Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

Abstract

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

A Review of the Antimalarial, Antitrypanosomal, and Antileishmanial Activities of Natural Compounds Isolated From Nigerian Flora

“…Other compounds with significant antitrypanosomal activity included calycosin ( 91 ; EC 50 ± SEM = 10.0 ± 0.44 μg/mL), 6-prenylnaringenin ( 92 ; EC 50 ± SEM = 11.4 ± 0.34 μg/mL), and riverinol ( 93 ; EC 50 ± SEM = 16.24 ± 0.24 μg/mL). Interestingly, none of the compounds showed cross-resistance to pentamidine and melarsoprol but 93 was significantly more active against two multi-drug resistant strains (3.6 and 2.5-fold, respectively ( p < 0.001) (Omar et al, 2016 ), from which either the TbAT1/P2 (Carter et al, 1999 ) or the HAPT1/AQP2 drug transporter (Alghamdi et al, 2020 ) had been deleted (for the structures of compounds 84 – 93 , see Figure 14 ).…”

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins

“…Parasitology potentially acting as a highly selective inhibitor of AQP2 (Fig. 2) (Alghamdi et al, 2020). However, consideration of structural features of the pore do support TbAQP2 acting as a channel for larger and more structurally flexible solutes including pentamidine (Petersen and Beitz, 2020).…”

“…The opposing membrane uptake model proposes that pentamidine, and potentially melarsoprol, are taken up via the intrinsic channel properties of TbAQP2. Indeed, a recent report demonstrates that drug permeation is possible due to a highly conserved amino acid motif in the central pore architecture of TbAQP2, facilitating the passage of 'high' molecular weight solutes (Alghamdi et al, 2020). This was demonstrated by TbAQP3 mutants containing the amino acids of the selectivity pore from TbAQP2 possessing increased capacity for pentamidine uptake (Alghamdi et al, 2020).…”

“…Indeed, a recent report demonstrates that drug permeation is possible due to a highly conserved amino acid motif in the central pore architecture of TbAQP2, facilitating the passage of 'high' molecular weight solutes (Alghamdi et al, 2020). This was demonstrated by TbAQP3 mutants containing the amino acids of the selectivity pore from TbAQP2 possessing increased capacity for pentamidine uptake (Alghamdi et al, 2020). Moreover, pentamidine permeation through TbAQP2 seems to be further aided by the intrinsic membrane potential and is not abrogated by partially blocking endocytic uptake (Alghamdi et al, 2020;, albeit at a rate that is considerably slower than for lower molecular weight solutes, which in essence implies a leak in the AQP2 permeability barrier.…”

The Strong Anti-Kinetoplastid Properties of Bee Propolis: Composition and Identification of the Active Agents and Their Biochemical Targets

“…Propolis is active against trypanosome various Trypanosoma strains including those that are highly resistant to current first-line drugs, as shown by our previous studies [14,15,112]. We determined the activities of compounds isolated from the ethanolic extracts of propolis collected from two regions in Nigeria against a panel of T. brucei strains including (i) T. brucei Lister 427 wild type (WT), which is the standard drug-sensitive control; (ii) an aquaglyceroporin2/3-null (AQP2/3-KO) strain, from which the TbAQP2/AQP3 locus was deleted [116], coding for the critical drug transporter HAPT1 [117,118] and, consequently, resistant to pentamidine and melarsoprol [119]; (iii) a multidrug-resistant strain, B48, adapted from Lister 427WT by deletion of the TbAT1/P2 drug transporter [120] and subsequent adaptation to very high concentrations of pentamidine in vitro [121], making the strain highly resistant to all diamidine-and melaminophenyl arsenical-based drugs.…”