Search for: All records

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a lethal disease caused by two vector-borne parasites:Trypanosoma brucei gambienseandTrypanosoma brucei rhodesiense. The limited number of antitrypanosomal therapies for treating these deadly parasites suffer from toxicity, poor efficacy, and unspecified targets; thus, more and better medicines are needed. We usedin silicomethods to predict features of the bioactive compound AZ960 that make it an ortholog-specific inhibitor for the extracellular-signal regulated kinase 8 ofT. brucei(TbERK8). Our homology models showed that the TbERK8 ATP binding pocket was smaller and more hydrophobic than that of human ERK8 (HsERK8). Molecular docking studies predicted six FDA-approved compounds that would be orthologue-specific inhibitors of HsERK8 or TbERK8. Experimental testing of these compounds identified prednisolone as an HsERK8-specific inhibitor. Sildenafil inhibited TbERK8, as predicted by our binding model. Its impact on TbERK8 activity supports our hypothesis that designing compounds that can exploit differences in the orthologs as buildable scaffolds and expand the repertoire of ortholog-specific antitrypanosomal agents. 

Abstract Proliferating cell nuclear antigen (PCNA) is a homo‐trimeric protein complex that clamps around DNA to tether DNA polymerases to the template during replication and serves as a hub for many other interacting proteins. It regulates DNA metabolic processes and other vital cellar functions through the binding of proteins having short linear motifs (SLiMs) like the PIP‐box (PCNA‐interacting protein‐box) or the APIM (AlkB homolog 2 PCNA‐interacting motif) in the hydrophobic pocket where SLiMs bind. However, overproducing TbPCNA or human PCNA (hPCNA) in the pathogenic protistTrypanosoma bruceitriggers a dominant‐negative phenotype of arrested proliferation. The mechanism for arrestingT. bruceiproliferation requires the overproduced PCNA orthologs to have functional intact SLiM‐binding pocket. Sight‐directed mutagenesis studies showed thatT. bruceioverproducing PCNA variants with disrupted SLiM‐binding pockets grew normally. We hypothesized that chemically disrupting the SLiM‐binding pocket would restore proliferation inT. brucei, overproducing PCNA orthologs. Testing this hypothesis is the proof‐of‐concept for aT. brucei‐based PCNA screening assay. The assay design is to discover bioactive small molecules that restore proliferation inT. bruceistrains that overproduce PCNA orthologs, likely by disrupting interactions in the SLiM‐binding pocket. The pilot screen for this assay discovered two hit compounds that linked to predetermined PCNA targets. Compound#1, a known hPCNA inhibitor, had selective bioactivity to hPCNA overproduced inT. brucei, validating the assay. Compound#6had promiscuous bioactivity for hPCNA and TbPCNA but is the first compound discovered with bioactivity for inhibiting TbPCNA.