Bhambid, M. | Search bioRxiv

bioRxiv2025

The SV40 T-ag nuclear localization signal affects Toxoplasma gondii viability by targeting importin α

Nucleocytoplasmic transport is essential in eukaryotes and mediated by importin (Imp) /{beta} receptors that recognize nuclear localization signals (NLSs) on cargo proteins. The SV40 large T-antigen NLS (SV40-NLS), studied extensively, binds importin with high affinity in the nanomolar range, while modified versions, the Bimax peptides, bind in the picomolar range. Bimax peptides impede nuclear import and reduce viability in yeast and human cells, highlighting the potential of NLS peptides as inhibitors of nuclear transport. In this study, we investigated the potential of the SV40-NLS to target Toxoplasma gondii importin (TgImp). Expression of the SV40-NLS fused to a GFP reporter led to cytotoxicity in T. gondii tachyzoites; this depended on the SV40-NLS sequence and its position within the protein. Over-expression of TgImp rescued parasites from the SV40-NLS-induced cytotoxicity, confirming that the mechanism of action involves disruption of nuclear import. Importantly, the same construct did not affect cell viability in mammalian cells, suggesting a selective vulnerability in importin -mediated nuclear transport in the parasite. The SV40-NLS peptide offers an advantage over small-molecule inhibitors by targeting large interaction surfaces of importin with high specificity, minimizing off-target effects. This study lays the groundwork for a novel peptide-based therapeutic strategy employing NLS motifs to selectively inhibit nuclear import in T. gondii.

molecular biology↗

bioRxiv2024

Importin alpha inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

Protozoan parasites of the phylum Apicomplexa, including Plasmodium falciparum and Toxoplasma gondii, cause widespread disease in humans. New drugs and protein targets are required for the treatment of these diseases, particularly therapies targeting multiple stages of the parasite life cycles. Nuclear import, carried out by the transporters importin (IMP) and {beta} subunits, is a valid target for the discovery of lead compounds against these protozoan parasites: small molecules were identified that inhibit interactions between IMP and nuclear localisation signals in vitro and also inhibit the growth of the rapidly-dividing stages of P. falciparum and T. gondii (asexual stages and tachyzoites) in culture. In this report, we add another small molecule (Bay 11-7082) to the panel of inhibitors of IMP and test the ability of these inhibitors to first, inhibit nuclear transport in the rapidly dividing stages and, next, the maturation of differentiated stages of both parasites. We show that GW5074 and CAPE inhibit nuclear transport in the P. falciparum blood stages, while Bay 11-7085 inhibits nuclear transport in T. gondii tachyzoites. Interestingly, CAPE strongly inhibits gametocyte maturation, the sexual stages of P. falciparum, and Bay 11-7085 weakly inhibits bradyzoite differentiation, the latent stages of T. gondii. As differentiation of both these stages is dependent on activation of gene expression, triggered by the nuclear translocation of transcription factors, our work provides a "proof of concept" that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of them recalcitrant to existing drugs.

molecular biology↗