bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Short format paper

Activity of epigenetic inhibitors against Plasmodium falciparum asexual and

sexual blood stages.

Leen Vanheer a, Björn F.C. Kafsack a,#

a Department of Microbiology & Immunology, Weill Cornell Medicine, New York, NY,

USA

# Address correspondence to Björn F.C. Kafsack, [email protected]

Abstract: Regulation of gene expression by epigenetic processes is critical for

malaria parasite survival in multiple life stages. To evaluate the suitability of targeting

these pathways we screened 350 epigenetic inhibitors against asexual blood stages

and gametocytes of P. falciparum. We observed ≥90% inhibition at 10 µM for 28% of

compounds, of which a third retained ≥90% inhibition at 1 µM. These results suggest

epigenetic regulation as a promising target for the development of new multi-stage

anti-malarials.

1 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Despite substantial progress in reducing malaria infections and deaths over the past

two decades, the disease remains among the greatest global health challenges, with

219 million cases and 435,000 deaths in 2017 (1). The emergence of drug and

insecticide resistance now threatens to reverse these gains and highlights the need

for new classes of anti-malarials for use in combination therapies (2). To minimize the

emergence and spread of resistance, these new classes should have independent

modes of action from existing therapies and be effective against multiple parasite

stages, including the asexual blood stages responsible for the disease’s clinical

manifestation and gametocytes, the sexual blood stages that mediate transmission.

Recent studies have demonstrated the essential function of multiple genes involved

with epigenetic regulation of gene expression in asexual blood stages (3-7). Many of

these genes likely also play key roles during the substantial chromatin remodeling that

occurs during the early stages of gametocytogenesis (8, 9). Earlier studies involving a

limited number of epigenetic inhibitors found activity against malaria parasites (10-14)

and several have already been approved for clinical use or are currently in clinical

trials for treatment of various cancers (15). To evaluate the promise of targeting

epigenetic processes more broadly, we decided to screen the two largest

commercially available libraries of epigenetic inhibitors against both asexual blood

stages and gametocytes of Plasmodium falciparum, the most widespread and virulent

human malaria parasite.

Small compound libraries of 209 and 141 epigenetic inhibitors at 10 mM in DMSO

were obtained from Selleckchem (Houston, TX) and Cayman chemicals (Ann Arbor,

MI), respectively. Libraries were aliquoted and were further diluted in DMSO to 2 mM

and 0.2 mM in V-bottom 96-well plates using a Tecan Freedom EVO 150 liquid

2 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

dispenser (HTSRC, Rockefeller University) and stored at -80°C. A P. falciparum NF54

strain expressing the tandem dimeric tomato red fluorescent protein under control of

a peg4 gametocyte promoter (16) was used for cell-based activity screens against

both asexual and early gametocytes and maintained at low parasitemia using

established methods (17). The activity against asexual blood stages was determined

by 72 hour SYBR Green assays (18) as previously described and normalized to

solvent-treated controls (included in triplicate on each plate) (19). Activity against

developing gametocytes was determined using a flow cytometric assay, as previously

described (16). Briefly, highly synchronous asexual cultures were grown for one cycle

at 3% haematocrit and 8-9% parasitemia to induce sexual commitment. Percoll-

sorbitol isolated schizonts were allowed to invade fresh RBCs for 3-4 hours and

remaining late stages were then removed with a second Percoll-sorbitol gradient. The

3-4-hour early rings, both asexual and committed, were seeded into flat-bottom 96-

well plates containing compounds at a final 1% haematocrit, 4% parasitemia, 50 mM

GlcNAc (Alfa Aesar, Haverhill, MA) in 200 µL and 0.5% DMSO. Cultures were

maintained for 6 days before quantifying gametocytemia by flow cytometry (Cytek

DxP12). EC50 values were calculated the nlmLS function of the minpack.lm package

(v1.2-1) of the R statistical package (v3.6.0).

We screened 350 small molecules known to target epigenetic processes from two

commercially available libraries at 10 µM and 1 µM against asexual and early

gametocyte blood stages of P. falciparum. Seventeen compounds were present more

than once, differing only by vendor or counterion. Since responses to repeat

compounds showed only minimal variation in response, their mean response is

reported (Fig. S1), leaving 332 unique compounds.

3 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Number of >50% inhbition at 10 µM >50% inhbition at 1 µM Target Class compounds asexuals gametocytes asexuals gametocytes Histone Acetylation 10 1 (10%) 1 (10%) 0 (0%) 0 (0%) Histone Deacetylation 89 43 (48.3%) 38 (42.7%) 25 (28.1%) 26 (29.2%) Histone Methylation 52 33 (63.5%) 24 (46.2%) 11 (21.2%) 12 (23.1%) Histone Demethylation 18 9 (50%) 7 (38.9%) 1 (5.6%) 2 (11.1%) Histone Phosphorylation 67 41 (61.2%) 38 (56.7%) 13 (19.4%) 13 (19.4%) Histone PARPylation 24 5 (20.8%) 4 (16.7%) 0 (0%) 0 (0%) Histone Reader Domains 28 6 (21.4%) 3 (10.7%) 0 (0%) 0 (0%) DNA Methylation 15 3 (20%) 1 (6.7%) 1 (6.7%) 1 (6.7%) Other 29 5 (17.2%) 4 (13.8%) 3 (10.3%) 1 (3.4%) Total 332 146 (44%) 120 (36%) 54 (16%) 55 (17%) Table 1. EC50 Activity of epigenetic inhibitors tested grouped by reported epigenetic process targeted in higher eukaryotes.

Number of >90% inhbition at 10 µM >90% inhbition at 1 µM Target Class compounds asexuals gametocytes asexuals gametocytes Histone Acetylation 10 0 (0%) 0 (0%) 0 (0%) 0 (0%) Histone Deacetylation 89 34 (38.2%) 34 (38.2%) 15 (16.9%) 14 (15.7%) Histone Methylation 52 19 (36.5%) 19 (36.5%) 9 (17.3%) 8 (15.4%) Histone Demethylation 18 4 (22.2%) 6 (33.3%) 1 (5.6%) 1 (5.6%) Histone Phosphorylation 67 25 (37.3%) 26 (38.8%) 5 (7.5%) 7 (10.4%) Histone PARPylation 24 4 (16.7%) 3 (12.5%) 0 (0%) 0 (0%) Histone Reader Domains 28 1 (3.6%) 1 (3.6%) 0 (0%) 0 (0%) DNA Methylation 15 1 (6.7%) 1 (6.7%) 1 (6.7%) 1 (6.7%) Other 29 4 (13.8%) 3 (10.3%) 1 (3.4%) 1 (3.4%) Total 332 92 (28%) 93 (28%) 32 (10%) 32 (10%) Table 2. EC90 Activity of epigenetic inhibitors tested grouped by reported epigenetic process targeted in higher eukaryotes.

Of the compounds screened, 148 had greater than half-maximal activity against at

least one stage (Fig. 1 & Fig S3. Also see Fig. S2 and Data Set 1 for activity of all

compounds tested). 44% (146) and 16% (54) of compounds had greater than half-

maximal activity against asexual stages at 10 µM and 1 µM, respectively (Table 1).

Activity against early gametocyte stages was similar, with 36% (120) and 17% (55) of

compounds resulting in greater than 50% inhibition at 10 µM and 1 µM, respectively.

Greater than 90% inhibition was observed for 28% (92) against asexual blood stages

at 10 µM, with 10% (32) retaining ≥90% activity even at 1 µM (Table 2). Against early

gametocyte stages 28% (93) and 10% (32) had EC90s below 10 µM and 1 µM,

4 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

respectively. Despite differences in methodology and parasite strains, these agree

well with results for 8 of these compounds that had been screened against either

asexual stages or gametocytes in earlier studies (10-14). Thirty-one of the most active

compounds (EC90s < 1 µM) were selected for more detailed dose-response studies

(Figure 1B). The majority showed similar potency against both blood stages but we

found that twelve compounds exhibited at least 2-fold difference in activity against the

two parasite stages tested (Fig. 1C). Of the eight compounds more active against

asexual stages, seven were (HDAC) inhibitors while two histone

methyltransferase inhibitors, the DNA methyltransferase (DNMT) inhibitor SGI-1027,

and the Pan-Jumonji Histone demethylase (HDM) inhibitor JIB-04 (20) were more

effective against early gametocytes.

When grouped based on their reported epigenetic targets in higher eukaryotes,

compounds that affect the deacetylation, methylation, and phosphorylation of histone

in other organisms each had hit rates between 35-40% at 10 µM for both asexual

blood stages and gametocytes (Table 1). Genome-wide mutagenesis studies in P.

falciparum and the rodent malaria parasite P. berghei have indicated the essentially

of multiple genes encoding histone modifying enzymes (6, 7). While phosphorylation

of histone tails has been observed in P. falciparum blood stages (21), it remains

unclear whether the observed activity of these kinase inhibitors is the result of

diminished histone phosphorylation, as the kinases implicated in modification of

histone tails in higher eukaryotes also perform other critical functions (see Fig. S3 for

kinase inhibitor results).

5 asexuals gametocytes

CUDC−101 SRT1720 Coumarin−SAHA HC Toxin AR−42 Dacinostat CAY10398 CAY10603 M 344 4−iodo−SAHA Nexturastat A CUDC−907 Pyroxamide LMK−235

Ricolinostat HDAC Tubastatin A SRT3025 Thiomyristoyl 3,3−Diindolylmethane AGK7 TMP269 HPOB CBHA Quercetin Tenovin−6 PCI 34051 4SC−202 MC 1568 Oxamflatin asexuals gametocytes Citarinostat Santacruzamate A CUDC−101 MS−275 Apicidin Tucidinostat Trichostatin A Tubacin SRT1720 Garcinol Coumarin−SAHA HAT HC Toxin JIB−04 AR−42 ML324 Dacinostat IOX1 Givinostat OG−L002 Quisinostat a−Hydroxyglutarate Panobinostat Methylstat HDM Abexinostat ORY−1001 Pracinostat GSK−J4 CAY10398 SP2509 Vorinostat CAY10603 UNC0642 Belinostat BIX01294 M 344 UNC0631 4−iodo−SAHA UNC0638 Nexturastat A LLY−507 CUDC−907 UNC0379 Pyroxamide UNC0646 LMK−235 MI−463 Resminostat MI−136

Ricolinostat HDAC Chaetocin Tubastatin A 3−Deazaneplanocin A SRT3025 GSK126 Scriptaid Sinefungin Thiomyristoyl Neplanocin A 3,3−Diindolylmethane BRD4770 AGK7 OICR−9429 TMP269 MS023

Mocetinostat Pinometostat HMT bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8,HPOB 2019. The copyright holder for this preprintUNC1999 (which was not CBHA SAM certified by peer review) is the author/funder, who has granted bioRxiv a licenseQuercetin to display the preprint in perpetuity.MM It−102 is made available under aCC-BY-NC 4.0 InternationalTenovin− 6license. EPZ005687 PCI 34051 SGC0946 4SC−202 CPI−360 MC 1568 A−366 Oxamflatin GSK503 Citarinostat EPZ004777 Santacruzamate A MI−3 MS−275 MI−2 Tucidinostat UNC0224 Tubacin GSK343 asexuals gametocytes asexual gametocyte asexual gametocyteGarcinol MI−503 HAT asexual gametocyte A CUDC−101 JIB−04 SGI−1027 Apicidin ML324 DNMT Trichostatin A IOX1 5−Azacytidine SRT1720 OG−L002 DNMT Coumarin−SAHA HDM a−Hydroxyglutarate Amodiaquine HC Toxin Methylstat HDM AR−42 ORY−1001 Dacinostat GSK−J4 LW 6 Givinostat SP2509 G007−LK Quisinostat I−BRD9 Panobinostat UNC0642 GSK4112 Abexinostat BIX01294 CPI−637 Pracinostat UNC0631 other GSK6853

CAY10398 UNC0638 A−966492 Other Vorinostat LLY−507 CAY10603 UNC0379 (+)−JQ1 Belinostat UNC0646 MS436 M 344 MI−463 AZ6102 4−iodo−SAHA MI−136 Nexturastat A Chaetocin OTX015 CUDC−907 3−Deazaneplanocin A 10 1 10 1 HDAC Pyroxamide GSK126 10 1 10 1 LMK−235 Sinefungin concentration (µM) Resminostat Neplanocin A concentration (µM)

Ricolinostat HDAC BRD4770 Tubastatin A HMT OICR−9429 SRT3025 MS023 % inhibition

Scriptaid Pinometostat HMT Thiomyristoyl UNC1999 0 25 50 75 100 3,3−Diindolylmethane SAM AGK7 MM−102 TMP269 EPZ005687 0 25 50 75 100 Santacruzamate A SGC0946 Mocetinostat CPI−360 HPOB A−366 CBHA GSK503 Quercetin EPZ004777 % inhibition Tenovin−6 MI−3 PCI 34051 MI−2 4SC−202 UNC0224 MC 1568 GSK343 Oxamflatin Etoposide Citarinostat MI−503 Tucidinostat 10 1 10 1 SGI−1027 Tubacin Gemcitabine concentration (µM) 5−Azacytidine Garcinol DNMT HAT Amodiaquine HAT 10 1 10 1 JIB−04 Mitomycin C ML324 Pirarubicin concentration (µM) IOX1 LW 6 OG−L002 B G007−LKasexuals gametocytes a−Hydroxyglutarate I−BRD9 Methylstat GSK4112 CUDC−101 HDM ORY−1001 CPI−637 Apicidin C GSK−J4 GSK6853 Trichostatin A SP2509 A−966492 Other SRT1720 Target Class Rucaparib Coumarin−SAHA 500 ● UNC0642 (+)−JQ1 HDAC HC Toxin BIX01294 HDAC MS436 ● HDM UNC0631 AZ6102 AR−42 CAY10398 Dacinostat UNC0638● HDM Niraparib ● HMT LLY−507 OTX015 Givinostat Quisinostat 400 MI-136UNC0379 HDAC ● UNC0646 HMT10 1 10 1 DNMT ● Panobinostat MI−463 concentration (µM) Abexinostat MI−136 DNMT Pracinostat Chaetocin CAY10398 3−Deazaneplanocin A Vorinostat 300 GSK126 Coumarin-SAHA Sinefungin % inhibition CAY10603 0 25 50 75 100 Belinostat ● Neplanocin A BRD4770 M 344 OICR−9429 4−iodo−SAHA 200 MS023 AR-42 JIB−04 ● Pinometostat HMT HDM UNC1999 CUDC-101 SAM JIB-04 UNC0642 ● ● BIX01294 AbexinostatMM−102

Gametocyte EC50 (nM) ● ● EPZ005687 UNC0631 100 SGC0946 UNC0638 Apicidin ● CPI−360 LLY−507 UNC0646A−366 HMT UNC0379 ● GSK503 UNC0646 ● UNC0379 EPZ004777 HC Toxin MI−463 0 SGI-1027 MI−3 MI−2 MI−136 UNC0224 Chaetocin GSK343 0 100 Etoposide200 300 400 500 DNMT SGI−1027 MI−503 1000 360 120 40 13 1000 360 120 40 13 AsexualSGI−1027 EC50 (nM) concentration (nM) Gemcitabine 5−Azacytidine Figure 1. Epigenetic Inhibitors withDNMT activity against P. falciparum blood stages. Amodiaquine Mitomycin C (A) Compounds with ≥50%Pirarubicin inhibition against asexual or early gametocyte blood LW 6 stages at 10 µM. HeatmapG007−LK of mean percent inhibition of asexual replication and early I−BRD9 GSK4112 gametocyte maturationCPI at−637 10 and 1 µM compared to solvent-treated controls (n=2, see GSK6853

Table S1 for completeA−966492 data). CompoundsOther are grouped based on the reported Rucaparib (+)−JQ1 epigenetic process affectedMS436 in higher eukaryotes: Histone deacetylation (HDAC), AZ6102 histone acetylation (HAT),Niraparib histone methylation (HMT), Histone Demethylases (HDM), OTX015 DNA methylation10 1 10(DNMT), 1 and “other”. Grey color indicates values excluded due to significant concentration hemolysis (µM) at 10 µM. (B) Additional analysis of dose response for 31 compounds with sub-micromolar EC90s (n=2-3). (C) Of these, twelve compounds had % inhibition a greater than 20 -fold25 50 difference75 100 (indicated by dotted lines) in activity against between asexual blood stages and gametocytes.

6 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Hit rates were lower for compounds targeting processes involved in demethylation,

acetylation, binding of histone modifications (histone readers), and DNA methylation.

P. falciparum encodes one or more of genes involved in these pathways and lower hit

rates against these may indicate greater divergence from their mammalian homologs

or non-essentially of these pathways in blood stages. For example, all but three of the

29 inhibitors of histone readers interfere with the recognition of acetylated histones by

Bromo-domains. Earlier studies noted the divergence of these domains in P.

falciparum while also demonstrating their essentiality for asexual growth. Interestingly,

we found that DNA methyltransferase inhibitor SGI-1027 had EC50 values in the low

nanomolar range against both stages, despite the fact that the lone DNA

methyltransferase in malaria parasites was found to be dispensable for asexual growth

in P. falciparum (6). When combined with the fact that SGI-1027 contains a quinoline

group common in many anti-malarials, this makes an alternative target likely. Overall,

our results show that epigenetic regulation of gene expression in malaria parasites is

a promising target for interfering with multiple stages of the parasite life cycle.

Acknowledgments: We thank the High Throughput and Spectroscopy Resource

Center at Rockefeller University for technical assistance, Prof. Photini Sinnis (Johns

Hopkins University) for generously providing the NF54 peg4-tdTomato reporter

parasites, and Prof. Elisabeth Martinez (UT Southwestern) for additional JIB-04

inhibitor. We also thank L. Kirkman for valuable feedback on the manuscript.

This work was supported by a Bohmfalk Charitable Trust Research Grant and NIH

R01AI141965 to BK, and a Belgian American Educational Foundation post-doctoral

fellowship to LV.

7 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

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Figure S1 asexuals gametocytes Selleck_S1053 Entinostat Cayman_13284

Selleck_S7294 PFI−2 Cayman_14678

Selleck_S4125 Phenylbutyrate Cayman_11323

Selleck_S7189 I−BET762 Cayman_10676

Selleck_S4246 2−PCPA Cayman_10010494

Selleck_S5001 Tofacitinib Selleck_S2789

Selleck_S7079 SGC0946 Cayman_13967

Selleck_S2012 100 PCI 34051 Cayman_10444 75 Selleck_S7062 Pinometostat Cayman_16175 50 Selleck_S7595 Santacruzamate A 25 Cayman_15403 % inhibition

Selleck_S2627 0 Tubastatin A Cayman_15785 Cayman_10559

Cayman_13828 3−Deazaneplanocin A Cayman_11102

Selleck_S2018 ENMD−2076 Selleck_S1181

Selleck_S1047 Vorinostat Cayman_10009929

Selleck_S1515 Pracinostat Cayman_10443

Selleck_S2170 Givinostat Cayman_11045

Selleck_S1095 Dacinostat Cayman_16427 10 1 10 1 concentration (µM) Figure S1. Similar activity of compounds that differed by counterion or vendor. % inhibition 0 25 50 75 % inhibition asexuals asexuals gametocytes gametocytes % inhibition at 10 µM % inhibition at 1 µM % inhibition at 10 µM % inhibition at 1 µM 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 UNC669 ● ● ● ● AZD2461 ● ● ● ● KD 5170 ● ● ● ● Selisistat ● ● ● ● WDR5−0103 ● ● ● ● DMOG ● ● ● ● EX−527 ● ● ● ● NU1025 ● ● ● ● BG45 ● ● ● ● ● ● ● ● Daprodustat ● ● ● ● Phenylbutyrate ● ● ● ● CPI−0610 ● ● ● ● PFI−3 ● ● ● ● GSK−J2 ● ● ● ● Splitomicin ● ● ● ● 2−hexyl−4−Pentynoic Acid ● ● ● ● Salermide ● ● ● ● 2−PCPA ● ● ● ● Tranylcypromine ● ● ● ● Suramin ● ● ● ● Filgotinib ● ● ● ● SRT2104 ● ● ● ● ME0328 ● ● ● ● Procainamide ● ● ● ● A−769662 ● ● ● ● Fasudil ● ● ● ● RG−108 ● ● ● ● AMI−1 ● ● ● ● Entacapone ● ● ● ● Isoliquiritigenin ● ● ● ● Iniparib ● ● ● ● trans−Resveratrol ● ● ● ● GSK2879552 ● ● ● ● PNU−74654 ● ● ● ● Butyrate ● ● ● ● Butyrolactone 3 ● ● ● ● AMG−900 ● ● ● ● SIRT1/2 Inhibitor IV ● ● ● ● Daptomycin ● ● ● ● Divalproex ● ● ● ● GSK−J1 ● ● ● ● CPI−455 ● ● ● ● 2−Methoxyestradiol ● ● ● ● MG149 ● ● ● ● ● ● ● ● ● ● ● ● IOX2 ● ● ● ● TMP195 ● ● ● ● ● ● ● ● 1−Naphthoic Acid ● ● ● ● Tasquinimod ● ● ● ● Mirin ● ● ● ● UNC3866 ● ● ● ● Resveratrol ● ● ● ● MLN8054 ● ● ● ● Droxinostat ● ● ● ● SRT2183 ● ● ● ● Salvianolic acid B ● ● ● ● MK−8617 ● ● ● ● N−Oxalylglycine ● ● ● ● ZM 39923 ● ● ● ● AG−490 ● ● ● ● F−Amidine ● ● ● ● HPI−4 ● ● ● ● Epigallocatechin Gallate ● ● ● ● 2,4−Pyridinedicarboxylate ● ● ● ● ● ● ● ● INO−1001 ● ● ● ● Piceatannol ● ● ● ● Tofacitinib ● ● ● ● UPF 1069 ● ● ● ● ● ● ● ● NMS−P118 ● ● ● ● Daminozide ● ● ● ● Octyl−alpha−ketoglutarate ● ● ● ● I−BET762 ● ● ● ● 2,3,5−triacetyl−5−Azacytidine ● ● ● ● Alisertib ● ● ● ● CAY10669 ● ● ● ● HLCL−61 ● ● ● ● SAH ● ● ● ● Daphnetin ● ● ● ● Ruxolitinib ● ● ● ● WP1066 ● ● ● ● (+)−Abscisic Acid ● ● ● ● RGFP966 ● ● ● ● Ellagic Acid ● ● ● ● I−BET151 ● ● ● ● Metformin ● ● ● ● Veliparib ● ● ● ● Lomeguatrib ● ● ● ● PJ34 HCl ● ● ● ● 3−amino Benzamide ● ● ● ● Nullscript ● ● ● ● SGC707 ● ● ● ● GSK2801 ● ● ● ● AG−14361 ● ● ● ● Sirtinol ● ● ● ● BSI−201 ● ● ● ● Roxadustat ● ● ● ● Enzastaurin ● ● ● ● Delphinidin ● ● ● ● MI−nc ● ● ● ● Valproic Acid ● ● ● ● Sotrastaurin ● ● ● ● CPI−1205 ● ● ● ● OF−1 ● ● ● ● GSK−LSD1 ● ● ● ● SirReal2 ● ● ● ● (−)− ● ● ● ● AZD5153 ● ● ● ● Thioguanine ● ● ● ● CCG−100602 ● ● ● ● GSK591 ● ● ● ● KC7F2 ● ● ● ● PFI−4 ● ● ● ● Tenovin−1 ● ● ● ● AGK2 ● ● ● ● AK−7 ● ● ● ● ITSA−1 ● ● ● ● PFI−2 ● ● ● ● ● ● ● ● MK−5108 ● ● ● ● Zebularine ● ● ● ● FG−2216 ● ● ● ● HNHA ● ● ● ● PFI−1 ● ● ● ● MK−8745 ● ● ● ● Fisetin ● ● ● ● BI−7273 ● ● ● ● EPZ020411 ● ● ● ● Anacardic Acid ● ● ● ● JGB1741 ● ● ● ● CPI−169 ● ● ● ● Phenformin ● ● ● ● MC 1568 ● ● ● ● E7449 ● ● ● ● Cl−Amidine ● ● ● ● SGC−CBP30 ● ● ● ● SGC2085 ● ● ● ● UNC1215 ● ● ● ● CPTH2 ● ● ● ● BGP−15 2HCl ● ● ● ● RSC−133 ● ● ● ● RG2833 ● ● ● ● A−196 ● ● ● ● CAY10591 ● ● ● ● PX−478 ● ● ● ● Phthalazinone pyrazole ● ● ● ● BML−210 ● ● ● ● (R)−PFI−2 ● ● ● ● CI−994 ● ● ● ● BRD73954 ● ● ● ● I−BET726 ● ● ● ● C646 ● ● ● ● AZD1208 ● ● ● ● ETC−1002 ● ● ● ● MS049 ● ● ● ● EED226 ● ● ● ● XL019 ● ● ● ● S−Ruxolitinib ● ● ● ● EPZ015666 ● ● ● ● Entinostat ● ● ● ● Pimelic Diphenylamide 106 ● ● ● ● I−CBP112 ● ● ● ● Mivebresib ● ● ● ● RVX−208 ● ● ● ● Lificiguat ● ● ● ● Go6976 ● ● ● ● UNC0321 ● ● ● ● ● ● ● ● compound Curcumol 5−Methylcytidine ● ● ● ● NVP−TNKS656 ● ● ● ● AICAR ● ● ● ● Remodelin ● ● ● ● 5−Methyl−2−deoxycytidine ● ● ● ● Momelotinib ● ● ● ● EI1 ● ● ● ● Bromosporine ● ● ● ● CPI−203 ● ● ● ● Santacruzamate A ● ● ● ● Picolinamide ● ● ● ● (−)−JQ1 ● ● ● ● 6−Thioguanine ● ● ● ● Tacedinaline ● ● ● ● Clevudine ● ● ● ● PF−CBP1 ● ● ● ● MS436 ● ● ● ● Tucidinostat ● ● ● ● CPI−637 ● ● ● ● MS−275 ● ● ● ● MI−2 ● ● ● ● ORY−1001 ● ● ● ● GSK4112 ● ● ● ● Quercetin ● ● ● ● Oclacitinib ● ● ● ● OTX015 ● ● ● ● 5−Azacytidine ● ● ● ● 4SC−202 ● ● ● ● 3−Deazaneplanocin A ● ● ● ● KW−2449 ● ● ● ● CYC116 ● ● ● ● a−Hydroxyglutarate ● ● ● ● GSK6853 ● ● ● ● OICR−9429 ● ● ● ● MS023 ● ● ● ● AGK7 ● ● ● ● SAM ● ● ● ● IOX1 ● ● ● ● Tubacin ● ● ● ● JNJ−7706621 ● ● ● ● Baricitinib ● ● ● ● (+)−JQ1 ● ● ● ● CPI−360 ● ● ● ● AT9283 ● ● ● ● G007−LK ● ● ● ● PCI 34051 ● ● ● ● EPZ005687 ● ● ● ● GSK343 ● ● ● ● AZD1480 ● ● ● ● GSK503 ● ● ● ● Decernotinib ● ● ● ● TMP269 ● ● ● ● MI−3 ● ● ● ● Garcinol ● ● ● ● MI−503 ● ● ● ● Etoposide ● ● ● ● Neplanocin A ● ● ● ● Methylstat ● ● ● ● Cerdulatinib ● ● ● ● PHA−680632 ● ● ● ● CEP−33779 ● ● ● ● BMS−911543 ● ● ● ● MM−102 ● ● ● ● HPOB ● ● ● ● 3,3−Diindolylmethane ● ● ● ● Peficitinib ● ● ● ● bioRxiv preprintTozasertib doi: https://doi.org/10.1101/694422; this version posted July 8, 2019.● The copyright holder● for this preprint (which was not ● ● certified byGemcitabine peer review) is the author/funder, who has granted bioRxiv a license● to display the preprint in perpetuity. It is made● available ● ● Aurora A Inhibitor I under aCC-BY-NC 4.0 International● license. ● ● ● OG−L002 ● ● ● ● Lestaurtinib ● ● ● ● Chaetocin ● ● ● AZ 960 ● ● ● ● A−366 ● ● ● ● LW 6 ● ● ● ● Barasertib ● ● ● ● SP2509 ● ● ● ● ZM 447439 ● ● ● ● SNS−314 ● ● ● ● Sinefungin ● ● ● ● Rucaparib ● ● ● ● Danusertib ● ● ● ● SMI−4a ● ● ● ● WHI−P154 ● ● ● ● UNC0224 ● ● ● ● Reversine ● ● ● ● TAK−901 ● ● ● ● GSK1070916 ● ● ● ● Gandotinib ● ● ● ● Tubastatin A ● ● ● ● Mocetinostat ● ● ● ● Hesperadin ● ● ● ● Niraparib ● ● ● ● TG101209 ● ● ● ● HC Toxin ● ● ● ● FLLL32 ● ● ● ● Vorinostat ● ● ● ● Tenovin−6 ● ● ● ● HTH−01−015 ● ● ● ● Apicidin ● ● ● ● Coumarin−SAHA ● ● ● ● Givinostat ● ● ● ● SGC0946 ● ● ● ● CUDC−907 ● ● ● ● SRT1720 ● ● ● ● CUDC−101 ● ● ● ● 4−iodo−SAHA ● ● ● ● Bisindolylmaleimide IX ● ● ● ● MI−136 ● ● ● ● Abexinostat ● ● ● ● Scriptaid ● ● ● ● Go 6983 ● ● ● ● GSK−J4 ● ● ● ● Resminostat ● ● ● ● Pacritinib ● ● ● ● ENMD−2076 ● ● ● ● Quisinostat ● ● ● ● Pracinostat ● ● ● ● SRT3025 ● ● ● ● UNC0638 ● ● ● ● I−BRD9 ● ● ● ● Pinometostat ● ● ● ● EPZ004777 ● ● ● ● BI−847325 ● ● ● ● Panobinostat ● ● ● ● MI−463 ● ● ● ● UNC0646 ● ● ● ● A−966492 ● ● ● ● CAY10603 ● ● ● ● UNC1999 ● ● ● ● BRD4770 ● ● ● ● Pyroxamide ● ● ● ● Oxamflatin ● ● ● ● Ricolinostat ● ● ● ● Mitomycin C ● ● ● ● CAY10398 ● ● ● ● AR−42 ● ● ● ● UNC0631 ● ● ● ● Fedratinib ● ● ● ● Nexturastat A ● ● ● ● Belinostat ● ● ● ● M 344 ● ● ● ● Dacinostat ● ● ● ● SGI−1776 ● ● ● ● Citarinostat ● ● ● ● Amodiaquine ● ● ● ● Thiomyristoyl ● ● ● ● Trichostatin A ● ● ● ● NVP−BSK805 ● ● ● ● WZ4003 ● ● ● ● UNC0379 ● ● ● ● GSK126 ● ● ● ● LLY−507 ● ● ● ● AZ6102 ● ● ● ● CX−6258 ● ● ● ● UNC0642 ● ● ● ● BIX01294 ● ● ● ● Pirarubicin ● ● ● ● LMK−235 ● ● ● ● SGI−1027 ● ● ● CBHA ● ● ● ● JIB−04 ● ● ● ● ML324 ● ● ● ● 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 % inhibition asexualsgametocytes Garcinol HAT HAT CUDC−101 Apicidin Trichostatin A SRT1720 Coumarin−SAHA HC Toxin AR−42 Dacinostat Givinostat Quisinostat Panobinostat Abexinostat Pracinostat CAY10398 Vorinostat CAY10603 Belinostat M 344 4−iodo−SAHA Nexturastat A CUDC−907 Pyroxamide LMK−235 Resminostat

HDAC Ricolinostat Tubastatin A SRT3025 Scriptaid Thiomyristoyl 3,3−Diindolylmethane AGK7 TMP269 Mocetinostat HPOB CBHA Quercetin Tenovin−6 bioRxiv preprint doi: PCIhttps://doi.org/10.1101/694422 34051 ; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review)4SC− 202is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available MC 1568 under aCC-BY-NC 4.0 International license. Oxamflatin Citarinostat Santacruzamate A MS−275 Tucidinostat Tubacin

JIB−04 ML324 IOX1 OG−L002 a−Hydroxyglutarate Methylstat HDM ORY−1001 GSK−J4 SP2509 asexuals gametocytes

UNC0642 DNMT SGI−1027 BIX01294 UNC0631 CUDC−101 UNC0638 Apicidin Figure S2. Mean PercentLLY −Inhibition507 of all 337 compounds at 10 µM and 1 µM againstTrichostatin asexual A Amodiaquine SRT1720 blood stages and gametocytes.UNC0379 The dotted and dashed lines indicate 50% and 90%Coumarin inhibition,−SAHA UNC0646 HC Toxin respectively. CompoundsMI− 463are ordered by increasing activity against asexual blood stagesAR−42 at 10 MI−136 Dacinostat µM. Error bars are standardChaetocin error of n=2. Givinostat 3−Deazaneplanocin AHDAC Quisinostat GSK126 Panobinostat Sinefungin Abexinostat Neplanocin A Pracinostat BRD4770 CAY10398 OICR−9429 Vorinostat MS023 CAY10603 HMT Pinometostat Belinostat UNC1999 M 344 SAM 4−iodo−SAHA MM−102 EPZ005687 HDM JIB−04 SGC0946 CPI−360 UNC0642 A−366 BIX01294 GSK503 UNC0631 EPZ004777 UNC0638 Figure S3 MI−3 LLY−507 MI−2 HMT UNC0379 UNC0224 UNC0646 GSK343 MI−463 Etoposide MI−136 A asexual gametocyteMI−503 B asexual gametocyte Chaetocin SGI−1776 SGI−1776 CX−6258 CX−6258 GSK1070916 GSK1070916 BI−847325 BI−847325 ZM 447439 Kinase ZM 447439 Fedratinib Fedratinib TG101209 TG101209 Lestaurtinib Lestaurtinib Aurora A Inhibitor I Barasertib Barasertib ENMD−2076 1000 360 120 40 13 1000 360 120 40 13 Hesperadin AZD1480 concentration (nM) Pacritinib NVP−BSK805 AZ 960 TAK−901 AT9283 % inhibition SNS−314 Go 6983 0 25 50 75 100 WZ4003 100 HTH−01−015 Bisindolylmaleimide IX Kinase CYC116 75 Cerdulatinib PHA−680632 50 Peficitinib CEP−33779 Tozasertib 25 Oclacitinib % inhibition Danusertib Gandotinib JNJ−7706621 0 SMI−4a Decernotinib Go6976 KW−2449 BMS−911543 Baricitinib WHI−P154 Reversine FLLL32 10 1 10 1 SGI−1027 concentration (µM) Mitomycin C Pirarubicin Gemcitabine LW 6 G007−LK I−BRD9 GSK4112 Figure S3. Activity of putativeCPI−637 kinase inhibitors. GSK6853

(A) CompoundsOther with ≥50%A−966492 inhibition against asexual or early gametocyte blood stages at 10µM. 5−Azacytidine Heatmap of mean percentRucaparib inhibition of asexual replication and early gametocyte maturation is (+)−JQ1 shown at 10 and 1 µM comparedMS436 to solvent-treated controls (n=2, see Table S1 for complete AZ6102 data). (B) Additional analysisNiraparib of dose response for kinase inhibitors with EC90 values below 1 OTX015 µM (n=210 µM-3). 1 µM 10 µM 1 µM

% inhibition 0 25 50 75 100 bioRxiv preprint doi: https://doi.org/10.1101/694422; this version posted July 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Data Set 1. Mean percent inhibition for all 350 compounds at 10 µM and 1 µM

against asexual blood stages and gametocytes, with their observed EC50 range or

value.

11