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.

31Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to 32 pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively 33 selected for increased pore size from a common ancestor aquaporin. We demonstrate that 34 TbAQP2's unique architecture permits pentamidine permeation through its central pore and 35 show how specific mutations in highly conserved motifs affect drug permeation. Introduction 36 of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. 37 Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically 38 favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally 39 strictly impermeable for ionic species. We also identify the structural determinants that make 40 pentamidine a permeant but exclude most other diamidine drugs. Our results have wide-41 ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. 42Moreover, these new insights in aquaporin permeation may allow the pharmacological 43 exploitation of other members of this ubiquitous gene family. 44 45 Keywords: 46 Drug transport / Aquaporin / evolution of membrane proteins / Trypanosoma brucei / 47 109 endocytosis rate and the rate of pentamidine uptake. Our results unequivocally show that 110 pentamidine permeates directly through the central pore of TbAQP2 and that uptake is 111 dependent on the microbial membrane potential. Having identified the essential 112 characteristics that allow the transport of large, flexible molecules through TbAQP2, this 113 should now allow the evaluation of aquaporins in other species for similar adaptations.114 7 Results 115 116 12. Investigation of the structural determinants of AQP2 for pentamidine transport 117 1.1. Positive selection for pore size 118 In T. brucei, the AQP2 and AQP3 genes are arranged as a tandem pair on chromosome 10 119 and have 74% amino acid identity. Whereas TbAQP2 clearly mediates pentamidine uptake, 120 TbAQP3 does not (Baker et al, 2012; Munday et al, 2014), nor do various chimeric AQP2/3 121 rearrangements that give rise to pentamidine resistance (Munday et al, 2014; Graf et al, 122 2015). To investigate the origin of the AQP2 gene, a phylogenetic analysis of AQPs in 123 African trypanosomes was performed. The number of aquaporin genes varies: there is a 124 single aquaporin in T. vivax and T. congolense, two in T. suis and three in T. brucei and its 125 derivatives (Supplemental Fig. 1A). The most probable tree (Supplemental Fig. 1B) is 126 consistent with the evolutionary history of the four species (Hutchinson & Gibson, 2015) and 127 indicates AQP1 as the ancestral AQP present in all trypanosome species. A duplication 128 occurred in the common ancestor of T. suis and T. brucei after divergence from T. 129 congolense and a further duplication, to form AQP2 and AQP3, in the ancestor of T. 130 brucei after divergence from T. suis. Multiple alignment (Supplemental Fig. 1A) shows that 131 the classical NPA/NPA a...

Artemisinin combination therapies (ACTs) have been used as the first-line treatments against Plasmodium falciparum malaria for decades. Recent advances in chemical proteomics have shed light on the complex mechanism of action of semi-synthetic artemisinin (ARTs), particularly their promiscuous alkylation of parasite proteins via previous heme-mediated bioactivation of the endoperoxide bond. Alarmingly, the rise of resistance to ART in South East Asia and the synthetic limitations of the ART scaffold have pushed the course for the necessity of fully synthetic endoperoxide-based antimalarials. Several classes of synthetic endoperoxide antimalarials have been described in literature utilizing various endoperoxide warheads including 1,2-dioxanes, 1,2,4-trioxanes, 1,2,4-trioxolanes, and 1,2,4,5-tetraoxanes. Two of these classes, the 1,2,4-

Synthetic endoperoxide antimalarials, such as 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes, are promising successors for current front-line antimalarials, semisynthetic artemisinin derivatives. However, limited solubility of second-generation analogues in biological-relevant media represents a barrier in clinical development. We present methodology for the synthesis of nonlinear analogues of second-generation tetraoxane antimalarials E209 and N205 to investigate reduced molecular symmetry on in vitro antimalarial activity and physicochemical properties. While maintaining good antimalarial activity and metabolic stability, head-to-head comparison of linear and nonlinear counterparts showed up to 10-fold improvement in FaSSIF solubility for three of the four analogues studied. Pharmacokinetic studies in rats comparing a selected nonlinear analogue 14a and its parent N205 showed improvement on oral absorption and exposure in vivo with more than double the AUC and a significant increase in oral bioavailability (76% versus 41%). These findings provide support for further in vivo efficacy studies in preclinical animal species.

Decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1) is a critical flavoenzyme in Mycobacterium tuberculosis, catalyzing a vital step in the production of lipoarabinomannan and arabinogalactan, both of which are essential for cell wall biosynthesis. Due to its periplasmic localization, DprE1 is a susceptible target, and several compounds with diverse scaffolds have been discovered that inhibit this enzyme, covalently or noncovalently. We evaluated a total of ∼1519 DprE1 inhibitors disclosed in the literature from 2009 to April 2022 by performing an in-depth analysis of physicochemical descriptors and absorption, distribution, metabolism, excretion, and toxicity (ADMET), to gain new insights into these properties in DprE1 inhibitors. Several molecular properties that should facilitate the design and optimization of future DprE1 inhibitors are described, allowing for the development of improved analogues targeting M. tuberculosis.