Biology subjects

Poulsen, S.-A.

Publications and source records attributed to Poulsen, S.-A..

4 recordsLinked to original sources

bioRxiv2025

Thymidine kinase-independent click chemistry DNADetect™ probes as an EdU alternative for mammalian cell DNA labelling

Nucleoside analogues have been powerful tools to study DNA synthesis, cell cycle progression and cellular fate for decades. EdU (5-ethynyl-2'-deoxyuridine), which contains an alkyne handle that allows copper-catalyzed azide-alkyne cycloaddition (CuAAC) to a fluorescent azide in a "click" chemistry reaction, is one of the most frequently utilised nucleoside analogue probes. EdU is transported into cells by nucleoside transporters followed by phosphorylation by thymidine kinase, the first step in thymidine metabolism leading to DNA synthesis. As some organisms like malaria parasites lack the thymidine kinase enzyme and cannot be labelled by EdU or related thymidine analogue probes, we previously designed and validated DNADetect EdU analogues as chemical probes. These probes comprise a masked monophosphate on the 5'-hydroxyl group of the nucleoside sugar moiety that is metabolised directly to EdU monophosphate, bypassing the need for thymidine kinase. Here, we demonstrate that DNADetect probes can be used to label DNA in mammalian cells. DNADetect probes are incorporated into proliferating HeLa cells as efficiently as EdU and outcompeted by thymidine. Additionally, we implement a protocol for best practice use of metabolic chemical probes by using a specifically designed inactive control probe for each active probe. While this approach is commonly applied with chemical probes that modulate protein function, it is yet to be commonly applied with metabolic chemical probes.

cell biology↗

bioRxiv2025

Natural product-mediated reaction hijacking mechanism validates Plasmodium aspartyl-tRNA synthetase as an antimalarial drug target

Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5'-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2 phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.

biochemistry↗

bioRxiv2025

Assessment of native mass spectrometry as a screening method to identify and characterize RNA-targeting small molecules

The intentional targeting of RNA with small molecules is recognized as a viable pathway to new therapeutics with potential to vastly expand the proportion of the human genome considered druggable. The practical considerations to deliberately target RNA are largely under development, including optimal screening methods to identify small molecules for binding to RNA. Native mass spectrometry (nMS) is established as a valuable biophysical method for identifying small molecule hits against diverse biomolecular targets, most frequently proteins and protein-protein interactions. Herein we assess nMS for studying the binding of small molecules with RNA aptamers as a model for nMS screening of RNA as a drug target. We first develop workflows for characterizing the binding of cognate RNA aptamer ligands and then establish a nMS method to screen a small molecule library against the RNA aptamers. nMS analysis permitted identification of binders, quantitation of binding strength and generation of structure-activity relationships with some dependence on the aptamer class. This work demonstrates the utility of nMS as a complementary and efficient target-based biophysical screening method that can characterize RNA-small molecule interactions and the potential of nMS becoming a powerful enabling tool in RNA-targeting drug discovery.

biophysics↗

bioRxiv2023

Thymidine kinase-independent click chemistry DNADetect probes for DNA proliferation assessment in malaria parasites

Metabolic chemical probes are small molecule reagents that utilise naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells however is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labelling of replicating asexual intraerythrocytic P. falciparum parasites, as determined by flow cytometry using copper catalysed azide-alkyne cycloaddition (CuAAC) to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

microbiology↗