Research Article: Identification of inhibitors of an unconventional Trypanosoma brucei kinetochore kinase

Date Published: May 31, 2019

Publisher: Public Library of Science

Author(s): Leah S. Torrie, Fabio Zuccotto, David A. Robinson, David W. Gray, Ian H. Gilbert, Manu De Rycker, Giovanni Maga.

http://doi.org/10.1371/journal.pone.0217828

Abstract

The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 μM and 102 μM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.

Partial Text

Kinetochores are multiprotein complexes that associate with the centromere of a chromosome during cell division, ensuring faithful transmission of genetic material to daughter cells [1, 2]. While kinetochores are evolutionary quite plastic, most eukaryotic kinetochores share the same canonical kinetochore machinery [3]. The kinetochores of the protozoan parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis [4], are an exception to this. The discovery of 20 novel kinetochore proteins in T. brucei [5–7], with no detectable homology to conventional eukaryotic kinetochore proteins, raises not only an interesting area of research to investigate the evolutionary history of these proteins, but also the possibility of exploiting these unique proteins in the search for new therapeutics, in particular as these proteins are conserved across the kinetoplastida order. This includes Leishmania spp. and Trypanosoma cruzi, the causative agents of leishmaniasis and Chagas disease [8, 9]. These neglected and devastating diseases affect some of the world’s poorest populations with current drug treatments suffering from high cost, host toxicity and emerging resistance [10, 11]. In the search for new therapeutics for these diseases, targeting novel, but essential, enzymes within the parasite provides an attractive start point for drug discovery. The finding that four of the kinetoplastid kinetochore proteins (KKTs) are kinases offers an opportunity to exploit a known, druggable enzyme family, whilst also targeting proteins unique to the parasite.

KKT19 was previously identified as an unconventional kinetochore kinase in T. brucei [5]. Kinetic characterisation of this protein revealed it was enzymatically active as a kinase, with a substrate specificity profile similar to other reported CLK kinases (consensus sequence R-X-X-S) [26, 30, 31]. In addition, TbKKT19 was inhibited by the pan-kinase inhibitor staurosporine as well as hypothemycin, a natural product known to inhibit kinases with a cysteine residue before the DXG motif [13], as seen for TbKKT19. Hypothemycin was previously shown to inhibit TbKKT10 and the IC50 generated here for TbKKT19, in presence of 80 μM ATP, as well as the ATP-competitive profile, are highly comparable to the data published for TbKKT10 (IC50 = 150 nM when assayed using 100 μM ATP [13]). This is not unexpected as both enzymes are closely related, and may well have redundant functions, it is therefore reassuring from a drug discovery perspective that dual inhibition of TbKKT10 and TbKKT19 is possible.

 

Source:

http://doi.org/10.1371/journal.pone.0217828

 

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