Brucei Rhodesiense Research Articles

New derivatives of 7-chloroquinolin-4-amine with antiprotozoal activity

Novel ω-aminoacyl and -alkyl derivatives of 7-chloroquinolin-4-amine were prepared and their structures confirmed by NMR spectroscopy. Their antiprotozoal activities were examined in vitro against the sensitive NF54 strain as well as against the multiresistant K1 strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900). The results were compared with the activities of clinically used drugs. Their antitrypanosomal activities were only moderate whereas their antiplasmodial activities looked very promising. Some were equal or slightly more active than chloroquine against the sensitive strain. However, in comparison to chloroquine, the activity of the new compounds was decreased much less in the resistant strain. Several possessed activity against both strains in low nanomolar concentration.

Buxus alkaloids with antiparasitic activity: Cyclovirobuxein B shows strong and selective toxicity against Trypanosoma brucei rhodesiense

Buxus alkaloids with antiparasitic activity: Cyclovirobuxein B shows strong and selective toxicity against Trypanosoma brucei rhodesiense

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

ABSTRACTThe biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89 mg g−1 DW; equivalent to 63.3 mg L−1), taxodione (1.15 mg g−1 DW; equivalent to 17.4 mg L−1), 15-deoxy-fuerstione (2.15 mg g−1 DW; equivalent to 32.5 mg L−1), and 7-(2′-oxohexyl)-taxodione (0.076 mg g−1 DW; equivalent to 1.1 mg L−1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC50 = 0.05 µM with high selectivity, selectivity index (SI) = 38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1 µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC50 values ranging from 0.62 to 194.7 µM) and lower selectivity (SI value ranged from 0.4 to 5.0).

Natural Sesquiterpene Lactones as Potential Trypanocidal Therapeutic Agents: A Review

Chagas' disease and Human African Trypanosomiasis are parasitic diseases that remain major health problems, mainly among the poorest and the most marginalized communities from Latin America and Africa. The scarcity of effective chemotherapy, due to the low investment in the research and development (R&D) of new drugs, together with a high incidence of side effects, and the emergence of drug resistance phenomena emphasize the urgent need for new prophylactic and therapeutic agents. Over the ages, humans have employed natural products to treat a wide spectrum of diseases. Recently, the pharmaceutical industry has focused on plant research and a large body of evidence has been collected to demonstrate the immense potential of medicinal plants as a source of bioactive compounds and lead molecules. In the field of parasitic diseases, drug development from plants has been successful for the sesquiterpene lactone (STL) artemisinin, which is employed as an antimalarial agent. STLs are a large group of naturally occurring terpenoids derived from plants that mostly belong to the Asteraceae family which exhibit a variety of skeletal arrangements and are the largest and most diverse category of natural products with an a- m6thylene-lactone motif. STLs display a broad spectrum of biological activities such as antitumor, cytotoxic, antibacterial, anthelmintic, uterus contracting, antimalarial, neurotoxic, antiprotozoal and allergic (contact dermatitis) activities. In this context, the purpose of the present review is to provide an overview of the trypanocidal activity reported for STLs against Trypanosoma cruzi and T. brucei rhodesiense over the period 1993-2015.

Purpureone, an antileishmanial ergochrome from the endophytic fungus Purpureocillium lilacinum

Abstract The ethyl acetate extracts prepared from the mycelia of three endophytic fungi Purpureocillium lilacinum, Aspergillus sp., and Fusarium sp., isolated from the roots of Rauvolfia macrophylla (Apocynaceae) were screened for their antiprotozoal activity in vitro against Plasmodium falciparum (NF54), Leishmania donovani, Trypanosoma brucei rhodesiense, and Trypanosoma cruzi. Amongst these extracts, the one from P. lilacinum showed potent antileishmanial activity against L. donovani (IC50 value of 0.174 μg mL−1) with good selectivity (SI=94.9) toward the L6 cell line, whereas the other extracts were inactive and not selective. The fractionation and purification of the active extract from P. lilacinum by column chromatography over silica gel yielded a new ergochromone derivative (1), together with six known compounds: (22E,24R)-stigmasta-5,7,22-trien-3-β-ol (2), (22E,24R)-stigmasta-4,6,8(14),22-tetraen-3-one (3), emodin (4), chrysophanol (5), aloe-emodin (6), and palmitic acid, whose structures were elucidated spectroscopically. Compound 1 was tested in vitro for its antiparasitic activities against the above listed parasites and for its antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Escherichia coli, Providencia stuartii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The compound displayed potent antileishmanial activity against L. donovani with an IC50 value of 0.63 μg mL−1 (0.87 μm) with good selectivity (SI=49.5) toward the L6 cell line. It also exhibited good antibacterial activity against three of the tested microbial strains B. cereus, E. coli ATCC879, and P. stuartii ATCC29916 with minimum inhibitory concentrations below 62.6 μg mL−1. Compound 1 is thus a promising active compound that could be investigated for antileishmanial and antimicrobial drug development.

Scoping Studies into the Structure‐Activity Relationship (SAR) of Phenylephrine‐Derived Analogues as Inhibitors of Trypanosoma brucei rhodesiense

AbstractHuman African Trypanosomiasis (HAT) is a disease caused by the parasite Trypanosoma brucei and is classified as a neglected tropical disease of concern in sub‐Saharan Africa. A scoping study has been undertaken to develop a preliminary structure activity relationship of a tetrahydroisoquinoline scaffold. Fourteen compounds based around this core scaffold were synthesised and evaluated for their activity against Trypanosoma brucei rhodesiense in vitro. Initial results are promising with a number of analogues showing low micromolar inhibition of T.b.rhodesiense with acceptable selectivity over mammalian cells. The most promising is a secondary amine analogue showing the most potent inhibition of T.b.rhodesiense, with an IC50 value of 0.25 ± 0.02 μM, while also showing low cytotoxicity to mammalian cells.

Ent-Pimarane and ent-Kaurane Diterpenes from Aldama discolor (Asteraceae) and Their Antiprotozoal Activity.

Aldama discolor (syn.Viguiera discolor) is an endemic Asteraceae from the Brazilian “Cerrado”, which has not previously been investigated for its chemical constituents and biological activity. Diterpenes are common secondary metabolites found in Aldama species, some of which have been reported to present potential antiprotozoal and antimicrobial activities. In this study, the known ent-3-α-hydroxy-kaur-16-en-18-ol (1), as well as three new diterpenes, namely, ent-7-oxo-pimara-8,15-diene-18-ol (2), ent-2S,4S-2-19-epoxy-pimara-8(3),15-diene-7β-ol (3) and ent-7-oxo-pimara-8,15-diene-3β-ol (4), were isolated from the dichloromethane extract of A. discolor leaves and identified by means of MS and NMR. The compounds were assayed in vitro against Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, Plasmodium falciparum and also tested for cytotoxicity against mammalian cells (L6 cell line). The ent-kaurane 1 showed significant in vitro activity against both P. falciparum (IC = 3.5 M) and L. donovani (IC = 2.5 M) and ent-pimarane 2 against P. falciparum (IC = 3.8 M). Both compounds returned high selectivity indices (SI >10) in comparison with L6 cells, which makes them interesting candidates for in vivo tests. In addition to the diterpenes, the sesquiterpene lactone budlein A (5), which has been reported to possess a strong anti-T. b. rhodesiense activity, was identified as major compound in the A. discolor extract and explains its high activity against this parasite (100% growth inhibition at 2 g/mL).

The antiprotozoal potencies of newly prepared 3-azabicyclo[3.2.2]nonanes.

3-Azabicyclo[3.2.2]nonanes are already reported as antiprotozoal agents. Structural variations were performed by attachment of several basic side chains, being part of drugs in use, to the ring nitrogen. The structures of the new compounds were established using one and two dimensional NMR measurements. All compounds were investigated for their antiplasmodial and antitrypanosomal activities against Plasmodium falciparum K 1 (multiresistant) and Trypanosoma brucei rhodesiense. Their cytotoxicity was assessed against L6 cells. The results are compared to the activities of formerly synthesized compounds. Structure-activity relationships are discussed.

New diaryl-substituted azabicyclo[3.2.2]nonanes and their antiprotozoal potencies

Several new diaryl-substituted azabicyclo[3.2.2]nonanes with varying substitution patterns of the aromatic rings and differing amino substitution at a bridgehead atom of the bicyclic ring system were synthesized from bicyclo[2.2.2]octan-2-ones via Schmidt or Beckmann reactions and subsequent reduction. The bicyclic octanones were prepared in a one-pot synthesis from acyclic starting materials or in one step from properly functionalized diarylcyclohexanones which were accessible by a catalyzed Diels–Alder reaction. The characterization of the new compounds was done by UV/Vis spectroscopy, FT-IR spectroscopy, and HRMS. Structural elucidation of isomers was done by NMR spectroscopy using 1D and 2D techniques as well as NOE measurements. The antiprotozoal activities against Plasmodium falciparum K 1 , resistant to chloroquine and pyrimethamine, and against Trypanosoma brucei rhodesiense as well as the cytotoxicities of the new compounds were determined and compared to those of reported compounds. Structure–activity relationships are discussed.

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.

Synthesis and potent antiprotozoal activity of mono/di amidino 2-anilinobenzimidazoles versus Plasmodium falciparum and Trypanosoma brucei rhodesiense

A series of mono and dicationic new 2-anilinobenzimidazole carboxamidines were prepared in a four step process starting from 4-amino-3-nitrobenzonitrile and corresponding o-phenylenediamines. Their antiparasitic activity against Plasmodium falciparum (P. falciparum) and Trypanosoma brucei rhodesiense (T.b. rhodesiense) were evaluated in vitro. Some of the dicationic compounds (10,12,14) showed equal or very close activity against T.b. rhodesiense with melarsoprol and also showed promising activity against P. falciparum as compared to chloroquine. Among the monocationic derivatives compound 21 exhibited best inhibitory activity against P. falciparum.

Antiprotozoal activity of bicycles featuring a dimethylamino group at their bridgehead

Several dimethylamino-derivatives of the new compound-class 3-azabicyclo[3.2.2]nonanes were prepared. For better comparison of activity also a few analogues of bicyclo[2.2.2]octanes and 2-azabicyclo[3.2.2]nonanes were synthesized. Their activities were examined in vitro against the multiresistant K1 strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900). A couple of the newly synthesized compounds showed promising antiprotozoal activity and selectivity. The results of the biological tests of the novel compounds were compared with the activities of already synthesized compounds and of drugs in use. Structure–activity relationships were discussed.

Loop-Mediated Isothermal Amplification for Detection of the 5.8S Ribosomal Ribonucleic Acid Internal Transcribed Spacer 2 Gene Found in Trypanosoma brucei gambiense.

The loop-mediated isothermal amplification (LAMP) assay with its advantages of cost effectiveness, rapidity, and simplicity, has evolved as a sensitive and specific method for the detection of African trypanosomes. Highly sensitive LAMP reactions specific for Trypanosoma brucei rhodesiense or that recognize but do not discriminate between Trypanosoma brucei brucei, T. b. rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma evansi have been developed. A sensitive LAMP assay targeting the T. b. gambiense 5.8S ribosomal RNA internal transcribed spacer 2 (5.8S-ITS2) gene is also available but this assay does not target binding sites that span the CCCA (C3A) (557-560 bps) insertion site that further differentiates T. b. gambiense from T. b. brucei Here we describe 5.8S-ITS2-targeted LAMP assay that fit these criteria. The LAMP primer sets containing the T. b. gambiense-specific C3A tetranucleotide at the start of the outer forward primer sequences showed high specificity and sensitivity down to at least 0.1 fg T. b. gambiense genomic DNA.

ChemInform Abstract: Design, Synthesis and Antitrypanosomal Activities of 2,6‐Disubstituted‐4,5,7‐trifluorobenzothiophenes.

AbstractNew trifluorobenzothiophene derivatives (IIIb) and (Vb) show significant antitrypanosomal activities against Trypanosoma brucei rhodesiense and no toxicity towards mammalian (MCF7) cells.

Tsetse Control and the Elimination of Gambian Sleeping Sickness.

Tsetse Control and the Elimination of Gambian Sleeping Sickness.

Apolipoprotein L1 Variant Associated with Increased Susceptibility to Trypanosome Infection.

ABSTRACTAfrican trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity.

Synthesis of novel amide and urea derivatives of thiazol-2-ethylamines and their activity against Trypanosoma brucei rhodesiense

2-(2-Benzamido)ethyl-4-phenylthiazole (1) was one of 1035 molecules (grouped into 115 distinct scaffolds) found to be inhibitory to Trypanosoma brucei, the pathogen causing human African trypanosomiasis, at concentrations below 3.6μM and non-toxic to mammalian (Huh7) cells in a phenotypic high-throughput screen of a 700,000 compound library performed by the Genomics Institute of the Novartis Research Foundation (GNF). Compound 1 and 72 analogues were synthesized in this lab by one of two general pathways. These plus 10 commercially available analogues were tested against T. brucei rhodesiense STIB900 and L6 rat myoblast cells (for cytotoxicity) in vitro. Forty-four derivatives were more potent than 1, including eight with IC50 values below 100nM. The most potent and most selective for the parasite was the urea analogue 2-(2-piperidin-1-ylamido)ethyl-4-(3-fluorophenyl)thiazole (70, IC50=9nM, SI>18,000). None of 33 compounds tested were able to cure mice infected with the parasite; however, seven compounds caused temporary reductions of parasitemia (⩾97%) but with subsequent relapses. The lack of in vivo efficacy was at least partially due to their poor metabolic stability, as demonstrated by the short half-lives of 15 analogues against mouse and human liver microsomes.

Structural characterization of the C-terminal coiled-coil domains of wild-type and kidney disease-associated mutants of apolipoprotein L1.

Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease.

Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol–pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu131 in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-016-2173-6) contains supplementary material, which is available to authorized users.

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N'-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.