Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Structural role of the flanking DNA in mariner transposon excision.

During cut-and-paste mariner/Tc1 transposition, transposon DNA is cut precisely at its junction with flanking DNA, ensuring the transposon is neither shortened nor lengthened with each transposition event. Each transposon end is flanked by a TpA dinucleotide: the signature target site duplication of mariner/Tc1 transposition. To establish the role of this sequence in accurate DNA cleavage, we have determined the crystal structure of a pre-second strand cleavage mariner Mos1 transpososome. The structure reveals the route of an intact DNA strand through the transposase active site before second strand cleavage. The crossed architecture of this pre-second strand cleavage paired-end complex supports our proposal that second strand cleavage occurs in trans. The conserved mariner transposase WVPHEL and YSPDL motifs position the strand for accurate DNA cleavage. Base-specific recognition of the flanking DNA by conserved amino acids is revealed, defining a new role for the WVPHEL motif in mariner transposition and providing a molecular explanation for in vitro mutagenesis data. Comparison of the pre-TS cleavage and post-cleavage Mos1 transpososomes with structures of Prototype Foamy Virus intasomes suggests a binding mode for target DNA prior to Mos1 transposon integration.

Cloning, expression and characterisation of FKB-6, the sole large TPR-containing immunophilin from C. elegans.

We have cloned, expressed, purified and characterised ceFKB-6, the only large tetratricopeptide repeat motif-containing immunophilin in Caenorhabditis elegans which is similar to the human orthologues FKBP51 and FKBP52. It shows increased peptidyl prolyl isomerase activity, the measured k(cat)/K(m) of 1.3 x 10(6) M(-1) s(-1)is twofold greater than that of hFKBP12 and hFKBP51. NMR studies of the interaction between FKB-6 and the C-terminal DAF-21 pentapeptide MEEVD show interactions consistent with those found between the large human immunophilin TPR domains and human Hsp90. In vivo localisation studies show that the fkb-6 gene is expressed in all stages from embryo to adult with predominant expression being noted in the adult dorsal and ventral nerve cords.

Structures of immunophilins and their ligand complexes.

This review includes an analysis of available X-ray and NMR structures of both members of the immunophilin family; cyclophilins and the FK-506 binding proteins (FKBPs). Available structures are compared and contrasted to highlight different structural features seen both within and between species. Each immunophilin family has been structurally characterised with a variety of small molecule ligands, principally immunosuppressive drugs and their analogues and an overview of these complexes is also presented. Currently the Protein Data Base contains over 60 entries for cyclophilins and over 40 entries for FKBPs. A number of FKBP related structures are also available including structures of MIP (Macrophage Infectivity Potentiator protein) from Legionella pneumophila and Trypanosoma cruzi and Trigger Factor from Mycoplasma genitalium. For all structures discussed in the review a summary of the available biological data is also presented.

A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90.

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.