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Potent Plasmodium falciparum gametocytocidal activity of lead anti-malaria chemotype, 1

diaminonaphthoquinones, identified in an anti-malaria compound screen 2

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Takeshi Q Tanaka1, W. Armand Guiguemde2, David S. Barnett2, Maxim I. Maron3, Jaeki Min2, 4

Michele C. Connelly2, Praveen Kumar Suryadevara2, R. Kiplin Guy2, Kim C. Williamson1,3# 5

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1Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious 7

Diseases, Bethesda, MD 20892, USA; 2Department of Chemical Biology and Therapeutics, St. 8

Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; 9

3Department of Biology, Loyola University, 1032 W. Sheridan Rd., Chicago, IL 60660, USA 10

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Running title: Gametocytocidal activity of anti-malaria chemotypes 12

# Corresponding author: Kim C. Williamson, Email: kwilli4@luc.edu 13

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AAC Accepts, published online ahead of print on 15 December 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.01930-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Forty percent of the world’s population is threatened by malaria, which is caused by Plasmodium 18

parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. 19

Drug-resistance has been reported for all commonly used anti-malarials and prompted screens to 20

identify new drug candidates. However, many of these new candidates have not been evaluated 21

against the parasite stage responsible for transmission, gametocytes. If P. falciparum 22

gametocytes are not eliminated patients continue to spread malaria for weeks after asexual 23

parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for 24

transmission and a safe, effective gametocytocidal agent could also be used in community wide 25

malaria control programs. Here, we identify 15 small molecules with nanomolar activity against 26

late stage gametocytes. Fourteen are diaminonaphthoquinones (DANQ) and one is a 2-imino-27

benzo[d]imidazole (IBI). One of the identified DANQs is a lead anti-malarial candidate, 28

SJ000030570. In contrast, 94% of the 650 compounds tested are inactive against late stage 29

gametocytes. Consistent with the ineffectiveness of most approved anti-malarials against 30

gametocytes, of the 19 novel compounds with activity against known anti-asexual targets, only 3 31

had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the 32

transmission stages and emphasize the importance of screening for gametocytocidal activity. The 33

potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual 34

parasites provides leads for the development of anti-malarials with the potential to prevent both 35

symptoms and the spread of malaria. 36

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Introduction 41

Effective chemotherapy remains a critical component of current malaria control strategies 42

and is essential to treat severe malaria (1). The introduction of artemisinin combination therapies 43

(ACTs) has successfully lowered malaria mortality but does not effectively control disease 44

spread because ACTs do not eliminate the sexual stages of the parasite that are required for 45

malaria transmission (2, 3). As a consequence, patients remain infectious for over a week after 46

asexual parasite clearance and the cessation of symptoms. Moreover, the identification of 47

parasite lines with delayed parasite clearance following ACT treatment have spurred the effort to 48

identify new anti-malarials (4). Several recent screens of novel small molecule libraries against 49

asexual parasites have expanded the repertoire of potential candidates for treating acute malaria, 50

but the analysis of their effects on the sexual stages is just beginning and has been focused on the 51

400 molecules included in the malaria box (5-13). Only 12 of the 260 anti-malaria compounds 52

analyzed in this study are also present in the malaria box. 53

Both gametocytes and asexual parasites develop within the erythrocyte but have distinct 54

developmental patterns that contribute to their differential sensitivity to common anti-malarials 55

(14, 15). While P. falciparum asexual stages undergo 4-5 rounds of DNA replication to produce 56

16-32 new parasites over the course of 48 hrs, gametocytes differentiate through 5 57

morphologically distinct stages (I-V) into a single male or female gametocyte over 10-12 days 58

(16). To completely block transmission, all these stages need to be eliminated during the course 59

of treatment. The lack of DNA replication during gametocyte development provides resistance 60

to drugs that target nucleic acid production, such as sulfadoxine/pyrimethamine, atovaquone, and 61

dihydroorotate dehydrogenase inhibitors (17). Additionally, stage III-V gametocytes are no 62

longer affected by compounds that block hemoglobin digestion, such as the 4-aminoquinolines 63

and cysteine protease inhibitors (17, 18) Gametocytes are also resistant to sorbitol lysis, 64

4

suggesting a reduction in permeability pathways such as the plasmodial surface anion channel 65

(PSAC) (19-21). The lack of PSAC could affect drug accessibility as shown for blasticidin and 66

leupeptin (22). Likewise, gametocytes are not cleared by antibacterial agents that target the 67

apicoplast, such as clindamycin and tetracycline analogs (23). Additional apicoplast-specific 68

enzyme systems have not yet been evaluated in gametocytes (24). In contrast, proteasome and 69

protein synthesis inhibitors are quite effective against all parasite stages (18, 25-27), including 70

late stage gametocytes, which indicates the presence of shared pathways that could be targets of 71

drugs with activities against both asexual and sexual stage parasites. 72

Here, we used the gametocyte viability assay we developed (28) and validated (29) to 73

screen a library of 260 lead-like compounds with activity against asexual parasites. The results 74

indicate that the majority of the anti-asexual compounds tested were inactive (> 80% viability 75

after treatment), including novel inhibitors of hemozoin formation and pyrimidine synthesis. 76

This finding is consistent with the limited gametocytocidal activity of commonly used anti-77

malarials and also demonstrates the specificity of the assay for late stage gametocytes. However, 78

nine percent of the compounds (23/260) did decrease gametocyte viability more that 50%, 79

suggesting the presence of targets that are important for both asexual and sexual development. 80

These 23 gametocytocidal compounds are members of five different chemotypes: the 81

diaminonaphthoquinones (DANQ), dihydropyridines (DHP), bisphenylbenzimidazoles (BPBI), 82

carbazoleaminopropanols (CAP), and iminobenzimidazoles (IBI). Two of these scaffolds, 83

DANQ and DHP, have been identified as leads against asexual parasites (30) . Follow up studies 84

screened 390 additional compounds to define structure-gametocytocidal activity profiles 85

identified 15 compounds with nanomolar EC50s. 86

87 Materials and methods 88

Chemical preparation 89

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All compounds used in these studies were purchased from vendors and used without further 90

purification. Prior to use, the identity of each compound was confirmed by UPLC/MS, and their 91

purities were confirmed to be greater than 95% by UPLC/ELSD/UV/MS. Stock solutions were 92

prepared at a nominal concentration of 10 mM in DMSO, and the concentrations were confirmed 93

by CLND prior to use. 94

P. falciparum gametocytocidal assay 95

AlamarBlue Viability Assay: The gametocyte induction and gametocytocidal assays were 96

performed using P. falciparum strain 3D7 as described (28). Briefly, parasite cultures were 97

maintained in complete RPMI (RPMI 1640, 25 mM HEPES, 25 mM NaHCO3 (pH 7.3), 100µg ml-1 98

hypoxanthine, and 5 µg ml-1 gentamycin (KD Biomedical, Columbia, MD) supplemented with 99

10% human serum (Interstate Blood Bank, Memphis, TN). Gametocyte cultures were set up at 100

0.2% parasitemia and a 6% hematocrit. On day three the hematocrit was reduced to 3% by 101

increasing the media added during the daily feed. Following N-acetyl glucosamine (NAG, 50 102

mM) treatment on days 10-12 to eliminate asexual parasites, stage III/IV/V gametocytes were 103

purified on a 65% Percoll gradient and returned to culture. The next day, the parasites were 104

resuspended at 10% gametocytemia, 0.5% hematocrit and aliquoted into a 96-well plate 105

containing the test compounds or positive (30 nM epoxomicin) and negative (DMSO) controls. 106

After incubation at 37oC for 3 days, 1/10 volume of the fluorescent viability indicator dye, 107

alamarBlue was added, and 24 hrs later the fluorescence was determined at 590/35 nm following 108

excitation at 530/25 nm. For compounds that interfere with alamarBlue reduction, wash steps 109

were added before alamarBlue addition to dilute the compound 2,500-fold. To do this, 100 μl of 110

incomplete media was added to each well at the end of the incubation period instead of 111

alamarBlue, resulting in a 2-fold dilution. After, centrifugation at 1,860 x g for 2 min, 150 μl of 112

supernatant was removed from each well and 200 μl of incomplete media was added, resulting in 113

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a 5-fold dilution. After centrifugation, 200 μl of supernatant was removed and replaced with 200 114

μl of incomplete media, resulting in another 5-fold dilution, and this procedure was repeated 115

twice more. After the last centrifugation, 200 μl of supernatant was removed and 50 μl/well of 116

complete media (10% human serum) was added, resulting in a 2-fold dilution, for a final dilution 117

of 2,500 (2x5x5x5x5x2) before the addition of 1/10 volume of alamarBlue. 118

Gametocytocidal confirmation assays: Zero, 12, 24, 48 and 72 hr after the addition of the 119

indicated compound, samples (5% gametocytemia, 2-3% hematocrit) were washed 3 times with 120

complete RPMI and analyzed using alamarBlue, Giemsa-stained smears, or MitoProbe DiIC1(5), 121

a membrane-potential-sensitive cyanine dye (Life Technologies). Samples probed with 122

alamarBlue were incubated for 24 hrs before the fluorescent signal was determined as previously 123

described. For MitoProbe DiIC1(5) staining 20 µl of the washed, compound-treated sample was 124

diluted to 200 µl with buffer containing 1.67 mg ml–1 glucose; 8 mg ml–1 NaCl; 8 mM Tris-Cl 125

(pH 8.2) and incubated with 50nM MitoProbe DiIC1(5) for 30 min prior to flow cytometry 126

(AccuriC6, BD). Uninfected RBCs incubated with MitoProbe DiIC1(5) and unstained P. 127

falciparum infected RBCs were used as controls to determine the threshold for MitoProbe 128

DiIC1(5) positive, single, intact cells (640 nm laser excitation and FL4 emission filter (675/25 129

nm). All experiments were done in triplicate. 130

Exflagellation assay: Twenty four to 48 hrs after Percoll purification, gametocytes were diluted 131

to 10% parasitemia using fresh human RBCs. Parasites and resuspended to 0.5% hematocrit with 132

complete RMPI 1640 media containing 10% human serum and the indicated compound 133

concentration or carrier alone. The cultures were gassed with 90% N2, 5% O2, 5% CO2 and 134

allowed to incubate at 37oC for 72 hrs. To measure exflagellation, a 500 µl aliquot was pelleted 135

by centrifugation (900 x g) and resuspended in 10 μl room temperature human serum with 100 136

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µM xanthurenic acid. Following a 15 minute incubation, 5 μl was applied to a hemocytometer 137

and the number of exflagellation centers counted in 50 fields using a 40x objective. 138

P. falciparum asexual growth assay: Asynchronous parasites were maintained in culture based 139

on the method of Trager (31). Parasites were grown in presence of fresh group O-positive 140

erythrocytes (Key Biologics, LLC, Memphis, TN) in Petri dishes at a hematocrit of 4-6% in 141

complete RPMI 1640 supplemented with 0.5% AlbuMAX II (Life Technologies). Cultures were 142

incubated at 37°C in a gas mixture of 90% N2, 5% O2, 5% CO2. For EC50 determinations, 20µl of 143

RPMI 1640 with 5µg ml-1 gentamycin were dispensed per well in a 384-well assay plate 144

(Corning 8807BC). An amount of 40 nl of compound, previously serial diluted in a separate 384-145

well white polypropylene plate (Corning, 8748BC), was dispensed to the assay plate by 146

hydrodynamic pin transfer (V&P Scientific Pin Head, FP1S50H) and then an amount of 20 µl of 147

a synchronized culture suspension (1% rings, 4% hematocrit) was added per well, thus making a 148

final hematocrit and parasitemia of 2% and 1%, respectively. Assay plates were incubated for 72 149

hr, and the parasitemia was determined by a method previously described (32): Briefly, an 150

amount of 10µl of the following solution in PBS (10X SYBR Green I, 0.5% v/v Triton X-100, 151

0.5 mg ml-1 saponin) was added per well. Assay plates were shaken for 1min, incubated in the 152

dark for 90min, then read with the EnVision spectrophotometer at Ex/Em of 485nm/535nm. 153

EC50s were calculated with the robust investigation of screening experiments (RISE) with four-154

parameter logistic equation. 155

Drug susceptibility assay on human cell lines 156

BJ and HepG2 cell lines were purchased from the American Type Culture Collection (ATCC, 157

Manassas, VA) and were cultured according to their recommendations. Cell culture media were 158

purchased from ATCC. Cells were routinely tested for mycoplasma contamination using the 159

MycoAlert Mycoplasma Detection Kit (Lonza). Exponentially growing cells (BJ:1000 cells/25 160

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μl/well; HepG2:400 cells/25 μl/well), were plated in Corning 384 well white custom assay plates 161

and incubated overnight at 37º C in a humidified, 5% CO2 incubator. DMSO inhibitor stock 162

solutions were added the following day to a maximum final concentration of 25 μM, 0.25% 163

DMSO and then diluted 1/3 for a total of ten testing concentrations. Cytotoxicity was 164

determined following a 72 hr incubation using Promega CellTiter Glo Reagent according to the 165

manufacturer’s recommendation. Luminescence was measured on an Envision plate reader 166

(Perkin Elmer). 167

Data analysis 168

Dose-response curves were calculated from normalized percent activity values and log10-169

transformed concentrations using the proprietary Robust Interpretation of Screening Experiments 170

(RISE) application written in Pipeline Pilot (Accelrys, v. 8.5) and the R program (33) 171

(http://www.R-project.org/). Briefly, non-linear regression was performed using the R drc 172

package with the four-parameter log-logistic function (LL2.4) (34). The median value from 173

replicates for each compound was fit three separate times by varying the parameters that were 174

fixed during regression: (1) all parameters free, (2) high response fixed to 100, (3) low response 175

fixed to 0. The best fit from these three nested models was selected using the anova.drc function. 176

Confidence intervals of 95% were produced based on this fit. Dose-response curves were 177

assigned a quality score according to the following heuristic. Compounds that failed to fit to any 178

curve, or with curves having efficacy <25% or >150% or hill slope <0.5 or >25 were designated 179

class ‘D1’. Compounds passing this first criteria with curves having efficacy <50%, calculated 180

EC50 > the highest concentration tested, lower and upper EC50 confidence limits > 10-fold EC50, 181

or slope at the highest concentration tested >75% (non-saturating) were designated class ‘C1’. 182

Compounds passing previous criteria with curves having lower and upper EC50 confidence limits 183

>5-fold EC50 or slope at the highest concentration tested >25% (not completely saturating) were 184

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designated class ‘B1’. All remaining curves were designated ‘A1’, which is indicative of ideal, 185

well-behaved sigmoidal response. In general only A-class curves were assigned potencies for 186

this manuscript. Curves that were inverted (activity decreased as concentration increased) were 187

prefixed with the letter ‘N’, such as ‘NA1’. In tabulating data, a single EC50 was reported only 188

for A1 and B1 class curves. C1 and D1 curves were assigned an arbitrary value of greater than 189

the highest concentration tested. 190

191

Results 192

Primary screening with anti-asexual compounds 193

For primary screening, 260 anti-malarial compounds were selected from 309,474 compounds in 194

the St. Jude chemical library tested against asexual P. falciparum parasites (5). These 260 195

compounds inhibited asexual growth >80% at a concentration of 2 μM in the original screen. To 196

evaluate their efficacy against late stage III-V gametocytes, they were tested at a single 197

concentration (10 μM), and 24 compounds were found to decrease viability to <55% (Fig. 1 & 198

Supplemental Table S1). Importantly, gametocytes were insensitive (>80% viability) to the 199

majority of the 260 anti-asexual compounds (200/260) demonstrating the distinct biology of late 200

stage gametocytes as well as the specificity of the assay for gametocytes (Fig. 1 and 201

Supplemental Table S1). The library included 19 compounds that have targets previously shown 202

not to affect gametocyte viability (dihydroorotate dehydrogenase, dihydrofolate reductase, 203

cytochrome bc1 complex or hemozoin formation) (5, 35) and only one of these, 204

bisphenylbenzimidazole (SJ000111341), an inhibitor of hemozoin formation, decreased 205

gametocyte viability to <22% (Supplemental Fig. S1). Twelve of the 260 compounds were 206

included in the malaria box and none of these decreased viability to <70% (Supplemental Table 207

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2S). In all, derivatives from just 3 molecular scaffolds, DANQ, IBI, and BPBI decreased 208

gametocyte viability to <22% at 10 μM. The 8 most effective compounds were DANQ 209

derivatives and will be discussed separately below. Based on the first screen, 390 additional 210

compounds were selected as structural analogs to five chemotypes (Supplemental Table S2). 211

Fifteen derivatives, at 6.25 µM, decreased viability to <55% (Supplemental Table S2). In total 212

the structure activity relationship of 479 unique compounds were analyzed, 22 (4.6%) of which 213

decreased gametocyte viability to <50% (Table 1, Supplemental Fig. S2). 214

Gametocytocidal activity and human cell cytotoxicity of DANQ derivatives 215

In the initial 260 compound screen, the 8 most potent compounds were all DANQ 216

derivatives (Supplemental Table S1). However, the fluorescent signals were lower than positive 217

control wells that contained 30 nM epoxomicin, a potent gametocytocidal agent, raising concern 218

that the compounds were affecting the alamarBlue indicator. Consequently, a series of wash 219

steps were included in the protocol and two approaches were taken to confirm the 220

gametocytocidal activity of the compounds (Fig 2). Gametocyte viability was tested immediately 221

after drug addition using the modified alamarBlue protocol that included wash steps and a new 222

flow cytometry protocol using a membrane-potential sensitive dye (MitoProbe DiIC1(5)) (Fig 2). 223

In contrast to the 24 h incubation period needed for alamarBlue, MitoProbe DiIC1(5) staining 224

only requires 30 minutes allowing more rapid screening of gametocyte viability. The results 225

indicate that gametocyte viability remains high 12 hrs after drug addition and then gradually 226

decreases until viable gametocytes are no longer detected at 72 hrs. At 24 hrs both assays 227

detected a ~20-30% reduction in viability for SJ000030570 (71±3% viability, alamarBlue & 228

80±5% viability DiIC1(5)) & SJ000024933 (70±2% viability, alamarBlue & 71±6% viability 229

DiIC1(5)), indicating that following the wash steps at early time points alamarBlue could detect 230

viable gametocytes even when using high DANQ concentrations SJ000030570 (18 µM) & 231

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SJ000024933 (23 µM). In contrast, at 72 hrs none of the gametocytes were viable in either assay 232

or Giemsa-stained smears. The time course of gametocyte elimination was similar for 233

SJ000030570, SJ000024933 and epoxomicin and this modified alamarBlue protocol was then 234

used to determine the structure-activity relationship of the DANQ scaffold using 21 DANQ 235

derivatives (Table 2, Fig 3). The asexual parasite EC50s were also determined using the SYBR 236

green assay (Table 2). Derivative SJ000030570 showed the best potency against both 237

gametocytes and asexual stage parasites (Gametocytocidal activity, EC50 = 0.061 μM) (Table 2), 238

while three additional derivatives had EC50s of 0.1 µM. The dose response curves are shown in 239

Supplemental Fig S3. 240

All 21 DANQ derivatives were also assayed for cytotoxicity against 2 mammalian cell 241

lines, BJ and HepG2 (Table 2). Four of the five most effective compounds (SJ000030570, 242

SJ000024933, SJ000022283, SJ000024948, SJ000032726) were over 55-fold more potent 243

against gametocytes than BJ or Hep2G cells with the most effective compound, SJ000030570, 244

demonstrating 180- and 80-fold selectivity, respectively. These five compounds also had 245

nanomolar activity against asexual parasites (Table 2) indicating a potential to be used to both 246

treat patients and block malaria transmission. However, there was poor correlation between the 247

anti-gametocyte and anti-asexual potency of the compounds suggesting different modes of action 248

in these two intraerythrocytic parasite stages (Table 2). 249

Structure/activity relationship DANQ derivatives 250

Analysis of the structures of gametocytocidal DANQ derivatives shows two structural variations 251

of the DANQ scaffold that display gametocytocidal activity (Fig. 3 and Table 2). The 252

compounds with the most potent activity are analogs of asexual lead compound SJ000030570, 253

which are defined by an acetyl group on the aniline nitrogen and a dimethylamine moiety (Fig. 254

3A). For this series, anti-malarial activity is retained with either electron-donating 255

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(SJ000030570) or electron-withdrawing (SJ000024933, SJ000022283, SJ000024948) 256

substitutions on the aniline ring, suggesting the electronics of this ring are not important. The 257

tertiary amide DANQ scaffold retained potency when the dimethylamine was replaced with a 258

morpholine moiety (Fig. 3B). For the morpholine series, the hydrocarbon chain length of the 259

aniline ether significantly influenced gametocytocidal activity: reduction from a five carbon 260

chain (SJ00032726) to a four carbon chain (SJ00032719) decreased activity 10-fold for the 261

morpholine analogs. Hydrocarbon chain length did not have a consistent effect on potency 262

against asexual stage parasites. A fluorine substitution in the para position on the aniline 263

(SJ000001054) was found to retain gametocytocidal and asexual activity, while replacement of 264

the aniline with an alkyl substituent negatively affected both gametocytocidal and asexual 265

activity as shown by SJ000154238. 266

A second DANQ scaffold characterized by a non-acetylated secondary amine aniline 267

nitrogen with a piperidine moiety as the second amine substituent also displayed activity against 268

both sexual and asexual stage parasites (Fig. 3C). Following the previously described series, 269

gametocytocidal compounds with long-chain alkyl ether substitutions in the para position of the 270

aniline (SJ000032721 and SJ000032714) were identified. In addition, analogs containing 271

electron-withdrawing substitutions (SJ000294509, SJ000021272 and SJ000044720) and 272

cyclohexane the substituted analog (SJ000294518) showed gametocytocidal activity. Another 273

variation on the second series of the DANQ scaffold containing benzotriazole substitutions, 274

SJ000244625 and SJ000244627, also provided compounds with gametocytocidal and asexual 275

potency (Table 2). 276

During the lead optimization of SJ000030570 it was observed that compounds containing 277

dialkylamine moieties display an inherent sensitivity to light. In the case of dimethylamine 278

analogs (Fig 3A), prolonged exposure to light results in the photolysis of one of the methyl 279

groups on the amine resulting in the formation of the monomethylamine analog SJ000541602. 280

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Furthermore, this analog is capable of cyclizing to form the corresponding imidazolium analog 281

SJ000561981. The resulting secondary amine and the corresponding imidazolium have shown to 282

be more stable to light and provide elevated levels of antimalarial activity against asexual stages 283

of malaria. To test this directly, SJ000030570 and two photostable derivatives were re-evaluated 284

taking precautions to decrease light exposure. In both the alamarBlue and MitoProbe DiIC1 assay 285

light-protected SJ0000305070 (EC50 0.319 ± 0.050 µM and 0.235 ± 0.093 µM, respectively) was 286

less active than derivatives SJ000561981 (EC50 0.093 ± 0.027 µM and 0.090 ± 0.064 µM, 287

respectively) and SJ000541602 (EC50 0.220 ± 0.034 µM and 0.240 ± 0.041 µM, respectively) 288

when tested against stage III-V gametocytes (Fig. S3). Importantly, all three compounds 289

completely inhibited exflagellation; the derivatives at concentrations >0.1 µM and light-290

protected SJ0000305070 at >0.3 µM concentrations, confirming the biological activity of these 291

compounds. 292

Validation of the activity data with light protected SJ000030570 and its photostable 293

analogs has validated the gametocytocidal activity of the DANQ series. However, the potential 294

exists for the other active compounds, which contain a dialkylamine moiety, to be partially 295

degraded at the time of analysis. Therefore, the reported activity for these compounds may be 296

overestimated by the presence of more potent degradation products. Conversely, the potency 297

may be underestimated by the degradation of the active constituent. Moving forward, further 298

analysis will be conducted on analogs from the photostable series. 299

300

IBI activity and cytotoxicity 301

A second class of compounds currently being investigated for anti-asexual activity also 302

consistently inhibited late stage gametocyte viability (Table 1 & Supplemental Fig S2). Over 303

half of the five 2-imino-benzo[d]imidazole derivatives (IBI) tested had >50% gametocytocidal 304

activity with EC50 ranging between 1 - 4 µM. The most effective compound (SJ000016864) had 305

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a similar EC50 against asexual parasites and was > 44-fold less toxic to the BJ human fibroblast 306

cell line. Additional compounds from this promising chemotype will have to be screened to 307

better define the structure activity relationship. 308

Discussion 309

Fifteen compounds with gametocytocidal activity in the nanomolar range were identified 310

in a screen of a total of 650 compounds, including 260 lead-like anti-malarial compounds 311

discovered in a whole cell screen against the asexual stages of the parasite life cycle. These 15 312

gametocytocidal compounds were derived from just 2 scaffolds: 14 were DANQ derivatives and 313

one was an IBI. The most effective compound, SJ000030570, was initially found to be >100-fold 314

more effective against gametocytes than the BJ human fibroblast cell line (EC50 >11 µM). In fact 315

all fourteen DANQ compounds had a therapeutic window >7 times when compared to BJ cells. 316

DANQ is one of the 3 scaffolds selected from the St. Jude Chemical Library for lead 317

optimization as new anti-malarials and possesses the best gametocytocidal potency of the 3 lead 318

compounds (30). 319

Each of these 3 anti-malaria leads [DANQ, DHP and dihydroisoquinoline (DHIQ)], as 320

well as IBI, are hypothesized to have novel mechanisms of action because they are structurally 321

distinct from previous anti-malarials and do not inhibit or bind to known asexual targets 322

including PfDHOD, PFDHFR, cytochrome bc1, falcipain 2 or hemozoin (5). In contrast to the 323

DANQs, the hydroxynaphthoquinone, atovaquone, inhibits cytochrome bc1 and does not reduce 324

gametocyte viability even at 10 µM (36). The dual anti-asexual and gametocytocidal activity of 325

DANQ and IBI suggest they interfere with pathways that are essential for both these 326

intraerythrocytic stages, while DHP and DHIQ target critical asexual-specific pathways. 327

However, the lack of correlation between the asexual and gametocytocidal potency of the DANQ 328

derivatives suggests their modes of action may differ in these two intraerythrocytic parasite 329

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stages. The structural differences between the DANQ derivatives could directly influence the 330

binding of the compound to a specific target or alter access of the compound to the parasite or 331

host red blood cell. RBC permeability has been shown to be enhanced in asexual-infected RBCs, 332

but not gametocyte infected RBCs (19, 20), and this difference could lead to differential uptake 333

of distinct compounds. Marked phenotypic and transcriptomic differences exist between the two 334

life cycle stages (37-41). For example, late stage female gametocytes contain a large set of 335

translationally repressed transcripts that are not expressed until the gametocyte is taken up in a 336

blood meal by a mosquito (42). A number of P. falciparum genes also have stage-specific 337

homologues, including diaminopeptidase (DPAP2) and plasmepsins (VI-IX) (39, 41). It is 338

possible that homologues expressed at different stages could have subtly different affinities for 339

compounds such as the DANQ derivative series that result in different activity profiles. The 340

presence of distinct targets in different parasite stage would also suggest that both genes would 341

have to acquire mutations for the parasite to become completely resistant to the compound. 342

The lack of gametocytocidal activity of the majority of the anti-asexual compounds tested 343

(237/260, 91%) further demonstrates the distinct sensitivities of late stage gametocytes and 344

asexual parasites and confirms the gametocyte specificity of the assay. As previously reported, 345

pathways involved in hemoglobin digestion, hemozoin formation, DNA replication, apicoplast 346

activity and increased RBC permeability were shown not to be essential for gametocyte 347

maturation (14, 17). Elucidating the mechanisms of action of these 237 novel anti-asexual 348

specific compounds will further increase the understanding of pathways required for asexual 349

growth, but not gametocyte viability. In contrast, the targets of DANQ and IBI are expected to be 350

required for the viability of both asexual and sexual stage parasites. Both stages develop within 351

the confines of an erythrocyte, and in silico profiling of proteomic data into broadly defined 352

functional classes indicate the presence of common pathways, including glutathione metabolism 353

16

and protein expression and degradation (43). Additional screening of the remaining compounds 354

from the St. Jude chemical library without asexual activity will be of interest to reveal 355

gametocyte specific compounds and their corresponding targets. 356

In summary, two scaffolds, DANQ and IBI that effectively block both asexual growth 357

and late stage gametocyte viability have been identified. One of the DANQs, SJ000030570, has 358

already been selected for anti-malaria lead optimization (30) resulting in the identification of two 359

photo stable analogs (SJ000541602 and SJ000561981) that were also found to have potent 360

gametocytocidal activity. In contrast, 625 other novel compounds were inactive against late stage 361

gametocytes (>50% viability). Differences between asexual and sexual stage parasites were also 362

observed in the structure-activity analysis of DANQ derivatives, as well as the other 6 363

chemotypes that had measurable activity against both parasite stages. Whether these structural 364

differences reflect stage-specific targets or access to the parasite remain to be determined. The 365

results clearly demonstrate the need to test both asexual and sexual stages to identify compounds 366

with the potential to inhibit the symptoms and spread of malaria. 367

Acknowledgements 368

This research was supported by the Intramural Research Program of the National Institute of 369

Allergy and Infectious Diseases, National Institutes of Health, the American Lebanese Syrian 370

Associated Charities (ALSAC), St. Jude Children’s Research Hospital (SJCRH), and Public 371

Health Service grant AI101396 from the National Institute of Allergy and Infectious Diseases. 372

TQT is a JSPS Research Fellow in Biomedical and Behavioral Research at NIH. 373

We thank Dr. S. Desai for use of the fluorescent plate reader, Dr. B. Grimberg for 374

suggesting MitoProbe DilC1(5) and Dr. C. Magle for critical reading of the manuscript. 375

376

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509

Figure Legends 510

Figure 1: Gametocyte viability. 511

Late stage gametocyte viability after incubation with the indicated compounds (10 µM) was 512

assayed using fluorescent indicator, alamarBlue as described in Methods. Gametocyte viability is 513

presented as the percent of carrier (DMSO) only control signal after subtracting the background 514

fluorescence signal remaining after treatment with 30 nM epoxomicin. The dashed green line 515

indicates 80% gametocyte viability, the red line indicates 55% gametocyte viability and the 516

dashed red line indicates 22% gametocyte viability. 517

518

Figure 2: Gametocytocidal activities of DANQ derivatives 519

20

Cultures were assayed for viability using flow cytometry (solid bars), alamarBlue fluorescence 520

(striped bars) (A) and Giemsa-stained blood smears (B) at the indicated times after the addition 521

of epoxomicin (light gray bar), SJ000024933 (blue bars), and SJ000030570 (dark gray bars). The 522

data is presented as the percent of the DMSO vehicle control value. 523

524

Figure 3: DANQ Structure-Activity analysis 525

The gametocytocidal EC50s of three series of DANQ derivatives were determined using the 526

alamarBlue viability assay to allow the comparison of chemical structure and activity. 527

528

529

21

Table 1. Gametocytocidal activities of selected chemotypes. The chemotype (group) and 530

backbone structure (backbone) are listed in addition to the number of derivatives of each 531

chemotype that were tested and reduced gametocyte viability <50% or 50-70%. 532

 

   Group Backbone Tested <50% viability 50%-70% viability

DANQ

23 12 0

DHIQ

23 0 3

DHP

27 0 8

Scaffold A 133 3 0

Scaffold B

188 2 8

CAP

82 4 0

IBI

5 3 0

533

534

22

Table 2.  535 Biological activities of 1,4-dioxo-1,4-dihydronaphlene (DANQ) derivatives.

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

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560

Continued on next page561

  R  R1  R2 Asex EC50 (µM) 

Gcyt  EC50 (µM) 

Cytotoxicity EC50 (µM) 

BJ  HepG2 

SJ000001054  

‐F  O.077  0.44  3.14  3.87 

SJ000030569  

 5.6  4.29  >26.0  22.6 

SJ000032719    

0.220  1.84  >31.2  3.89 

SJ000032726      

0.072  0.18  >36.1  22.5 

SJ000032725      

0.18  0.98  >25.3  9.76 

SJ000032718    

0.459  0.80  >28.4  13.7 

SJ000019400  

‐H  ‐Br  0.014  6.67  >29.2  >29.2 

SJ000032714  

‐H  

0.052  0.74  >20.0  >20.0 

SJ000294509  

‐H  

0.042  2.73  >37.9  >33.9 

SJ000021272  

‐H  ‐I  0.021  0.95  >25.9  21.8 

SJ000032721  

‐H  0.057  0.83  >20.0  >20.0 

SJ000022283  

‐F  0.030  0.10  3.83  0.71 

SJ000024948  

‐I  0.039  0.10  7.92  5.89 

SJ000030570     0.032  0.061  >30.0  >26.0 

SJ000244625 

 ‐H  ‐Br  0.006  0.88  >24.8  >24.8 

SJ000244627 

 ‐H 

 0.038  0.6  >24.5  >24.5 

23

Continued from previous page 562

Table 2. Biological activities of 1,4-dioxo-1,4-dihydronaphlene (DANQ) derivatives. 563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

DANQ  R  R1  R2 Asex EC50 (µM) 

Gcyt  EC50 (µM) 

Cytotoxicity EC50 (µM) 

BJ  HepG2 

SJ000044720  

‐H  0.10  1.68  >20.0  >20.0 

SJ000024933    

0.093  0.10  10.3  5.69 

SJ0000154238    

  5.4  1.29  >30.5  >30.5 

SJ000294518  

‐H  0.036  1.74  >29.9  >29.9 

SJ000294499      

4.0  2.88  >35.1  >35.1 

SJ000541602    

0.001  0.092  13.7  7.6 

SJ000561981  0.001  0.122  1.9  9.7