Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic ...stage of the disease, urgently calling for the identification of new targets and drug candidates.
Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzob,f1,4oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzob,f1,4thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.
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•Trypanosoma parasites cause severe disease and peroxisomal transport (PEX) is a valid drug target.•PEX5-PEX14 interaction inhibitors are delivered and SAR information is provided.•The developed small molecule PEX14 inhibitors kill Trypanosoma parasites in a cell-based assay.•Provided inhibitors offer a starting point for the development of pharmacologically relevant trypanocidal compounds.
PEX19 binding sites are essential parts of the targeting signals of peroxisomal membrane proteins (mPTS). In this study, we characterized PEX19 binding sites of PEX11, the most abundant peroxisomal ...and glycosomal membrane protein from
Trypanosoma brucei
and
Saccharomyces cerevisiae
.
Tb
PEX11 contains two PEX19 binding sites, one close to the N-terminus (BS1) and a second in proximity to the first transmembrane domain (BS2). The N-terminal BS1 is highly conserved across different organisms and is required for maintenance of the steady-state concentration and efficient targeting to peroxisomes and glycosomes in both baker’s yeast and
Trypanosoma brucei
. The second PEX19 binding site in
Tb
PEX11 is essential for its glycosomal localization. Deletion or mutations of the PEX19 binding sites in
Tb
PEX11 or
Sc
PEX11 results in mislocalization of the proteins to mitochondria. Bioinformatic analysis indicates that the N-terminal region of
Tb
PEX11 contains an amphiphilic helix and several putative TOM20 recognition motifs. We show that the extreme N-terminal region of
Tb
PEX11 contains a cryptic N-terminal signal that directs PEX11 to the mitochondrion if its glycosomal transport is blocked.
Highlights • The direct impact of lentiviral particles on cognate B cells in vitro was shown. • Activation events in B-cells after the contact with lentiviral particles. • Lentiviral particles ...initiated germinal center-like reactions without T-cell help. • T-independent differentiation into memory and IgG3 and IgA-producing cells. • Production of CD40L by cognate B-cells stimulated with lentiviral particles.
In animal models, lentiviral particles (LP) were shown to be promising HIV vaccine candidates. Since little is known about the direct impact of LP on antigen-specific B cells, we incorporated Hen Egg ...Lysozyme (HEL) into LP (HEL-LP) derived from HIV to study their effect on HEL-specific, B cell receptor-transgenic B-cells (HEL+B-cells)in vitro. We observed preferential binding of HEL-LP to HEL+B-cells and their efficient internalization. HEL-LP were able to effectively cross-link B-cell receptors as indicated by the loss of surface CD62L. In the absence of CD4+T-cells, other activation events induced by LP in cognate naïve B-cells included increased expression of activation and co-stimulatory molecules as well as an enhanced proliferative response. Additionally, the B-cell phenotype shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. The observed CD4+T-cell independent activation and differentiation may be due to LP-induced expression of CD40L by a subset of cognate B-cells. Thus, even in the absence of CD4+T-cells LP provide strong direct activation signals to cognate naïve B-cells, which may contribute to the strong humoral immune responses observed after LP immunization.
