Trypanosome outer kinetochore proteins suggest conservation of chromosome segregation machinery across eukaryotes

Kinetochores are multiprotein complexes that couple eukaryotic chromosomes to the mitotic spindle to ensure proper segregation. The model for kinetochore assembly is conserved between humans and yeast, and homologues of several components are widely distributed in eukaryotes, but key components are absent in some lineages. The recent discovery in a lineage of protozoa called kinetoplastids of unconventional kinetochores with no apparent homology to model organisms suggests that more than one system for eukaryotic chromosome segregation may exist. In this study, we report a new family of proteins distantly related to outer kinetochore proteins Ndc80 and Nuf2. The family member in kinetoplastids, KKT-interacting protein 1 (KKIP1), associates with the kinetochore, and its depletion causes severe defects in karyokinesis, loss of individual chromosomes, and gross defects in spindle assembly or stability. Immunopurification of KKIP1 from stabilized kinetochores identifies six further components, which form part of a trypanosome outer kinetochore complex. These findings suggest that kinetochores in organisms such as kinetoplastids are built from a divergent, but not ancestrally distinct, set of components and that Ndc80/Nuf2-like proteins are universal in eukaryotic division

New inhibitors induce a decrease of cellular uptake of <i>T. cruzi</i>.

<p>New inhibitors induce a decrease of cellular uptake of <i>T. cruzi</i>.</p

Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.

<p>Two new compounds are more potent inhibitors of <i>Tc</i>PRAC than PYC.</p

Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.

<p>Inhibition of <i>Tc</i>PRAC proline racemization measured by polarimetry.</p

Effects of <i>Tc</i>PRAC inhibition by OxoPA and BrOxoPA on parasite interaction with host cells in vitro.

<p>Vero cells cultures were infected for 17 h at 37°C with cultured trypomastigotes at a 10∶1 parasite/cell ratio then washed and incubated for 17 h with 0, 100 and 1000 µM of PYC, or 0, 10 and 30 µM OxoPA or Br-OxoPA in fresh medium. Total parasite numbers/culture were estimated by capture ELISA.</p

Transition path characteristics.

<p>(A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. Transition path characteristics. (A) Energy profile and metric quantities for the <i>Tc</i>PRAC transition path. The energy profile (full line) shows that the intermediate states have low energy and do not present any energy barriers. Dotted and dashed lines show the distance from the first structure (d_1,i) and the cumulative distance covered from the first structure (l_1,i), respectively (RMS in Å, see Material and Methods). Little swerving was necessary to avoid the energy barriers. The points corresponding to the intermediate structures used in the screening are marked by crosses. (B) Scores of known ligands, synthesized analogues, and new inhibitors when docked in the selected binding site models. Br-OxoPA could not be docked in the crystallographic structure and its score in the fourth conformation is circled. The score threshold that was chosen in the subsequent virtual screening phase for ligand selection is indicated by a dashed line and the exclusion region is striped. (C) Cavity volume and extension in transition path intermediates, and docked molecules properties. Volume and extension are calculated as explained in Material and Methods. The volume is displayed by the thin line curve. Cavity extension is displayed by the thick curve and crosses mark intermediates that were used for virtual screening. The extension of PYC is shown by the horizontal line, that of OxoPA in all-trans conformation is shown by the horizontal dashed line. The extension of BrOxoPA in all-trans conformation is given in dotted line for two extreme rotamers on the C4–C5 bond. The average molecular weight of the library compounds successfully docked in conformers 1, 4 and 10 is displayed by filled circles. For clarity, the average mass has been divided by 2 to fit the same scale as the cavity volume.</p

Structure of initial compounds and analogues.

<p>(1) PYC and its nitrogen-bearing analogues: (2) Imidazole-4-carboxylic acid, (3) 1-<i>H</i>-Imidazole-2-carboxylic acid and (4a) PZC, (4b) Cl-PZC and (4c) Br-PZC.</p

Selection of active site conformations for virtual screening.

<p>(A) Four of the 49 conformations defining the path were selected for virtual screening. Protein secondary structures are shown schematically as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060955#pone-0060955-g002" target="_blank">Figure 2</a>. Transparent green spheres show the enclosed void volume of the pocket, with the ligand inside the first three structures (opaque green). (B) Structure of the two identified novel inhibitors of <i>Tc</i>PRAC.</p

Kinetics of D-proline formation with time in the presence or absence of <i>Tc</i>PRAC inhibitors.

<p>(A) Concentrations of D-proline formed were determined by polarimetry in reaction assays containing 0.15 µM of <i>Tc</i>PRAC and 40 mM L proline (see Material and Methods). Optical rotation was measured every 10 s for 8 min, with 10 µM PYC competitive inhibitor (black circles), 10 µM OxoPA (black squares) and 10 µM Br-OxoPA (black triangles), or without inhibitor (white circles). (B) Percentage of residual activity after pre-incubation of the enzyme with different concentrations of OxoPA and BrOxoPA for 5 minutes (upper panel) and 10 minutes (lower panel).</p

Combined approaches for drug design points the way to novel proline racemase inhibitor candidates to fight Chagas' disease.

International audienceChagas' disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles