Towards new drugs for trypanosomatid diseases

based on specific high-affinity inhibitors for

Trypanosoma brucei RNA editing ligase 1
Stephan Zimmermann1, Victoria Feher2, Jesper Srensen2, Chris Smith3, Laurence
Hall1, Sean Riley4, Mike Greaney3, Rommie E. Amaro2, Achim Schnaufer1
1Centre of Immunity, Infection & Evolution, and Institute of Immunology & Infection Research, University of Edinburgh, UK
2Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

3School of Chemistry, University of Manchester, UK

4The Scripps Research Institute, San Diego, USA

Background LOPAC Hits

RNA editing by uridine insertion/deletion is essential for mitochondrial
gene expression in all trypanosomatids pathogens. 150
Myricetin
Editing is catalyzed by multiprotein complexes, the editosomes. Suramin
A key catalytic component of editosomes is RNA editing ligase 1 (REL1). To validate the suitability of the FRET-based REL1
REL1 has been validated as a drug target in T. brucei in vitro and in vivo 100
activity assay for HTS, the assay conditions were
adapted to 384-well format and used to carry out a
(Schnaufer et al., 2001). See also abstract #107. pilot screen against the LOPAC library (Z score =

% inhibition
0.74). Six of the seven compounds that showed the
The 1.2 crystal structure of REL1 revealed a deep ATP binding pocket strongest inhibition in the single-shot screen were
with numerous opportunities for specific binding of small molecule analysed by dose response curves to determine
IC50 values. The compounds myricetin, tyrphostin
inhibitors (Deng, Schnaufer et al., 2004). 50 Suramin IC50 = 2.94 M
AG 537, suramin, NF023 hydrate and
Methyl-3,4-dephostatin IC50 > 50 M
aurothioglucose could be confirmed, while the
There are no close REL1 homologs in the mammalian host and thus this Myricetin IC50 = 1.78 M initial hit methyl-3,4-dephostatin showed no
enzyme represents a target of potentially high specificity. Tyrphostin AG 537 IC50 = 0.25 M significant inhibition. Myricetin was also identified
as an inhibitor of T. brucei hexokinase 1 (Dodson
Computational screening efforts based on molecular dynamics 0 et al., 2011). A closely related compound,
-4 -5 -6 -7 -8 -9 quercertin, is toxic to T. b. gambiense in vitro with
simulations resulted in the identification of several REL1 inhibitors with compound [log (M)] an LD50 of 10 M without affecting human
IC50 values in the single-digit M range (Amaro, Schnaufer et al., 2008; hemopoietic cells (Mamani-Matsuda et al., 2004).
Durrant, Hall et al., 2010). These inhibitors are not drug-like, although
they share similarities with suramin.
Here we describe development of a high throughput screening (HTS)
Dundee HTS Series 7 Analogues With Increased Potency
compatible, fluorescence-based REL1 assay, identification with this Example Diverse Hits 100
assay of novel small compound REL1 inhibitors in large chemical IC50 (M)
libraries, and expansion of selected hits to increase potency.
75

% inhibition
series 2
13.6
A FRET-based RNA ligase assay 50

25
series 3
7.2

0
fluorophore-labelled
0.1 1 10 100
REL1 substrate
compound [M]

The Dundee DDU screen of 23,000 compounds resulted in the most diverse set of hits, the least
promiscuous hits and the most hits with approachable chemistry of our screens thus far. The hits
were classified into six series. Analogue purchases from commercial vendors for two series
resulted in seven compounds with IC50 < 500 nM and three compounds with IC50 < 100 nM.

NIH/TSRI Hits

Predicted binding of the TbREL1 catalytic domain to

nicked dsRNA. Modelled using TbREL1 structure
1XDN Deng, Schnaufer et al., 2004) and T4Rnl2
structure 2HVR (Nandakumar et al., 2006).

Four compounds had > 80% inhibition at 9.7 M. A PubChem search suggests these compounds are
promiscuous binders; each having activity in >100 other screens. These hits currently have low
priority for us for hit-to-lead development.

HTS Results
Donor Ex/Em
Compounds tested ~ 114,000*
Dose response curves 194#
*GSK Screen still being analyzed
IC50 < 10 M 16
IC50 < 1 M 1
Acceptor Ex/Em DDU Analogue Follow-Up Results
Medium TS Results Compounds tested 61
Compounds tested ~ 880 Dose response curves 34
Dose response curves 50 IC50 < 10 M 15
FRET IC50 < 10 M 6 IC50 < 0.5 M 7
IC50 < 1 M 1

Summary
HTS outcomes is largely dependent upon the screening collection. Four
Screening Activities
screening collections have been utilized thus far, LOPAC, Dundee Drug
High-throughput screening collaborations Discovery, NIH/TSRI Florida and GSK Tres Cantos Madrid.
- Scripps Institute San Diego (LOPAC Collection), 1,280 cpds
The DDU collection, known to be highly curated specifically for neglected disease
- Dundee Drug Discovery Unit (DDU), ~23,000 cpds
(Brent, R. et. al., 2008), has yielded the most useful hits to date.
- GlaxoSmithKline Tres Cantos Madrid (GSK), ~160,000 cpds
Analogues purchased to DDU have yielded two chemical series with a number of
Hit confirmation ongoing
hits less than 200 nM. A structured-based synthetic approach is being applied to
- The Scripps Research Institute Florida (NIH/TSRI), ~90,000 cpds increase potency for this series.
Medium-throughput in-house testing Other more general collections such as the NIH and LOPAC yielded hits, some
- GSK Published Kinase Inhibitor Set, ~360 cpds previously known such as suramin, however they are also inhibitors for many
- Analogue compounds acquired after analysis of DDU HTS by Amaro lab, ~520 cpds other proteins.

References [1] Amaro et al. (2008) PNAS 105(45):17278-83. [2] Deng et al. (2004) J Mol Biol 343:601-13. [3] Dodson et al. (2011) Exp Parasitol 127:423-8. [4] Durrant et al. (2010) PLoS Negl Trop Dis 4(8):e803. [5] Mamani-Matsuda et al. (2004)
Antimicrob Agents Chemother 48(3):924-9. [6] Nandakumar, Shuman, Lima (2006) Cell 127(1): 71-84. [7] Schnaufer et al. (2001) Science 291(5511):2159-62.