Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 May 10:3:fddsv.2023.1185679.
doi: 10.3389/fddsv.2023.1185679.

The critical role of mode of action studies in kinetoplastid drug discovery

Alan H Fairlamb  1 , Susan Wyllie  1
Affiliations

The critical role of mode of action studies in kinetoplastid drug discovery

Alan H Fairlamb et al. Front Drug Discov (Lausanne). .

Abstract

Understanding the target and mode of action of compounds identified by phenotypic screening can greatly facilitate the process of drug discovery and development. Here, we outline the tools currently available for target identification against the neglected tropical diseases, human African trypanosomiasis, visceral leishmaniasis and Chagas' disease. We provide examples how these tools can be used to identify and triage undesirable mechanisms, to identify potential toxic liabilities in patients and to manage a balanced portfolio of target-based campaigns. We review the primary targets of drugs that are currently in clinical development that were initially identified via phenotypic screening, and whose modes of action affect protein turnover, RNA trans-splicing or signalling in these protozoan parasites.

Keywords: chemical proteomics; chemical pulldown; drug resistance; leishmaniasis; metabolomics; target identification; trypanosomiasis; whole genome sequencing.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author AHF declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1. Schematic of RNAi approach to identifying essential genes and drug resistant mechanisms.
A plasmid library containing randomly sheared genomic fragments was transfected into bloodstream form T. brucei. After culturing under non-inducing or inducing conditions, or in the presence or absence of drug, genomic DNA was isolated and adaptor-ligated libraries prepared. After amplification, size selection and sequencing, the RNAi target fragments are mapped to the reference genome. The depth of sequence coverage of target fragments between induced and uninduced cultures reveals “cold spots” (essential genes) and the depth of coverage of target fragments between induced drug treated and RNAi-induced controls reveals “hot spots” (resistance genes). Where gene knock-down confers a selective advantage. Full details are available elsewhere (Alsford et al., 2011; Alsford et al., 2012).
Figure 2
Figure 2. Genome-wide target overexpression strategies.
Panel (A). Episomal cosmid expression strategy for Leishmania. Promastigotes are transfected with a shuttle cosmid vector pcosTL (Kelly et al., 1994) containing ~38 kb genomic DNA fragments under G418 selection (Hoyer et al., 2001). After the parasite cosmid library has undergone drug selection, cosmids are sequenced and mapped onto the reference genome (Corpas-Lopez et al., 2019). A similar approach is under development for T. cruzi at the University of Dundee. Panel (B). Expression from genes at the rRNA locus in T. brucei. Genomic DNA fragments were cloned in pRPaOEX, transfected into bloodstream form T. brucei and inserted at the rDNA locus under the control of a tetracycline-inducible ribosomal RNA (rRNA) promoter. Following drug selection, DNA was extracted from the surviving population and the overexpression inserts amplified by long range PCR, sequenced and mapped onto the reference genome (Corpas-Lopez et al., 2019).
Figure 3
Figure 3. Rescued growth in a T. brucei cell line overexpressing GSK3β short.
In the absence of tetracycline induction, cell growth (black symbols) follows a standard dose-response inhibition curve with an EC50 of 2.8 μM and slope 2.7. After tetracycline induction (red symbols) cell growth is stimulated up to maximum growth at 3 μM by DDD85893 (compound 4 m (Urich et al., 2014)) and inhibited thereafter with an EC50 of 9.1 μM and slope 3.6. Data redrawn from (Grimaldi, 2014). The dashed (growth stimulation) and solid (growth inhibition) lines are independent best non-linear fits to a dose-response equation at concentrations above and below 2 µM.
Figure 4
Figure 4. Schematic representation of proteomics approaches to drug target identification.
Panel (A). Standard chemical pulldown workflow. Linkers (commonly PEG) are attached to a permissable position on the compound of interest. Linker analogues are attached to resin to create a “drug-bead”. Drug beads are incubated in parasite cell lysate that has been preincubated in the presence or absence of parent compound. Proteins binding to drug beads in the presence or absence of competition from the parent compound are identified by mass spectrometry. Proteins whose binding has been reduced in the presence of the parent compound are considered to be specifically binding to the linker compound and considered putative drug targets (Smith et al., 2023). Panel (B). Functionalised linkers comprising a diazarine photoactivatable warhead and an alkyne handle to facilitate subsequent pulldown are attached to a permissbale position on the compound of interest. These functionalised linker analogues are incubated with live cells. Covalent attachment of the linker analogue to its molecular target is triggered by exposure to UV light. Interacting targets are enriched from the cell lysates using the bio-orthogonal handle and identified by mass spectrometry. Panel (C). Standard thermal proteome profiling (TPP) workflow. Parasites are preincubated in the presence of drug or vehicle (DMSO). Treated and control cell lysates are prepared, aliquoted and aliqots incubated at a range of temperatures. Following incubation, soluble proteins are harvested, processed, labelled with TMTs, pooled and analysed by LC-MS/MS (Corpas-Lopez and Wyllie, 2021).
Figure 5
Figure 5. Structures and primary targets of clinical candidates.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Allen CL, Goulding D, Field MC. Clathrin-mediated endocytosis is essential in Trypanosoma brucei. EMBO J. 2003;22(19):4991–5002. doi: 10.1093/emboj/cdg481. - DOI - PMC - PubMed
    1. Alpizar-Sosa EA, Ithnin NRB, Wei W, Pountain AW, Weidt SK, Donachie AM, et al. Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response. PLoS Negl Trop Dis. 2022;16(9):e0010779. doi: 10.1371/journal.pntd.0010779. - DOI - PMC - PubMed
    1. Alsford S, Eckert S, Baker N, Glover L, Sanchez-Flores A, Leung KF, et al. High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature. 2012;482(7384):232–236. doi: 10.1038/nature10771. - DOI - PMC - PubMed
    1. Alsford S, Turner DJ, Obado SO, Sanchez-Flores A, Glover L, Berriman M, et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011;21(6):915–924. doi: 10.1101/gr.115089.110. - DOI - PMC - PubMed
    1. Andreini C, Bertini I, Rosato A. Metalloproteomes: A bioinformatic approach. Accounts Chem Res. 2009;42(10):1471–1479. doi: 10.1021/ar900015x. - DOI - PubMed

LinkOut - more resources