Differential Effects of Paromomycin on Ribosomes of Leishmania mexicana and Mammalian Cells

Abstract

Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania

“…The LC 50 values obtained for the 6′-OH AGs (Paromomycin and G418) were in the μM range, and showed good agreement with previous published values for these compounds (18,35,36). The obtained LC 50 values for the 6′-NH 2 derivatives (Neomycin and Gentamicin) were higher than those obtained for the 6′-OH derivatives, and are also in good agreement with previously reported work demonstrating the lower potency of Neomycin B, compared with Paromomycin, for treatment of leishmaniasis (18). These results are also correlated well with the fact that 6′-NH 2 derivatives are less effective against AG resistant bacterial strains containing an A1408G substitution (30).…”

“…Although no direct evidence is currently available showing that G418 induces miscoding by leishmanial ribosomes, G418 has long been marked as a potent miscoding agent in eukaryotes (38). In addition, a recent work has demonstrated the ability of a structurally related derivative, Paromomycin, to induce miscoding in Leishmania (18). Our modeling data with Paromomycin indeed indicate that the two compounds share a rather great similarity in their binding mechanisms and both might cause the induction of an ON-state conformation upon binding (Fig.…”

“…However, the rRNA A site is highly conserved among organisms belonging to all five kingdoms of life (Fig. 1), and together with recent evidence showing that AGs interfere with the leishmanial protein synthesis machinery (9,18,19), this implies that the putative binding site of AGs in Leishmania is similar to that present in bacteria; namely, the leishmanial A site. Recent works by Kondo and coworkers (10,20) described the binding pattern of several natural and semisynthetic AGs to an A1408G mutant Mycobacterium A-site construct.…”

Ribosomes from Trypanosomatids: Unique Structural and Functional Properties

“…This antibiotic has shown strong anti-leishmania activity when used alone or in combination with other drugs [75]. In a recent work, advances on the molecular basis for the differential effect of paromomycin on Leishmania have been reported [4]. In this work it was demonstrated that paromomycin selectively inhibits the rate of in vitro protein synthesis by trypanosomatid ribosomes, comparing to mammalian extracts.…”

“…Interestingly, this region forms the decoding center, and is also the action site of aminoglycoside antibiotics. Moreover, differences in this region have shown to be responsible for the higher susceptibility of trypanosomatid ribosomes to the aminoglycoside paromomycin [4], as will be discussed below.…”