Physiologic Targets and Modes of Action for CBL0137, a Lead for Human African Trypanosomiasis Drug Development.

CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei Herein, we use a four-step strategy to 1) identify physiologic targets and 2) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase) and DNA-binding proteins [universal minicircle sequence binding protein 2, replication protein A1 (RPA1), replication protein A2 (RPA2)]. In chemical biology studies, CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiologic targets of the drug. CBL0137 inhibited 1) nucleus mitosis, 2) nuclear DNA replication, and 3) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNA interference (RNAi) against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiologic targets of CBL0137 can be deployed with different drugs in other biologic systems. SIGNIFICANCE STATEMENT: To distinguish drug-binding proteins from physiologic targets in the African trypanosome, we devised and executed a multidisciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.

How Physiologic Targets Can Be Distinguished from Drug-Binding Proteins.

In clinical trials, some drugs owe their effectiveness to off-target activity. This and other observations raise a possibility that many studies identifying targets of drugs are incomplete. If off-target proteins are pharmacologically important, it will be worthwhile to identify them early in the development process to gain a better understanding of the molecular basis of drug action. Herein, we outline a multidisciplinary strategy for systematic identification of physiologic targets of drugs in cells. A drug-binding protein whose genetic disruption yields very similar molecular effects as treatment of cells with the drug may be defined as a physiologic target of the drug. For a drug developed with a rational approach, it is desirable to verify experimentally that a protein used for hit optimization in vitro remains the sole polypeptide recognized by the drug in a cell. SIGNIFICANCE STATEMENT: A body of evidence indicates that inactivation of many drug-binding proteins may not cause the pharmacological effects triggered by the drugs. A multidisciplinary cell-based approach can be of great value in identifying the physiologic targets of drugs, including those developed with target-based strategies.

Design, Synthesis, and Evaluation of Novel Anti-Trypanosomal Compounds.

Human African trypanosomiasis (HAT) is a deadly neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. During the course of screening a collection of diverse nitrogenous heterocycles, we discovered two novel compounds that contain the tetracyclic core of the Yohimbine and Corynanthe alkaloids, were potent inhibitors of T. brucei proliferation and T. brucei methionyl-tRNA synthetase (TbMetRS) activity. Inspired by these key findings, we prepared several novel series of hydroxyalkyl δ-lactam, δ-lactam, and piperidine analogs and tested their anti-trypanosomal activity. A number of inhibitors are more potent against T. brucei than these initial hits with one hydroxyalkyl δ-lactam derivative being 25-fold more effective in our assay. Surprisingly, most of these active compounds failed to inhibit TbMetRS. This work underscores the importance of verifying, irrespective of close structural similarities, that new compounds designed from a lead with a known biological target engage the putative binding site.

AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on T. brucei using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.

Novel Effects of Lapatinib Revealed in the African Trypanosome by Using Hypothesis-Generating Proteomics and Chemical Biology Strategies.

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei Lapatinib, a human epidermal growth factor receptor (EGFR) inhibitor, can cure 25% of trypanosome-infected mice, although the parasite lacks EGFR-like tyrosine kinases. Four trypanosome protein kinases associate with lapatinib, suggesting that the drug may be a multitargeted inhibitor of phosphoprotein signaling in the bloodstream trypanosome. Phosphoprotein signaling pathways in T. brucei have diverged significantly from those in humans. As a first step in the evaluation of the polypharmacology of lapatinib in T. brucei, we performed a proteome-wide phosphopeptide analysis before and after drug addition to cells. Lapatinib caused dephosphorylation of Ser/Thr sites on proteins predicted to be involved in scaffolding, gene expression, and intracellular vesicle trafficking. To explore the perturbation of phosphotyrosine (pTyr)-dependent signaling by lapatinib, proteins in lapatinib-susceptible pTyr complexes were identified by affinity chromatography; they included BILBO-1, MORN, and paraflagellar rod (PFR) proteins PFR1 and PFR2. These data led us to hypothesize that lapatinib disrupts PFR functions and/or endocytosis in the trypanosome. In direct chemical biology tests of these speculations, lapatinib-treated trypanosomes (i) lost segments of the PFR inside the flagellum, (ii) were inhibited in the endocytosis of transferrin, and (iii) changed morphology from long and slender to rounded. Thus, our hypothesis-generating phosphoproteomics strategy predicted novel physiological pathways perturbed by lapatinib, which were verified experimentally. General implications of this workflow for identifying signaling pathways perturbed by drug hits discovered in phenotypic screens are discussed.

New chemical scaffolds for human african trypanosomiasis lead discovery from a screen of tyrosine kinase inhibitor drugs.

Human African trypanosomiasis (HAT) is caused by the protozoan Trypanosoma brucei. New drugs are needed to treat HAT because of undesirable side effects and difficulties in the administration of the antiquated drugs that are currently used. In human proliferative diseases, protein tyrosine kinase (PTK) inhibitors (PTKIs) have been developed into drugs (e.g., lapatinib and erlotinib) by optimization of a 4-anilinoquinazoline scaffold. Two sets of facts raise a possibility that drugs targeted against human PTKs could be "hits" for antitrypanosomal lead discoveries. First, trypanosome protein kinases bind some drugs, namely, lapatinib, CI-1033, and AEE788. Second, the pan-PTK inhibitor tyrphostin A47 blocks the endocytosis of transferrin and inhibits trypanosome replication. Following up on these concepts, we performed a focused screen of various PTKI drugs as possible antitrypanosomal hits. Lapatinib, CI-1033, erlotinib, axitinib, sunitinib, PKI-166, and AEE788 inhibited the replication of bloodstream T. brucei, with a 50% growth inhibitory concentration (GI50) between 1.3 µM and 2.5 µM. Imatinib had no effect (i.e., GI50>10 µM). To discover leads among the drugs, a mouse model of HAT was used in a proof-of-concept study. Orally administered lapatinib reduced parasitemia, extended the survival of all treated mice, and cured the trypanosomal infection in 25% of the mice. CI-1033 and AEE788 reduced parasitemia and extended the survival of the infected mice. On the strength of these data and noting their oral bioavailabilities, we propose that the 4-anilinoquinazoline and pyrrolopyrimidine scaffolds of lapatinib, CI-1033, and AEE788 are worth optimizing against T. brucei in medicinal chemistry campaigns (i.e., scaffold repurposing) to discover new drugs against HAT.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery.

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Pseudokinase NRP1 facilitates endocytosis of transferrin in the African trypanosome.

Trypanosoma brucei causes human African trypanosomiasis (HAT) and nagana in cattle. During infection of a vertebrate, endocytosis of host transferrin (Tf) is important for viability of the parasite. The majority of proteins involved in trypanosome endocytosis of Tf are unknown. Here we identify pseudokinase NRP1 (Tb427tmp.160.4770) as a regulator of Tf endocytosis. Genetic knockdown of NRP1 inhibited endocytosis of Tf without blocking uptake of bovine serum albumin. Binding of Tf to the flagellar pocket was not affected by knockdown of NRP1. However the quantity of Tf per endosome dropped significantly, consistent with NRP1 promoting robust capture and/or retention of Tf in vesicles. NRP1 is involved in motility of Tf-laden vesicles since distances between endosomes and the kinetoplast were reduced after knockdown of the gene. In search of possible mediators of NRP1 modulation of Tf endocytosis, the gene was knocked down and the phosphoproteome analyzed. Phosphorylation of protein kinases forkhead, NEK6, and MAPK10 was altered, in addition to EpsinR, synaptobrevin and other vesicle-associated proteins predicted to be involved in endocytosis. These candidate proteins may link NRP1 functionally either to protein kinases or to vesicle-associated proteins.

Hypothesis-generating proteome perturbation to identify NEU-4438 and acoziborole modes of action in the African Trypanosome.

NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438's molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.

Discovery of functional motifs in h-regions of trypanosome signal sequences.

N-terminal signal peptides direct secretory proteins into the ER (endoplasmic reticulum) of eukaryotes or the periplasmic space of prokaryotes. A hydrophobic core (h-region) is important for signal sequence function; however, the mechanism of h-region action is not resolved. To gain new insight into signal sequences, bioinformatic analysis of h-regions from humans, Saccharomyces cerevisiae, Trypanosoma brucei and Escherichia coli was performed. Each species contains a unique set of peptide motifs (h-motifs) characterized by identity components (i.e. sequence of conserved amino acids) joined by spacers. Human h-motifs have four identity components, whereas those from the other species utilize three identity components. Example of h-motifs are human Hs3 {L-x(2)-[AGILPV]-L-x(0,2)-L}, S. cerevisiae Sc1 [L-x(0,2)-S-x(0,3)-A], T. brucei Tb2 {L-x(1,2)-L-[AILV]} and E. coli Ec1 [A-x(0,2)-L-x(0,3)-A]. The physiological relevance of h-motifs was tested with a T. brucei microsomal system for translocation of a VSG (variant surface glycoprotein)-117 signal peptide. Disruption of h-motifs by scrambling of sequences in h-regions produced defective signal peptides, although the hydrophobicity of the peptide was not altered. We conclude that: (i) h-regions harbour h-motifs, and are not random hydrophobic amino acids; (ii) h-regions from different species contain unique sets of h-motifs; and (iii) h-motifs contribute to the biological activity of ER signal peptides. h-Regions are 'scaffolds' in which functional h-motifs are embedded. A hypothetical model for h-motif interactions with a Sec61p protein translocon is presented.

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome.

The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.

Glycosylphosphatidylinositol-specific phospholipase C regulates transferrin endocytosis in the African trypanosome.

GPI-PLC (glycosylphosphatidylinositol-specific phospholipase C) is expressed in bloodstream-form Trypanosoma brucei, a protozoan that causes human African trypanosomiasis. Loss of genes encoding GPI-PLC reduces the virulence of a pleomorphic strain of the parasite, for reasons that are not clear. In the present paper, we report that GPI-PLC stimulates endocytosis of transferrin by 300-500%. Surprisingly, GPI-PLC is not detected at endosomes, suggesting that the enzyme does not interact directly with the endosomal machinery. We therefore hypothesized that a diffusible product of the GPI-PLC enzyme reaction [possibly DAG (diacylglycerol)] mediated the biological effects of the protein. Two sets of data support this assertion. First, a catalytically inactive Q81L mutant of GPI-PLC, expressed in a GPI-PLC-null background, had no effect on endocytosis, indicating that enzyme activity is essential for the protein to stimulate endocytosis. Secondly, the exogenous DAGs OAG (1-oleyl-2-acetyl-sn-glycerol) and DMG (dimyristoylglycerol) independently stimulated endocytosis of transferrin. Furthermore, the DAG mimic PMA, a phorbol ester, also activated endocytosis in T. brucei. DAG-stimulated endocytosis is a novel pathway in the trypanosome. We surmise that (i) GPI-PLC regulates transferrin endocytosis in T. brucei, (ii) GPI-PLC is a signalling enzyme, and (iii) DAG is a second messenger for GPI-PLC. We propose that regulation of endocytosis is a physiological function of GPI-PLC in bloodstream T. brucei.

Post-translational import of protein into the endoplasmic reticulum of a trypanosome: an in vitro system for discovery of anti-trypanosomal chemical entities.

HAT (human African trypanosomiasis), caused by the protozoan parasite Trypanosoma brucei, is an emerging disease for which new drugs are needed. Expression of plasma membrane proteins [e.g. VSG (variant surface glycoprotein)] is crucial for the establishment and maintenance of an infection by T. brucei. Transport of a majority of proteins to the plasma membrane involves their translocation into the ER (endoplasmic reticulum). Thus inhibition of protein import into the ER of T. brucei would be a logical target for discovery of lead compounds against trypanosomes. We have developed a TbRM (T. brucei microsome) system that imports VSG_117 post-translationally. Using this system, MAL3-101, equisetin and CJ-21,058 were discovered to be small molecule inhibitors of VSG_117 translocation into the ER. These agents also killed bloodstream T. brucei in vitro; the concentrations at which 50% of parasites were killed (IC50) were 1.5 microM (MAL3-101), 3.3 microM (equisetin) and 7 microM (CJ-21,058). Thus VSG_117 import into TbRMs is a rapid and novel assay to identify 'new chemical entities' (e.g. MAL3-101, equisetin and CJ-21,058) for anti-trypanosome drug development.

Trypanosoma brucei: reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells.

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic "pulse-chase" analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.

Kinetoplast Division Factors in a Trypanosome.

Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes - kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts.

Improvement of Aqueous Solubility of Lapatinib-Derived Analogues: Identification of a Quinolinimine Lead for Human African Trypanosomiasis Drug Development.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC50)) was established, as part of the lead selection process. Increasing the sp3 carbon content of 1 resulted in 10e (0.19 µM EC50 against T. brucei and 990 µM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC50: 0.013 µM; aqueous solubility: 880 µM; and CEC50: 0.18 µM). Compound 22a reduced parasitemia 109 fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome.

The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.

Anilinoquinoline based inhibitors of trypanosomatid proliferation.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.