Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Toward Chemical Validation of Leishmania infantum Ribose 5-Phosphate Isomerase as a Drug Target.

Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely outdated, inadequate, and face mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalyzing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the nonoxidative branch of the pentose phosphate pathway, which catalyzes the interconversion of d-ribose 5-phosphate and d-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB (LiRpiB), performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several fragments were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as antileishmanial agents.

Substrate specificity of the neutral sphingomyelinase from Trypanosoma brucei.

The kinetoplastid parasite Trypanosoma brucei causes African trypanosomiasis in both humans and animals. Infections place a significant health and economic burden on developing nations in sub-Saharan Africa, but few effective anti-parasitic treatments are currently available. Hence, there is an urgent need to identify new leads for drug development. The T. brucei neutral sphingomyelinase (TbnSMase) was previously established as essential to parasite survival, consequently being identified as a potential drug target. This enzyme may catalyse the single route to sphingolipid catabolism outside the T. brucei lysosome. To obtain new insight into parasite sphingolipid catabolism, the substrate specificity of TbnSMase was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Recombinant TbnSMase was shown to degrade sphingomyelin, inositol-phosphoceramide and ethanolamine-phosphoceramide sphingolipid substrates, consistent with the sphingolipid complement of the parasites. TbnSMase also catabolized ceramide-1-phosphate, but was inactive towards sphingosine-1-phosphate. The broad-range specificity of this enzyme towards sphingolipid species is a unique feature of TbnSMase. Additionally, ESI-MS/MS analysis revealed previously uncharacterized activity towards lyso-phosphatidylcholine despite the enzyme's inability to degrade phosphatidylcholine. Collectively, these data underline the enzyme's importance in choline homoeostasis and the turnover of sphingolipids in T. brucei.

Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B.

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Antileishmanial Chemotherapy through Clemastine Fumarate Mediated Inhibition of the Leishmania Inositol Phosphorylceramide Synthase.

Current chemotherapeutics for leishmaniasis have multiple deficiencies, and there is a need for new safe, efficacious, and affordable medicines. This study describes a successful drug repurposing approach that identifies the over-the-counter antihistamine, clemastine fumarate, as a potential antileishmanial drug candidate. The screening for inhibitors of the sphingolipid synthase (inositol phosphorylceramide synthase, IPCS) afforded, following secondary screening against Leishmania major (Lmj) promastigotes, 16 active compounds. Further refinement through the dose response against LmjIPCS and intramacrophage L. major amastigotes identified clemastine fumarate with good activity and selectivity with respect to the host macrophage. On target engagement was supported by diminished sensitivity in a sphingolipid-deficient L. major mutant (ΔLmjLCB2) and altered phospholipid and sphingolipid profiles upon treatment with clemastine fumarate. The drug also induced an enhanced host cell response to infection indicative of polypharmacology. The activity was sustained across a panel of Old and New World Leishmania species, displaying an in vivo activity equivalent to the currently used drug, glucantime, in a mouse model of L. amazonensis infection. Overall, these data validate IPCS as an antileishmanial drug target and indicate that clemastine fumarate is a candidate for repurposing for the treatment of leishmaniasis.

Halogenated tryptophan derivatives disrupt essential transamination mechanisms in bloodstream form Trypanosoma brucei.

Amino acid metabolism within Trypanosoma brucei, the causative agent of human African trypanosomiasis, is critical for parasite survival and virulence. Of these metabolic processes, the transamination of aromatic amino acids is one of the most important. In this study, a series of halogenated tryptophan analogues were investigated for their anti-parasitic potency. Several of these analogues showed significant trypanocidal activity. Metabolomics analysis of compound-treated parasites revealed key differences occurring within aromatic amino acid metabolism, particularly within the widely reported and essential transamination processes of this parasite.

New Drugs for Human African Trypanosomiasis: A Twenty First Century Success Story.

The twentieth century ended with human African trypanosomiasis (HAT) epidemics raging across many parts of Africa. Resistance to existing drugs was emerging, and many programs aiming to contain the disease had ground to a halt, given previous success against HAT and the competing priorities associated with other medical crises ravaging the continent. A series of dedicated interventions and the introduction of innovative routes to develop drugs, involving Product Development Partnerships, has led to a dramatic turnaround in the fight against HAT caused by Trypanosoma brucei gambiense. The World Health Organization have been able to optimize the use of existing tools to monitor and intervene in the disease. A promising new oral medication for stage 1 HAT, pafuramidine maleate, ultimately failed due to unforeseen toxicity issues. However, the clinical trials for this compound demonstrated the possibility of conducting such trials in the resource-poor settings of rural Africa. The Drugs for Neglected Disease initiative (DNDi), founded in 2003, has developed the first all oral therapy for both stage 1 and stage 2 HAT in fexinidazole. DNDi has also brought forward another oral therapy, acoziborole, potentially capable of curing both stage 1 and stage 2 disease in a single dosing. In this review article, we describe the remarkable successes in combating HAT through the twenty first century, bringing the prospect of the elimination of this disease into sight.