RESEARCH ARTICLE
◥MALARIA
Validation of the protein kinase
PfCLK3 as a multistage cross-species
malarial drug target
Mahmood M. Alam^1 , Ana Sanchez-Azqueta^2 , Omar Janha^2 *, Erika L. Flannery^3 ,
Amit Mahindra^4 , Kopano Mapesa^4 , Aditya B. Char^5 , Dev Sriranganadane^6 ,
Nicolas M. B. Brancucci^7 , Yevgeniya Antonova-Koch^8 , Kathryn Crouch^1 ,
Nelson Victor Simwela^1 , Scott B. Millar^1 , Jude Akinwale^9 , Deborah Mitcheson^10 ,
Lev Solyakov^9 , Kate Dudek^9 , Carolyn Jones^9 , Cleofé Zapatero^11 , Christian Doerig^12 ,
Davis C. Nwakanma^13 , Maria Jesús Vázquez^11 , Gonzalo Colmenarejo^14 ,
Maria Jose Lafuente-Monasterio^11 , Maria Luisa Leon^11 , Paulo H. C. Godoi^6 ,
Jon M. Elkins^15 , Andrew P. Waters^1 , Andrew G. Jamieson^4 , Elena Fernández Álvaro^11 ,
Lisa C. Ranford-Cartwright^5 , Matthias Marti^1 , Elizabeth A. Winzeler^8 ,
Francisco Javier Gamo^11 , Andrew B. Tobin^2 †
The requirement for next-generation antimalarials to be both curative and transmission-
blocking necessitates the identification of previously undiscovered druggable molecular
pathways. We identified a selective inhibitor of thePlasmodium falciparumprotein kinase
PfCLK3, which we used in combination with chemogenetics to validatePfCLK3 as a drug
target acting at multiple parasite life stages. Consistent with a role forPfCLK3 in RNA
splicing, inhibition resulted in the down-regulation of more than 400 essential parasite
genes. Inhibition ofPfCLK3 mediated rapid killing of asexual liver- and blood-stage
P. falciparumand blockade of gametocyte development, thereby preventing transmission,
and also showed parasiticidal activity againstP. bergheiandP. knowlesi. Hence, our data
establishPfCLK3 as a target for drugs, with the potential to offer a cure—to be
prophylactic and transmission blocking in malaria.
D
espite artemisinin-based combination ther-
apies offering effective frontline treatment
for malaria, there are still more than 200
million cases of malaria worldwide each
year, resulting in an estimated 500,000
deaths. This, combined with the fact that there is
now clear evidence for the emergence of resistance
not only to artemisinin ( 1 , 2 ) but also to partner
drugs including piperaquine and mefloquine
( 3 , 4 ), means that there is an urgent need for
novel therapeutic strategies to cure malaria while
also preventing transmission. Global phospho-
proteomic studies on the most virulent species of
human malaria,Plasmodium falciparum, have
established protein phosphorylation as a key
regulator of a wide range of essential parasite
processes ( 5 – 8 ). Furthermore, of the 65 eukaryotic
protein kinases in the parasite kinome ( 9 ), more
than half have been reported to be essential for
blood-stage survival ( 8 – 12 ). These studies, together
with the generally accepted potential of target-
ing protein kinases in the treatment of numerous
human diseases ( 13 , 14 ), suggest that inhibition
of parasite protein kinases might offer a viable
strategy for the treatment of malaria ( 6 , 15 )
To directly test this hypothesis, we focused on
oneofthefourmembersoftheP. falciparum
cyclin-dependent–like (CLK) protein kinase fam-
ily,PfCLK3 (PF3D7_1114700), a protein kinase
essential for maintaining the asexual blood stage
of bothP. falciparum( 8 , 12 )andP. berghei( 10 , 11 ).
In mammalian cells, the CLK protein kinase fam-
ily and the closely related SRPK family are crucial
mediators of multiple phosphorylation events on
splicing factors, including serine-arginine–rich
(SR) proteins, which are necessary for the correctassembly and catalytic activity of spliceosomes
[reviewed in ( 16 )]. A key member of the human
CLK family is the splicing factor kinase PRP4
kinase (PRPF4B), which homology-based studies
have identified as the closest related human kinase
toPfCLK3 ( 17 , 18 ). PRPF4B plays an essential role
in the regulation of splicing by phosphorylation
of accessory proteins associated with the spliceo-
some complex ( 19 ). The finding thatPfCLK3 can
phosphorylate SR proteins in vitro ( 20 )supports
the notion thatPfCLK3, like the other members
ofthePfCLK family ( 17 ), plays an essential role in
parasite pre-mRNA processing ( 18 ).High-throughput screen identifies
selectivePfCLK3 inhibitors
We established high-throughput inhibition assays
for two essential members of thePfCLK family,
PfCLK1 andPfCLK3 (fig. S1). Both of these pro-
tein kinases were purified as active recombinant
proteins (fig. S2A) and were used in a high-
throughput time-resolved florescence resonance
energy transfer (TR-FRET)assay,showingrobust
reproducibility in 1536-well assay format (Z′>0.7)
(fig. S2, B to H). This assay was used to screen 24,619
compounds, comprising 13,533 compounds in
the Tres Cantos Anti-Malarial Set (TCAMS) ( 21 ),
1115 in the Protein Kinase Inhibitor Set (PKIS)
( 22 ), and 9970 in the MRCT index library ( 23 ),
at a single dose (10mM). Hits were defined as
those compounds that were positioned >3 stan-
dard deviations from the mean of the percent
inhibition distribution curve (Fig. 1, A and B) and
that also showed >40% inhibition. This identi-
fied 2579 compounds (consisting of MRCT = 250,
PKIS = 4, TCAMS = 2325), which, together with
the 259 compounds identified as“thekinasein-
hibitor set”from within the Medicines for Malaria
Venture (MMV) box, a collection of 400 anti-
malarial compounds ( 24 ), were used to generate
concentration inhibition curves (Fig. 1C and
table S1). On the basis of the selectivity criterion
of a difference of more than 1.5 log units in the
negative logarithm of the half-maximal inhibition
(pIC 50 ), 28% of the hits showed specific inhibition
ofPfCLK1 and 13% specifically inhibitedPfCLK3,
whereas 23% of the compounds inhibited both
PfCLK3 andPfCLK1; the remainder (36%) were
inactive (Fig. 1, C and D, and table S1). Exemplar
molecules from each of these three classes are
shown in fig. S3.
Highlighted in Fig. 1C is TCMDC-135051, which
showed the highest selectivity and potency for
inhibition ofPfCLK3. TCMDC-135051 also showed
lower activity against the closely related human
kinase CLK2 (29% sequence identity withPfCLK3)RESEARCH
Alamet al.,Science 365 , eaau1682 (2019) 30 August 2019 1of8
(^1) Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow G12 8QQ, UK. (^2) Centre for Translational Pharmacology, Institute of Molecular Cell and Systems Biology, University
of Glasgow, Glasgow G12 8QQ, UK.^3 Novartis Institute for Biomedical Research, Emeryville, CA 94608, USA.^4 School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.^5 Institute of
Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Science, University of Glasgow, Glasgow G12 8QQ, UK.^6 Structural Genomics Consortium,
Universidade Estadual de Campinas, Campinas, São Paulo 13083-886, Brazil.^7 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, 4051 Basel,
Switzerland.^8 Skaggs School of Pharmaceutical Sciences, UC Health Sciences Center for Immunology, Infection and Inflammation, University of California, San Diego, School of Medicine, La Jolla,
CA 92093, USA.^9 Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK.^10 Department of Molecular Cell Biology, University of Leicester, Leicester LE1 9HN,
UK.^11 Diseases of the Developing World, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain.^12 Biomedical Science Cluster, School of Health and Biomedical Sciences, Royal Melbourne Institute
of Technology, Melbourne, VIC 3000, Australia.^13 MRC Unit the Gambia, Fajara, Banjul, The Gambia.^14 Biostatistics and Bioinformatics Unit, IMDEA Food Institute, 28049 Madrid, Spain.
(^15) Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, UK.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected]