Abstract
Severe anemia is an important contributor to mortality in children with severe malaria. Anemia in malaria is a multi-factorial complication, since dyserythropoiesis, hemolysis and phagocytic ...clearance of uninfected red blood cells (RBCs) can contribute to this syndrome. High levels of oxidative stress and immune dysregulation have been proposed to contribute to severe malarial anemia, facilitating the clearance of uninfected RBCs. In a cohort of 552 Ugandan children with severe malaria, we measured the levels of xanthine oxidase (XO), an oxidative enzyme that is elevated in the plasma of malaria patients. The levels of XO in children with severe anemia were significantly higher compared to children with severe malaria not suffering from severe anemia. Levels of XO were inversely associated with RBC hemoglobin (ρ = − 0.25,
p
< 0.0001), indicating a relation between this enzyme and severe anemia. When compared with the levels of immune complexes and of autoimmune antibodies to phosphatidylserine, factors previously associated with severe anemia in malaria patients, we observed that XO is not associated with them, suggesting that XO is associated with severe anemia through an independent mechanism. XO was associated with prostration, acidosis, jaundice, respiratory distress, and kidney injury, which may reflect a broader relation of this enzyme with severe malaria pathology. Since inhibitors of XO are inexpensive and well-tolerated drugs already approved for use in humans, the validation of XO as a contributor to severe malarial anemia and other malaria complications may open new possibilities for much needed adjunctive therapy in malaria.
Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite
. There is currently a lack of effective and tolerable clinically available ...therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter.
glucokinase (
GlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of
GlcK inhibitors that also exhibited anti-
efficacy called the 3-nitro-2-phenyl-2
-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited
GlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the
infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds
and
were determined to inhibit
GlcK quite well with IC
values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2
-chromene analogues, two of which were
and
(compounds
and
, respectively). Twelve of these compounds had a one-point change from the chemical structure of
. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a)
(Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b)
(427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the
GlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC
determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-
-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.
Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for ...controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice.
To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria.
Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice.
Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.
Chagas disease is caused by the protozoan parasite
and affects over 6 million people worldwide. Development of new drugs to treat this disease remains a priority since those currently available have ...variable efficacy and frequent adverse effects, especially during the long regimens required for treating the chronic stage of the disease.
modulates the host cell-metabolism to accommodate the cell cytosol into a favorable growth environment and acquire nutrients for its multiplication. In this study we evaluated the specific anti-
activity of nine bio-energetic modulator compounds. Notably, we identified that 17-DMAG, which targets the ATP-binding site of heat shock protein 90 (Hsp90), has a very high (sub-micromolar range) selective inhibition of the parasite growth. This inhibitory effect was also highly potent (IC
= 0.27 μmol L
) against the amastigote intracellular replicative stage of the parasite. Moreover, molecular docking results suggest that 17-DMAG may bind
Hsp90 homologue Hsp83 with good affinity. Evaluation in a mouse model of chronic
infection did not show parasite growth inhibition, highlighting the difficulties encountered when going from in vitro assays onto preclinical drug developmental stages.
Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic ...potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.
Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this ...pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.
Chagas disease, caused by the parasite
, affects more than six million people worldwide, with its greatest burden in Latin America. Available treatments present frequent toxicity and variable ...efficacy at the chronic phase of the infection, when the disease is usually diagnosed. Hence, development of new therapeutic strategies is urgent. Repositioning of licensed drugs stands as an attractive fast-track low-cost approach for the identification of safer and more effective chemotherapies. With this purpose we screened 32 licensed drugs for different indications against
. We used a primary in vitro assay of Vero cells infection by
. Five drugs showed potent activity rates against it (IC
< 4 µmol L
), which were also specific (selectivity index >15) with respect to host cells.
inhibitory activity of four of them was confirmed by a secondary anti-parasitic assay based on NIH-3T3 cells. Then, we assessed toxicity to human HepG2 cells and anti-amastigote specific activity of those drugs progressed. Ultimately, atovaquone-proguanil, miltefosine, and verapamil were tested in a mouse model of acute
infection. Miltefosine performance in vitro and in vivo encourages further investigating its use against
.
Chagas disease is one of the world’s neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically ...available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with ICsub.50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal ICsub.50 determinations, and an assessment of structure–activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.
Herein, we describe the hit optimization of a novel diarylthioether chemical class found to be active against
; the parasite responsible for Chagas disease. The hit compound was discovered through a ...whole-cell phenotypic screen and as such, the mechanism of action for this chemical class is unknown. Our investigations led to clear structure-activity relationships and the discovery of several analogues with high
potency. Furthermore, we observed excellent activity during acute
efficacy studies in mice infected with transgenic
. These diarylthioether compounds represent a promising new chemotype for Chagas disease drug discovery and merit further development to increase oral exposure without increasing toxicity.
Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed ...artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite’s own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.
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•ATZ links ART to an anti-malarial PI•Malaria-activated ATZ installs the PI on parasite proteins•ATZ overcomes resistance to both its components•ATZ suppresses recrudescence of ART-resistant parasites in mice
Zhan et al. combined two anti-malarial agents—an artemisinin and a proteasome inhibitor—into a single compound called ATZ, which has an additional anti-malarial mechanism of action beyond that of its components. ATZ killed malaria resistant to either component or both.
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