The superfamily of protein kinases are emerging as valid therapeutic targets for anti-leishmanial drug development. The essential kinase GSK3β regulates cell proliferation, motility, glycogen metabolism and apoptosis. A peptide sequence derived from ubiquitin hydrolase (UBH5) is one of many substrates of GSK3β and was obtained from a strain of Leishmania Mexicana for recombinant expression and affinity purification. Radiometric kinase assays were then used to screen 320 small-molecule chemical inhibitors designed by Lifearc for their ability to inhibit recombinant GSK3β phosphorylation of UBH5. Three chemical small-molecule inhibitors were identified G18, J16 and J22 which inhibited UBH5 peptide substrate phosphorylation in a concentration- dependent manner with IC50 values of 3.0 µM, 1.4 µM and 3.2 µM, respectively. Two compounds Malabaricone B and Malabricone C were extracted from a plant called Myristica malabarica, previously identified as having anti-leishmanial properties, and investigated for their ability to inhibit GSK3β enzymatic activity. Both plant compounds induced concentration-dependent inhibition of GSK3β auto-phosphorylation and UBH5 peptide substrate phosphorylation. Malabaricone B showed an IC50 of 1.5 µM for auto-phosphorylation and an IC50 of 3.8 µM for substrate phosphorylation. Malabaricone C had IC50's of 1.6 µM and 4.2 µM, respectively. Both Malabaricones were type 1 ATP-competitive inhibitors. Leishmania MPK4 is an essential kinase involved in lesion formation and life cyclestage conversion. Recombinant MPK4 was not well expressed in bacteria therefore two expression plasmids were generated in order to express MPK4 in Leishmania for later purification and screening of inhibitors. The plasmids were constructed expressing C-terminal green fluorescent protein (GFP)-tagged LmxMPK4 or N-terminal GFP-tagged and His-tagged LmxMPK4 both containing the selective marker gene conferring blasticidin resistance. The expression plasmids were used for transfection with wild type Leishmania Mexicana promastigotes and a mutant form which contained no genomic copy of LmxMPK4 but instead carried a plasmid containing the LmxMPK4 gene. Appearance of the GFP during fluorescence microscopy suggested the LmxMPK4-GFP fusion protein was expressed. However, an attempt to replace the plasmid containing LmxMPK4 from the mutant form with a GFP-tagged version was unsuccessful indicating the fusion protein was inactive.
|Date of Award||17 Jan 2019|
- University Of Strathclyde
|Supervisor||Martin Wiese (Supervisor)|