Investigating the function of CLK1 kinase in Trypanosoma brucei and identifying other essential kinases

The work presented in this thesis was motivated primarily by trying to explore the potential of protein kinases as therapeutic targets for the treatment of HAT. The first part of the thesis was aimed at identifying novel protein kinase targets in T. brucei by conducting a high-throughput RNAi screen against a subset of the T. brucei kinome. This study validated our luciferase-based assay method as a suitable assay for high-throughput RNAi screens in T. brucei. More importantly, it identified two kinases, CRK12 and ERK8, that are essential for normal proliferation by the parasite, presenting new therapeutic avenues warranting further exploration. The second part of the thesis work was aimed at investigating the function of a previously identified essential kinase, TbCLK1. This work was motivated by the fact that information relating to a potential drug target can aid in the drug discovery process by providing valuable phenotypic readouts for inhibition studies, but also by helping to identify downstream substrates that may themselves become drug targets. In this work, evidence is presented that strongly suggests TbCLK1 is essential for cis-splicing in T. brucei and that it may potentially be involved in other functions

A global comparison of the human and T. brucei degradomes gives insights about possible parasite drug targets.

We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups (M32 and C51) that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at http://babbittlab.ucsf.edu/resources.html

Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [corrected].

Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context. DOI:http://dx.doi.org/10.7554/eLife.00712.001

A Global Comparison of the Human and <em>T. brucei</em> Degradomes Gives Insights about Possible Parasite Drug Targets

<div><p>We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of <em>Homo sapiens</em> and the human parasite <em>Trypanosoma brucei</em>. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups (“M32” and “C51”) that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at <a href="http://babbittlab.ucsf.edu/resources.html">http://babbittlab.ucsf.edu/resources.html</a>.</p> </div

Structure alignment of <i>T. brucei</i> C51 model (TbC51m) with a distant structure homolog, human Cathepsin F (CatF).

<p>The superposition shows these two proteins have some general, overall structural similarities, but also large differences near the active site. The TbC51 model is colored in light orange, and the human CatF is in light green. While the catalytic Cys-His dyads are closely superimposed (depicted in ball-and-stick), a striking difference is marked by an arrow indicating the predicted steric clash between the CatF vinyl sulfone inhibitor (red) and the helix of TbC51 that partially obstructs the active site.</p

Global view of predicted active proteases of human and <i>T. brucei</i> showing sequence similarity relationships.

<p>Protease sequences are represented as nodes, and similarity relationships between sequences better than the threshold (BLAST <i>E-</i>value ≤1e-5) are depicted as “edges” or lines between nodes. In the network are represented 594 human and 127 <i>T. brucei</i> sequences (total of 721 nodes and 10,188 edges). (A) Distribution by family of proteases. Nodes for human sequences are represented as circles and for <i>T. brucei</i> sequences as triangles, and are colored by MEROPS-associated family (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#s2" target="_blank">Methods</a>). Families of some of the larger clusters are labeled, and the parasite-specific C51 and M32 clusters are circled in red. (B) Structure coverage of sequence space is broad in human and <i>T. brucei</i>. The same sequence similarity network as in panel A is shown except that it is color-coded by species and nodes are enlarged and designated by different shapes to denote if a crystal structure or model exists for that sequence. Node shapes: square = crystal structure; triangle = ModBase model; diamond = ModWeb model; small circle = no structure.</p

Structure similarity network of human and <i>T. brucei</i> proteases using crystal structures and models.

<p>Nodes represent experimentally characterized (crystal structure) or modeled structures and edges represent pairwise structural similarity above the structural similarity threshold (FAST SN score ≥4.5). Nodes for 342 human and 71 <i>T. brucei</i> are shown in the network (total of 413 nodes and 7,234 edges). The two <i>T. brucei-</i>specific families (TbM32 and C51) highlighted in the sequence similarity network shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#pntd-0001942-g001" target="_blank">Figure 1</a> are circled in red. (A) Nodes are colored by MEROPS-associated family, revealing cross-family structural relationships. Human structures are represented as circles and <i>T. brucei</i> as triangles. (B) The same structure similarity network as in panel A is painted by species and structure representation. Nodes are color-coded by species and node shape corresponds to type of structure representation for that sequence: square = crystal structure; triangle = ModBase model; diamond = ModWeb model. In contrast to <i>T. brucei,</i> there are a large number of experimentally characterized (crystal) structures for humans, but many <i>T. brucei</i> structures can be modeled.</p

Distribution by catalytic type of peptidases predicted to be active in humans and <i>T. brucei</i>.

<p>In humans, proteases of catalytic type S (where the catalytic moiety is serine) is dominant, but metallo (type M) and cysteine (type C) peptidases are also abundant. In contrast, in <i>T. brucei,</i> serine peptidases are less abundant, and cysteine and metallo proteases are equally prominent. Other main catalytic types in each organism include the threonine (type T) and aspartatic (type A) proteases. Catalytic types were assigned by catalytic type designated in the family of the closest BLAST hits to MEROPS sequences.</p

TbM32 is inhibited by 28FII (ACE2 inhibitor) and not by lisinopril (ACE inhibitor).

<p>The chart shows results from a representative experiment with 1,10P (1,10 Phenanthroline, 100 µM), lisinopril (10 µM), and 28FII (10 µM). ** indicates significant difference from the control (DMSO vehicle) at p<0.005. The positive control 1,10P is a metal chelator that inhibits metallopeptidases.</p

Structural similarity network of human and <i>T. brucei</i> proteases labeled by clan.

<p>The same network as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#pntd-0001942-g003" target="_blank">Figure 3</a> is colored here by assigned MEROPS clan (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#s2" target="_blank">Methods</a>). One cluster is composed of multiple clans (MC, MF, MH, and CF).</p