Characterising Plasmodium falciparum cyclin dependent like kinase 1 (PfCLK1) as a potential antimalarial target

Niniola, Olaniyan (2025) Characterising Plasmodium falciparum cyclin dependent like kinase 1 (PfCLK1) as a potential antimalarial target. PhD thesis, University of Glasgow.

Abstract

The widespread and indiscriminate use of antimalarial drugs has contributed to recurring parasite resistance that threatens a global resurgence of malaria with increasing morbidity and mortality rate towards the verge of endemicity. The dual specificity protein kinase family, CLKs, play crucial roles in the regulation of transcript splicing by phosphorylating SR proteins. Global phospho-proteomic studies of the Plasmodium kinome have shown such splicing events to be an essential process across developmental stages of the parasite life cycle. Thus, members of the CLK family have been considered as potential therapeutic targets in present antimalarial drug development pipelines for new molecules which demonstrate the required efficacy and selective toxicity to the parasite without the propensity to induce parasite resistance. A member kinase PfCLK3 was validated as a cross species multistage drug target in a previous study by our group using a selective and specific small drug like inhibitor TCMDC-135051. However, to achieve poly-pharmacology or develop drugs for combination therapy as an approach to circumvent potential drug resistance, a second target PfCLK1 with similar therapeutic potential was identified. This thesis aimed to extend the previous study by characterising potent selective inhibitors for PfCLK1 and explore the role of the kinase in Plasmodium falciparum. The primary screen at GlaxoSmithKline from the previous study yielded four distinct chemical series of small molecule inhibitors with specific nanomolar activity towards PfCLK1. In this study, two potent members JZ208105-178D1 and HGC-0017530023-NX-1 were further characterised through enzymatic and cellular analyses to investigate their mode of inhibition and determine their interaction with the target kinase.

First, the enzymatic parameters of the biochemical assays were determined using recombinant PfCLK3 and kinase domain PfCLK1 proteins. In addition, a robust assay measuring ATP consumption was developed that proved suitable for high throughput screening. Substrates that were recognised and efficiently phosphorylated by both kinases were identified and used to establish optimal concentrations of protein and ATP for inhibition assay conditions. Second, a detailed analysis of the inhibition mechanism, target specificity, timing of peak activity during life cycle and the rate of parasite reduction was completed to identify the most potent inhibitor that could be optimised to the status of lead compound through further in vitro and in vivo studies. In vitro inhibition results showed that both compounds displayed low nanomolar inhibition of kinase domain PfCLK1 and further demonstrated selective inhibition towards PfCLK3 suggestive of an affinity for the highly conserved domain between both kinases. Additionally, JZ208105-178D1 demonstrated a mode of binding suggestive of ATP competitive inhibition, in contrast to the non-ATP competitive inhibition observed for HGC-0017530023-NX-1. Both inhibitors emerged effective at high nanomolar to low micromolar concentrations against asexual blood stages of P. falciparum 3D7 wild type. For chemical validation, a PfCLK1-like PfCLK3 mutant parasite, G449P demonstrated the same sensitivity as wild type parasites to both inhibitors suggesting PfCLK1 inhibition is linked to their parasitical activity.

Susceptibility profiling of P. falciparum 3D7 asexual blood stages to both inhibitors revealed a variety of stage-specific profiles that differentiated the modes of action including TCMDC-135051 and identified trophozoite-specific peak activity for JZ208105-178D1 and TCMDC-135051. Given that JZ208105- 178D1 conferred higher inhibitory effect in vitro and in vivo, further investigations were carried without HGC-0017530023-NX-1. The morphological analysis of asexual blood stages following treatment with JZ208105-178D1 confirmed that parasite development was completely arrested at the early trophozoite stage. This contrasted with the progression of untreated parasites to late trophozoites at comparable time points. In addition, JZ208105-178D1 impaired the maturation of P. falciparum NF54 gametocytes, stages responsible for transmission, by an ~11-fold reduction in gametocyte number at 1x EC50 concentration.

The parasite reduction rate assay was used to quantify the speed of therapeutic capacity, JZ208105-178D1 demonstrated activity levels similarly to TCMDC-135051 and standard antimalarial artemisinin. The combined data in this study suggests the malaria parasite's response to JZ208105-178D1 inhibition are PfCLK1 dependent and corroborate previous functional characterisation studies relating to PfCLK1 expression in the parasites. Thus, pointing to the PfCLK1 kinase as a promising target for antimalarial and transmission blocking chemotherapy strategies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Tobin, Professor Andrew and Marti, Professor Matthias
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85006
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 Apr 2025 10:04
• Last Modified: 08 Apr 2025 10:10
• Thesis DOI: 10.5525/gla.thesis.85006
• URI: https://theses.gla.ac.uk/id/eprint/85006

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