In Vitro Efficacy, Resistance Selection, and Structural Modeling Studies Implicate the Malarial Parasite Apicoplast as the Target of Azithromycin

Abstract

Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.Licensed antibiotics with antimalarial activity offer considerable promise as suitable partners for novel combination therapies directed against drug-resistant Plasmodium falciparuminfections. Clinical studies have demonstrated good clinical cure rates with treatments that use tetracycline, its more potent derivative doxycycline, or clindamycin, in combination with fast-acting antimalarials such as quinine (1-3). Whereas the mode of action of these antibiotics has until recently remained elusive for Plasmodium, against bacteria they act by binding to the 70 S ribosome (comprised of the 50 S and 30 S subunits), thereby inhibiting protein synthesis. Tetracycline and doxycycline bind to the 30 S subunit (comprising the 16 S rRNA and ribosomal proteins), whereas clindamycin binds to the 50 S subunit (comprising the 23 S rRNA, the 5 S rRNA, and ribosomal proteins including L4 and L22) (4). The World Health Organization recommends doxycycline as malaria prophylaxis for travelers to endemic areas. Tetracycline and doxycycline, however, manifest toxicity in children below the age of eight and in pregnant women, effectively precluding their use in the most important malaria patient populations (5).Another antibiotic that appears particularly promising for antimalarial combination therapy is azithromycin (AZ), 2 a 15-m...

Borrelidin, a potent antimalarial: stage-specific inhibition profile of synchronized cultures of Plasmodium falciparum

“…[20][21][22][23] Recently, it was reported that macrolide antibiotics, such as azithromycin, caused the 'delayed death' of P. falciparum and their target was the apicoplast. [24][25][26] Although borrelidin is also a macrolide, its mode of action may well be different because borrelidin did not show 'delayed death' in this experiment.…”

New approaches in antimalarial drug discovery and development: a review

“…Fosmidomycin inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in the synthesis of isoprenoids, which is essential for parasite survival but not for the human host (which synthesise isoprenoids through the mevalonate pathway) (Wiesner et al 2003). Azithromycin, a macrolide antibiotic, affects P. falciparum growth by targeting the apicoplast 50S ribosomal subunit and inhibits protein synthesis in this organelle (Sidhu et al 2007). Geldanamycin, a benzoquinone ansamycin antibiotic, also shows antimalarial activity by inhibiting the heat shock protein, HSP90, a molecular chaperone that is important for the maturation of proteins that promote the survival and the growth of dividing cells (Mout et al 2012).…”

Advances in understanding the genetic basis of antimalarial drug resistance

“…However, some circumstances will require alternative approaches to their identification, notably when predicting resistance to drugs used in new antimalarial regimens. Sidhu et al [20] generated P. falciparum lines resistant to azithromycin in vitro. By taking a candidate gene approach, they were able to identify a mutation in the apicoplast-encoded ribosomal protein L4, which on the basis of structural models and literature on azithromycin-resistant bacteria was predicted to confer resistance.…”

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