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Discovery and Validation of Lmj_04_BRCT Domain, a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis.

ABSTRACT: Since many of the currently available antileishmanial treatments exhibit toxicity, low effectiveness, and resistance, search and validation of new therapeutic targets allowing the development of innovative drugs have become a worldwide priority. This work presents a structure-based drug discovery strategy to validate the Lmj_04_BRCT domain as a novel therapeutic target in Leishmania spp. The structure of this domain was explored using homology modeling, virtual screening, and molecular dynamics studies. Candidate compounds were validated in vitro using promastigotes of Leishmania major, L. amazonensis, and L. infantum, as well as primary mouse macrophages infected with L. major. The novel inhibitor CPE2 emerged as the most active of a group of compounds against Leishmania, being able to significantly reduce the viability of promastigotes. CPE2 was also active against the intracellular forms of the parasites and significantly reduced parasite burden in murine macrophages without exhibiting toxicity in host cells. Furthermore, L. major promastigotes treated with CPE2 showed significant lower expression levels of several genes (α-tubulin, Cyclin CYCA, and Yip1) related to proliferation and treatment resistance. Our in silico and in vitro studies suggest that the Lmj_04_BRCT domain and its here disclosed inhibitors are new potential therapeutic options against leishmaniasis.

SUBMITTER: Pena-Guerrero J

PROVIDER: S-EPMC8508789 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated.We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites.Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and therefore could become a novel therapeutic and druggable target in leishmaniasis therapy. In addition, following comparative sequence analyses, we found the RAC/AKT-like proteins from Leishmania constitute a subgroup by themselves within a general AKT-like protein family.

Project description:BackgroundHepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide. PAFAH1B3 plays an important role on occurrence and development in a variety tumor. However, the function of PAFAH1B3 in HCC remains unclear.MethodsThe TIMER, ONCOMINE, Human Protein Atlas (HPA), GEPIA, The Cancer Genome Atlas (TCGA), HCCDB, UALCAN and LinkedOmics database were used to analyze the prognostic value, co-expression genes and regulator networks of PAFAH1B3 in HCC. siRNA transfections and inhibitor of PAFAH1B3 P11 were used to verify the anti-tumor effect on HCC cell lines. Gene expression was detected by qRT-PCR. The functions of PAFAH1B3 downregulation in HCC cell lines were investigated using cell cycle analysis, apoptosis detection, CCK8 assay and transwell assay. Western blot was used to evaluate the role of PAFAH1B3 on metabolic pathways in HCC cells.ResultsBased on the data from databases, the expression of PAFAH1B3 was remarkably increased in HCC patients. High expression of PAFAH1B3 was associated with poorer overall survival (OS) and disease-free survival (DFS). And PAFAH1B3 was notably linked to age, sex, grade, stage, race, and TP53 mutational status. Then, the functional network analysis showed PAFAH1B3 may be involved in HCC through cell cycle, cell metabolism, spliceosome, and RNA transport. Furthermore, the mRNA expression of PAFAH1B3 was also increased in HCC cell lines. Flow cytometry analysis showed that PAFAH1B3 manipulated apoptosis and cell cycle regulation. CCK8 assay showed that PAFAH1B3 silencing or pharmacologic inhibitor of PAFAH1B3 inhibited the proliferation of HepG2, Huh7 and MHCC-97H cells. Transwell assay results showed that PAFAH1B3 silencing also significantly impaired the invasion and migratory ability of HCC cells. In addition, PAFAH1B3 silencing significantly downregulated the expression of glycolysis and lipid synthesis signaling pathways.ConclusionOur findings suggested that PAFAH1B3 plays a critical role in progression of HCC. PAFAH1B3 as a prognosis marker and potential target for HCC has prospective clinical significance.

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Discovery and Validation of Lmj_04_BRCT Domain, a Novel Therapeutic Target: Identification of Candidate Drugs for Leishmaniasis.

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