IN-SILICO SCREENING AND IN-VITRO VALIDATION OF PROTEIN INHIBITORS WITH PHARMACOLOGICAL ACTIVITY AGAINST PLASMODIUM MALARIA

Abstract

The World Health Organization (WHO) documents malaria as one of the leading causes of high morbidity and mortality worldwide. The disease affects millions and kills thousands of people annually. Efforts to reduce the global burden of malaria have prompted WHO to recommend prevention strategies like using anti-malarial drugs and malaria vaccines. However, these strategies have been ineffective because of anti-malarial drug resistance and the inefficacy of malaria vaccines. The current recommended drug combination is Artemisinin-based Combination Therapy (ACT). However, the extended ACTs clearance times, linked to the emergence of artemisinin monotherapy resistance recorded most recently in Africa and the Great Mekong region, pose a danger to its efficacy. Similarly, the RTS,S/AS01 (Mosquirix) vaccine’s modest effectiveness against malaria at 36% among kids aged 5 to 17 months who need four doses, fails to aid malaria eradication. To address the limitations of current anti-malarial therapies and vaccines, this study aimed to: (1) identify novel PfHsp90 and PfCSP inhibitors with pharmacological activity against Plasmodium malaria through hierarchical virtual screening (HVS) using geldanamycin and monoclonal antibody L9 as reference ligands, respectively; (2) assess the stability of protein inhibitor complexes via molecular dynamics simulations (MDS); and (3) validate the inhibitory efficacy of selected compounds through in vitro assays. Since P. falciparum heat shock protein 90 (PfHsp90) and P. falciparum circumsporozoite surface protein (PfCSP) are well-characterized malaria drugs and vaccines targets, respectively, this study sought to use them in discovering better efficacious malaria drug and vaccine candidates. Geldanamycin (GDM), a potent Hsp90 inhibitor with well-characterized binding to ATP-binding domain, was used to identify PfHsp90 inhibitors by screening it against the ZINC20 database via the ZINCPHARMER web server. This virtual screening process resulted in 17 hits. Monoclonal antibody (mAb) L9, with its high-affinity binding to the central NANP repeat region, was used to identify PfCSP inhibitors, yielding 23 hits. These ZINCPHARMER hits were subjected to drug-likeness and pharmacokinetics properties analysis in the SwissADME web server, and 18 (9 for PfHsp90 and 9 for PfCSP) of them satisfied the requirements. The 18 ZINC compounds were docked with PfHsp90 and PfCSP using the PyRx software to understand their interactions. From the molecular docking results, ZINC09060002 ( 8.2 kcal/mol), ZINC72133064 (-7.8 kcal/mol), ZINC72163401 (-7.7 kcal/mol), ZINC72358537 ( 8.1 kcal/mol), and ZINC72358557 (-7.6 kcal/mol) had better binding affinities to PfHsp90 than GDM (-7.5 kcal/mol). Similarly, ZINC25374360 (-8.1 kcal/mol), ZINC40144754 (-8.3 kcal/mol), and ZINC71996727 (-8.9 kcal/mol) bound strongly to PfCSP with binding affinities of less than 8.0 kcal/mol. The stability of these molecularly docked protein-inhibitor complexes was assessed through MDS using GROMACS 2022. The PfHsp90 inhibitors (ZINC72163401, ZINC72358537, and ZINC72358557) and PfCSP inhibitors (ZINC25374360 and ZINC71996727) formed stable complexes with their respective target proteins. These five compounds were subjected to in vitro validation using the Sybr Green I fluorescence assay. Cultured P. falciparum parasites were exposed to serial dilutions of each inhibitor, and parasite viability was quantified based on DNA binding fluorescence intensity. IC₅₀ values were calculated to determine the inhibitory potency of each compound. They showed promising inhibition of parasite growth with IC50 values ranging between 5 – 150 ng/mL. In this regard, the three PfHsp90 inhibitors are anti-malarial candidates while the two PfCSP inhibitors are potential vaccine adjuvants that might increase the efficacy of the existing Mosquirix vaccine. However, further structural optimization studies and clinical (in vivo) tests are necessary to ascertain the antimalarial activity of these compounds in humans.