Toolkit of Approaches To Support Target-Focused Drug Discovery for Plasmodium falciparum Lysyl tRNA Synthetase

Abstract

There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase ( Pf KRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.

Chemical pulldown combined with mass spectrometry to identify the molecular targets of antimalarials in cell-free lysates

“… 3 , 4 The drug bead synthesis protocol specifically details the attachment of DDD02355221 ( Figure 1 ) onto an activated Sepharose resin. 1 Probe DDD02355221 is an analog of DDD01510706 , a known inhibitor of P. falciparum lysyl tRNA synthetase (KRS). 5 However, we have successfully used this procedure to prepare drug beads based upon other molecules of interest.…”

“…For example, DDD02355221-drug beads were prepared with 82% and 11% loading levels for the high- and low-loading drug beads respectively. 1 …”

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Figure 6 Graph representing data from a competitive pulldown experiment The graph, generated in Perseus, shows the log2 fold-change of proteins bound to DDD02355221-beads (high-loading level) incubated with P. falciparum lysate pre-treated with DMSO or competitor (DDD01510706) (Milne et al. 1 ). Protein intensity is represented on the y-axis (log10 scale).

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Current and emerging target identification methods for novel antimalarials

“…Furthermore, using multiple target identification methods in parallel can also provide confidence in targets that are consistently identified. Successful target identification programs have often combined multiple approaches, with complimentary techniques confirming both target engagement and the phenotypic relevance of identified targets ( Milne et al, 2022 ). For example, the plasmepsin inhibitors WM4, WM5 and WM382 utilised IVIEWGA to originally identify plasmepsin X as a potential target, and demonstrated that WM382 specifically bound to both plasmepsin IX and X through targeted chemoproteomic and CETSA assays ( Favuzza et al, 2020 ).…”

Developing, Choosing, and Using the Chemical Toolbox for Infectious Diseases Research

“…Chemical biology tools are often developed using chemical probes as starting points 12 and adding additional functionality such as a biotin handle, click handle, or fluorescent dye. The importance of the criteria outlined above for the generation of robust, well-characterized probes therefore directly influences the performance and utility of the resulting tools.…”