the required concentrations for a permanent inhibition produce subsequent sideeffects that hamper their clinical use. Besides, they only poorly modulate the resistance in protozoan parasites. We review here a rational strategy developed to overcome the MDR phenotype in Leishmania, consisting in: i) the selection of an MDR Leishmania tropica line that overexpresses a Pgp-like transporter, ii) the use of their cytosolic NBDs as new pharmacological targets, iii) the search of new natural compounds that revert the MDR phenotype in Leishmania bybinding to the TMDs, iv) the combination of subdoses of the above selected modulators directed to both targets in the transporter, NBDs and TMDs, to accumulate their reversal effects while diminishing their toxicity. In this way, we have reverted the MDR phenotype in Leishmania, including the resistance to the most promising new antileishmania agents, the alkyl-lysophospholipids. This approach might be extrapolated to be used in other eukaryotic cells.

The streptogramin antibiotics were discovered over 40 years ago but are only now emerging as important therapeutic agents for the treatment of infection caused by a variety of bacteria. The streptogramins consist of mixtures of two structurally distinct compounds, type A and type B, which are separately bacteriostatic, but bactericidal in appropriate ratios. These antibiotics act at the level of inhibition of translation through binding to the bacterial ribosome. Resistance to streptogramins occurs through a number of mechanisms including target modification, efflux, and enzyme catalyzed antibiotic modification. This review describes the current understanding of streptogramin function and resistance with emphasis on molecular mechanism and epidemiology.

Antibiotic resistance appearance and spread have been classically considered the result of a process of natural selection, directed by the use of antibiotics. Bacteria, that have to face the antibiotic challenge, evolve to acquire resistance and, under this strong selective pressure, only the fittest survive, leading to the spread of resistance mechanisms and resistant clones. Horizontal transference of resistance mechanisms seems to be the main way of antibiotic resistance acquisition. Nevertheless, recent findings on hypermutability and antibiotic-induced hypermutation in bacteria have modified the landscape. Here, we present a review of the last data on molecular mechanisms of hypermutability in bacteria and their relationship with the acquisition of antibiotic resistance. Finally, we discuss the possibility that antibiotics may act not only as selectors for antibiotic resistant bacteria but also as resistance promoters.