OKAFOR, ESTHER OGECHI and Covenant University, Theses (2023) CDPK5 AND MSP1 GENE MODULATION AND IN VITRO PARASITE INHIBITION BY MCL OR ITS COMBINATION WITH ARTEMISININ. Masters thesis, COVENANT UNIVERSITY.
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Abstract
Recent treatment failures in artemisinin-based combination therapy (ACT) have raised concerns about its efficacy against malaria and emphasize the need to discover new treatment targets and resistance-free drugs. A small molecule (MCULE-7146940834 - MCL) showed inhibitory potential against Plasmodium falciparum, targeting gene families crucial for red blood cell invasion in silico but has not been validated in vitro. This study assesses MCULE-7146940834 in vitro, evaluating its inhibitory concentration (IC50) and gene modulation effects on Merozoite Surface Protein 1 (PfMSP1) and Calcium-dependent Protein Kinase (PfCDPK5), both independently and in combination with Artemisinin (ART). PfMSP1 facilitates the attachment and binding of the merozoite to the host RBC, while PfCDPK5, facilitates the secretion of invasion-related proteins and motor function to drive penetration making them promising antimalarial drug targets. Plasmodium falciparum derived from field isolates and the 3D7 strain were cultured within O+ human red blood cells. This cultivation occurred in RPMI 1640 medium supplemented with 10% heat-inactivated human serum, 25 mM HEPES buffer, and 50 μg/ml penicillin-streptomycin. The entire process was carried out under controlled conditions of 5% CO2 at 37°C. Serially diluted drugs of ART, ART+MCL, and MCL were administered to 96-well microtitre plates, over 72 hours, with doses incrementing by a factor of 10 from 0 to 100μM. This procedure adhered to the WHO micro-test protocol and involved incubating the substances with parasite culture medium samples at a parasitemia level of 0.2% and a haematocrit of 4% for the same duration. The evaluation of parasitemia was performed microscopically using Giemsa-stained smears. RNA from cultured samples, pre- and post-treatment, was extracted, quantified, and analysed by real-time polymerase chain reaction using primers specific for the PfMSP1 and PfCDPK5 genes and PfGAPDH as an internal reference gene. All assays were carried out in duplicates and analyzed using graph pad prism software at p < 0.05. The outcomes from the analysis of half maximal inhibitory concentrations (IC50) using linear regression demonstrated an in vitro IC50 value of 24.68 μM for MCL, and a value of 5.006 μM for the combination of MCL with artemisinin. Relative gene expression (ΔCt) shows increased expression of PfMSP1 and PfCDPK5 relative to PfGAPDH. These results demonstrated that the MCULE-7146940834 holds promise as a potential candidate for antimalarial drug development, making it a valuable hit compound for subsequent optimization.
| Item Type: | Thesis (Masters) |
|---|---|
| Subjects: | Q Science > Q Science (General) Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology |
| Divisions: | Faculty of Engineering, Science and Mathematics > School of Chemistry |
| Depositing User: | AKINWUMI |
| Date Deposited: | 03 Oct 2023 11:21 |
| Last Modified: | 03 Oct 2023 11:21 |
| URI: | http://eprints.covenantuniversity.edu.ng/id/eprint/17326 |
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