Biology subjects

Crispim, M.

Publications and source records attributed to Crispim, M..

5 recordsLinked to original sources

bioRxiv2025

Antimalarial Potential of Synthetic Geraniol and Nerol Analogs

Drug resistance is a major threat to malaria control, and thus new drugs are required to fight against this parasitosis. To accelerate drug development, it is of special interest the exploration of natural compounds or repositioning drugs already employed for other diseases. Considering this, previous studies have found that diverse plant terpenes arrest Plasmodium parasites in vitro and in vivo models. However, most terpenes possess low toxicity for the parasite and/or face pharmacokinetic issues. Here, we report several new acyclic monoterpene analogs which possess great antiplasmodial activity in vitro (50% inhibitory concentration at low micromolar scale) against P. falciparum parasites. Also, in vitro studies using hepatocellular carcinoma cells (HepG2) demonstrated remarkable selectivity for malaria parasites. Furthermore, bioinformatic approaches revealed that these compounds possess acceptable pharmacological properties. All these results suggest that acyclic monoterpene analogues could serve as a promising starting point for the development of synthetic terpenes as antimalarial drugs.

microbiology↗

bioRxiv2025

4-Nitrobenzoate inhibits 4-hydroxybenzoate polyprenyltransferase in malaria parasites and enhances atovaquone efficacy

Ubiquinone (UQ) is a critical component of the electron transport chain in Plasmodium falciparum, the etiological agent of human malaria. The first step in UQ biosynthesis is the condensation of 4-hydroxybenzoate (4-HB) and an isoprenic chain by the enzyme 4-hydroxybenzoate polyprenyltransferase (4-HPT; COQ2 gene). Atovaquone (AV), an antimalarial drug, competes with ubiquinol (UQH2) for binding to the mitochondrial bc1 complex, preventing the redox recycling of UQ. In clinical practice, AV is combined with proguanil, a dehydrofolate reductase inhibitor, in a single formulation. However, parasitic resistance to this combination has been demonstrated, indicating the need for new pharmacological combinations to potentiate AV. Previously, 4-nitrobenzoate (4-NB) demonstrated the ability to inhibit UQ biosynthesis in P. falciparum parasites as well as potentiate AV efficacy in vitro. However, both its pharmacodynamics and whether this potentiation could be useful in vivo remained obscure. Here we show that 4-NB enhances AV antiplasmodial efficacy to kill parasites, increases its selectivity compared with animal cells, and preserves proguanil efficacy. 4-NB specifically inhibited the 4-HPT enzymatic activity in mutant strains of Saccharomyces cerevisiae complemented with PfCOQ2. Finally, 4-NB also improved AV antimalarial efficacy in mice infected with Plasmodium berghei parasites. Finally, work with various 4-HB analogs delineated the chemical requirements to potentiate AV activity. These findings clarify the importance of UQ biosynthesis for malaria parasites and suggest that PfCOQ2 could be a therapeutic target to enhance the efficacy of AV.

microbiology↗

bioRxiv2024

Hexosamine Biosynthesis Disruption Impairs GPI Production and Arrests Plasmodium falciparum Growth at Schizont Stages

UDP-N-acetylglucosamine (UDP-GlcNAc) is a crucial sugar nucleotide for glycan synthesis in eukaryotes. In the malaria parasite Plasmodium falciparum, UDP-GlcNAc is synthesized via the hexosamine biosynthetic pathway (HBP) and is essential for glycosylphosphatidylinositol (GPI) anchor production, the most prominent form of protein glycosylation in the parasite. In this study, we explore a conditional knockout of glucosamine-6-phosphate N-acetyltransferase (PfGNA1), a key HBP enzyme. PfGNA1 depletion led to significant disruptions in HBP metabolites, impairing GPI biosynthesis and causing mislocalization of the merozoite surface protein 1 (MSP1), the most abundant GPI-anchored protein in the parasite. Furthermore, parasites were arrested at the schizont stage, exhibiting severe segmentation defects and an incomplete rupture of the parasitophorous vacuole membrane (PVM), preventing egress from host red blood cells. Our findings demonstrate the critical role of HBP and GPI biosynthesis in P. falciparum asexual blood stage development and underscore the potential of targeting these pathways as a therapeutic strategy against malaria. Author SummaryMalaria remains a major cause of illness and death, particularly in sub-Saharan Africa, with increasing resistance to treatments highlighting the urgent need for new strategies. Malaria parasites rely on the hexosamine biosynthetic pathway to produce UDP-N-acetylglucosamine, an essential metabolite for glycosylphosphatidylinositol synthesis. Glycosylphosphatidylinositol molecules anchor vital proteins to the parasites surface and, as free glycolipids, serve as structural components of its membranes. Our study examined the effects of disrupting PfGNA1, a key enzyme in the hexosamine biosynthetic pathway, which is distinct from its human counterparts. Disruption of PfGNA1 blocked the production of glycosylphosphatidylinositol, leading to improper protein localization, developmental arrest, and failure of the parasites to mature or exit infected red blood cells. Our results underscore the central role of the hexosamine biosynthetic pathway and glycosylphosphatidylinositol biosynthesis, which are essential for parasite survival. This pathway represents a promising target for developing novel antimalarial therapies.

microbiology↗

bioRxiv2024

Elucidating the Transport Mechanisms and Metabolic Roles of Serine, Threonine, and Glycine in Trypanosoma cruzi

O_SCPLOWLC_SCPLOW-Serine (O_SCPLOWLC_SCPLOW-Ser) and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThreonine (O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr) have versatile roles in metabolism. In addition to their use in protein synthesis, these amino acids participate in the biosynthesis pathways of other amino acids and even phospholipids. Furthermore, O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr can be substrates for a Ser/Thr dehydratase (Ser/ThrDH), resulting in pyruvate (Pyr) and 2-oxobutyrate, respectively, thus being amino acids with anaplerotic potential. Trypanosoma cruzi, the etiological agent of Chagas disease, uses amino acids in several biological processes: metacyclogenesis, infection, resistance to nutritional and oxidative stress, osmotic control, etc. In this study, we investigated the import and metabolism of O_SCPLOWLC_SCPLOW-Ser, O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr, and Gly in T. cruzi. Our results demonstrate that these amino acids are transported from the extracellular environment into T. cruzi cells through a saturable transport system that fits the Michaelis-Menten model. Our results show that O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOWO_SCPCAP-C_SCPCAPThr can sustain epimastigote (Epi) cell viability under nutritional stress (NS) conditions and can stimulate oxygen consumption to maintain intracellular ATP levels. Additionally, our findings indicate that O_SCPLOWLC_SCPLOW-Ser plays a role in establishing the mitochondrial membrane potential ({Delta}{Psi}m) in T. cruzi. O_SCPLOWLC_SCPLOW-Ser is also involved in energy metabolism via the Ser-Pyr pathway, which stimulates the production and subsequent excretion of acetate and alanine. Our results demonstrate the importance of O_SCPLOWLC_SCPLOW-Ser and O_SCPLOWLC_SCPLOW-Thr in the energy metabolism of T. cruzi and provide new insights into the metabolic adaptations of this parasite during its life cycle.

biochemistry↗

bioRxiv2023

Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

A promising treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation, by competition. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional gene knockouts were created to investigate the biological importance of the FOH/GGOH salvage pathway. The knockout parasites were viable but more susceptible to fosmidomycin, and their sensitivity to MEP inhibitors could not be rescued by the addition of prenols. Moreover, the knockout parasites lost their ability to use prenols for protein prenylation. These results demonstrate that FOH/GGOH salvage is an additional source of isoprenoids by malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme whose inhibition may potentiate the antimalarial efficacy of drugs that affect isoprenoid metabolism.

biochemistry↗