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.

PKCθ plays an important role in T cell biology and is a validated target for a number of disease states. A series of potent and selective PKCθ inhibitors were designed and synthesized starting from a HTS hit compound. Cell activity, while initially a challenge to achieve, was built into the series by transforming the nitrile unit of the scaffold into a primary amine, the latter predicted to form a new hydrogen bond to Asp508 near the entrance of the ATP binding site of PKCθ. Significant improvements in physiochemical parameters were observed on introduction of an oxetane group proximal to a primary amine leading to compound 22, which demonstrated a reduction of symptoms in a mouse model of multiple sclerosis.

Trazodone is a drug that was introduced in the clinic almost 40 years ago. It is licensed to treat depression, but it is also commonly used off-label to treat insomnia. A recent study shows that it could be promising in preventing neurodegeneration in mice, and clinical trials to assess its possible beneficial effects on dementia and Alzheimer's disease are expected to start soon in humans. In this study, we describe the dose-dependent pharmacology of trazodone by carrying out pharmacokinetic simulations aiming to predict the brain concentrations of trazodone for different drug-dosing regimens and calculating occupancy for 28 different targets for which published trazodone-binding data are available. Our study indicates that low doses of trazodone (typically 50 mg daily) should suffice to block specific receptors responsible for the hypnotic effect, and to provide the protective effect against neuroinflammation and neurodegeneration that could be beneficial in dementia. Higher doses are required for an antidepressant effect. The occupancy of specific receptors at therapeutic doses also explains peculiar side effects reported by patients treated with trazodone (e.g. dry-mouth, hypotension and priapism).

Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.

SummaryThe Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1−/− T. brucei line. Out of a set of six reported SNPs from a reported ‘resistance allele’, none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1‐WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [3H]‐adenosine and [3H]‐diminazene, identical to the tbat1−/− control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [3H]‐diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

Drug resistance in pathogenic protozoa is very often caused by changes to the 'transportome' of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil(®)), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the "selectivity region" of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.

Cyclic nucleotide phosphodiesterases (PDEs) have been identified as important enzyme targets for drug development in both humans and Trypanosoma brucei, the causative agent of human African trypanosomiasis. With this in mind, we recently reported the profiling of a range of human phosphodiesterase inhibitors, showing that human PDE4 inhibitors tend to display the best potency against the trypanosomal phosphodiesterase TbrPDEB1. Among these was GSK‐256066, a potent inhibitor of human PDE4 and a weak inhibitor of TbrPDEB1. In this report, we describe the results of a structure–activity relationship study of this chemotype, leading to the discovery of analogs with improved potency against TbrPDEB1 and micromolar inhibition of T. brucei cellular growth. We rationalize the potency trends via molecular docking of the new inhibitors into a recently reported apo structure of TbrPDEB1. The studies in this article will inform future efforts in repurposing human PDE inhibitors as antitrypanosomal agents.

Assessment of the accuracy of experimental and theoretical methods of pKa determination for acids and bases as separate classes.

A medicinal chemistry exploration of the human phosphodiesterase 4 (hPDE4) inhibitor cilomilast (1) was undertaken in order to identify inhibitors of phosphodiesterase B1 of Trypanosoma brucei (TbrPDEB1). T. brucei is the parasite which causes African sleeping sickness, a neglected tropical disease that affects thousands each year, and TbrPDEB1 has been shown to be an essential target of therapeutic relevance. Noting that 1 is a weak inhibitor of TbrPDEB1, we report the design and synthesis of analogs of this compound, culminating in 12b, a sub-micromolar inhibitor of TbrPDEB1 that shows modest inhibition of T. brucei proliferation.

Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).

The identification of a novel series of PKCθ inhibitors and subsequent optimization using docking based on a crystal structure of PKCθ is described. SAR was rapidly generated around an amino pyridine-ketone hit; (6-aminopyridin-2-yl)(2-aminopyridin-3-yl)methanone 2 leading to compound 21 which significantly inhibits production of IL-2 in a mouse SEB-IL2 model.