Supplementary Figures and Tables - media.nature.com · 1.5 Kb 2.0 Kb 1.0 Kb M wt sampleA sampleB pfcdpk1 pfcdpk4 hdhfr b Supplementary Figure 1. Disruption of pfcdpk1 and pfcdpk4.

SUPPLEMENTARY INFORMATION for

Gene expression signatures and small molecule compounds link a protein kinase to

Plasmodium falciparum motility Nobutaka Kato1,3, Tomoyo Sakata3, Ghislain Breton1, Karine G. Le Roch3, Advait Nagle3, Carsten Andersen3, Badry Bursulaya3, Adele Godfrey-Certner4, Kerstin Henson1,3, Jeffrey Johnson1, Kota Arun Kumar5, Felix Marr1, Daniel Mason3, Case McNamara3, David Plouffe3, Vandana Ramachandran1, Muriel Spooner3, Tove Tuntland3, Yingyao Zhou3, Eric Peters3, Arnab Chatterjee3, Peter G. Schultz1, 3, Gary E. Ward4, Nathanael Gray3, Jeffrey Harper2, Elizabeth A. Winzeler1, 3 1Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, ICND202 La Jolla, CA 92037, USA. 2Biochemistry Department, University of Nevada, Reno, NV 89557, USA. 3Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA. 4 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA. 5Michael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

Corresponding Author: Elizabeth A. Winzeler The Scripps Research Institute 10550 North Torrey Pines Road. ICND 202 La Jolla, CA 92037 Tel. 858-784-9468 Fax. 858-784-9860 Email: [email protected]

Running title PfCDPK1 as an anti-malaria drug target

mailto:[email protected]

a

0.5 Kb

1.5 Kb

2.0 Kb

1.0 Kb

M wt sampleA sampleB

pfcdpk1

pfcdpk4

hdhfr

b

Supplementary Figure 1. Disruption of pfcdpk1 and pfcdpk4. (a) Cassette for the disruption of pfcdpk1. Tk = thymidine kinase. Dhfr = human dihydrofolate reductase. (b) PCR using primers in region flanking the ORFs was performed to independently monitor gene replacement at the pfcdpk1 and cdpk4 loci. Samples A and B represent genomic DNA extracted from parasites transfected with pfcdpk1 or pfcdpk4 disruption vectors, respectively. The analysis demonstrated that the pfcdpk1 ORF is still present in sample A but the pfcdpk4 ORF is replaced with hdhfr in sample B.

0

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0 50 100 150 200 250 300 350

well #

C P

M

staurosporine (500 nM)

DMSO

Supplementary Figure 2. The scintillation proximity assay delivered reproducible performance. PfCDPK1 reaction containing 500 nM of staurosporin (positive control) was compared with the same reaction containing DMSO (negative control) using the scintillation proximity assay in 384-well plate format. The separation between positive and negative controls resulted in a Z’ factor value of 0.69.

a. M G C S Q S S N V K D F K T R R S K F T N G N N Y G K S G N N K N S E D L A I N P G M Y V R K K E GK I G E S Y F K V R K L G S G A Y G E V L L C R E K H G H G E K A I K V I K K S Q F D K M K Y S I TN K I E C D D K I H E E I Y N E I S L L K S L D H P N I I K L F D V F E D K K Y F Y L V T E F Y E GG E L F E Q I I N R H K F D E C D A A N I M K Q I L S G I C Y L H K H N I V H R D I K P E N I L L EN K H S L L N I K I V D F G L S S F F S K D N K L R D R L G T A Y Y I A P E V L R K K Y N E K C D VW S C G V I L Y I L L C G Y P P F G G Q N D Q D I I K K V E K G K Y Y F D F N D W K N I S E E A K EL I K L M L T Y D Y N K R I T A K E A L N S K W I K K Y A N N I N K S D Q K T L C G A L S N M R K FE G S Q K L A Q A A I L F I G S K L T T L E E R K E L T D I F K K L D K N G D G Q L D K K E L I E GY N I L R S F K N E L G E L K N V E E E V D N I L K E V D F D K N G Y I E Y S E F I S V C M D K Q IL F S E E R L R D A F N L F D T D K S G K I T K E E L A N L F G L T S I S E Q M W N E V L G E A D KN K D N M I D F D E F V N M M H K I C D N K S S

b.

354.2?

397.1?

467.3?

468.1?

485.2?

581.4?582.4?

600.1?

a6+2H

y10-NH3+2H

b12-NH3-NH3+2H

y6+1y9+1

y3b3 b4y4 b5

y5

b6y6b7 y7

b8y8b9 b10

y10b11

L A Q A A I L F I G S KK S G I F L I A A Q A L

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 200 400 600 800 1000 1200

1232.24 AMU, +2 H (Parent Error: 1200 ppm)

parent-NH3

y1 b2 y2 b3 y3y4

b5y5

b6y6

b7 y7

Q I L F S E E RR E E S F L I Q

m/z

Rel

ativ

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tens

ity

0%

25%

50%

75%

100%

0 200 400 600 800 1000


355.1?

592.5?

y10-NH3-H2O+2H

y6+1b2

y2

b3y3

b4

y4

b5

y5

b6

y6 y7

b8

y8

b9

E L I E G Y N I L RR L I N Y G E I L E

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 200 400 600 800 1000 1200

1218.56 AMU, +2 H (Parent Error: -79 ppm)

b11+2H

b13-H2O-H2O+2Hy7+1 y8+1

parent-H2O

b3 b4

y2

b5

y3

b6

y4

b7

y5

b8

y6b9

y7

b10

y8

b11y9 y10 b12y11 y12

L G S G A Y G E V L L C+57 RR C+57 L L V E G Y A G S G L

m/z

Rel

ativ

e In

tens

ity

0%

25%

50%

75%

100%

0 200 400 600 800 1000 1200


c9+2Hy6-NH3

a5+1

a5+2

y6+1parent-NH3

b2y2

y3b3 y4b4

y5y6 y7

b7y8b8

b9y9 y10b10

F D E C+57 D A A N I M KK M I N A A D C+57 E D F

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Rel

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ity

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b5-H2O y5+1

b6-H2Ob7-H2O

b8-H2O

b3y2 b4 y3b5y4

y5

b6

y6

b7

y7

b8

y8 y9

b11y10

b12

L G T A Y Y I A P E V L RR L V E P A I Y Y A T G L

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100%

0 250 500 750 1000 1250


(K)QILFSEER(L)(K)ELIEGYNILR(S)

(K)FDEcDAANIMK(Q)(R)LGTAYYIAPEVLR(K)

(K)LAQAAILFIGSK(L)(K)LGSGAYGEVLLcR(E)

Supplementary Figure 3. Target confirmation using LC/MS/MS. (a) Sequence coverage of six unique tryptic peptides from PfCDPK1 identified by LC/MS/MS. (b) Tandem mass spectra of the six peptides identified by LC/MS/MS. The database searching conditions employed are specified in the Supplementary Methods.

Supplementary Figure 4. Classification of genes changing after treating mixed blood stage cultures of P. falciparum treated with purfalcamine. For this analysis P. falciparum genes were divided into 15 previously defined expression clusters that are roughly the result of differences in the time of expression of different genes in the parasite intraerythrocytic cycle. We then analyzed the distribution of genes increasing or decreasing after purfalcamine (1) treatment after 24 and 48 hours for each cluster. Only clusters that show statistically significant changes, which would not be obtained through permutation testing, are shown (Table 2). Red indicates up-regulated genes, and green, down-regulated while each black bar shows one of the genes in the cluster. Two independent cultures were analyzed. Of particular significance are changes that are observed after 48 hours where there is a decrease in the abundance of most merozoite (30 hours) and ring stage (36 hours) transcripts with a concomitant upregulation of late trophozoite stage transcripts.

a.

0

2.5

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7.5

10

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mia

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no drug

drug (450nM) added @ 12h



b.

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0h 6h 12h 18h 24h 32h 36h 40h 48h

schizonttrophring

Supplementary Figure 5. Effect of purfalcamine is independent from the time the compound is added. Purfalcamine (1) was added to synchronized culture at different time points (12, 24, and 36 hours), but the compound exhibited its potency only at the late schizont stage. (a) shows the change in parasitimia and (b) shows the change in stage composition.

a.

y7-98parent-196

parent+1-98

y2b3

b4y3

b5 y4 b6y5

b7

y6

b8

y7

y8b9

y9b10 y10

L A P S+80 S+80 I D E KE D I S+80 S+80 P A L G L

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Rel

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(K)LGLAPssIDEK(K)

b10-NH3-H2O

parent-NH3-196

parent-196

parent+2-196

parent-NH3-98

parent-98

parent+1-98

b3 y4b4 y5 b5y6

b6

y8b7 y9

y10

b9y11

y12b11y13 y14b12 b13y15

b14b15

b16y17 b17

F N E K S+80 S+80 G G K I S I D N AY S A N D I S I K G G S+80 S+80 K E

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500 750 1000 1250 1500 1750 2000


(R)IYFNEKssGGKISIDNASYNAR(K)

b. ▼▼

P. falciparum ----MKQECNVCYFNLPDPESTLGPYDNELNYFTWGPGFEYEPEPQRKPLSIEES----FENSEESEESVADIQQLEEKVDESDVRIYFNEKSSGGKISI P. knowlesi ----MEKQCPVCYYNLPDPESAIAPYDTELNYFMWGPGFEWQPEPEVKNILVEEEN---YEESEQSEESVAGLEELDEKVNRNEAKDMFNTKSSNGKLRI P. vivax MAAKMEKQCPVCYFNLPDPESAIAPYDTELNYFMWGPGFEWQPEPEVKNILVEEEN---FEESEQSEESVAGLEELDEKVDRSEAKTMFNEKSSNGKVSV P. berghei ----MEQQCHMCYFELPDAKTTIGPYDNELNYFMWGPGFEWKPEPVVKQISGEDT----YDETEESEESEHGFDELDEKVNKDDRKIYFDEKSSSGKISI P. yoelii ----MEQQCHACYFELPDPKTTIGPYDSELNYFMWGPGFEWKPEPVVSQISGEDT----YDETEESEESEHGFDELDEKVNKDDRKRYFDEKSSGGKISI T. gondii -MSKVEKKCPVCYQKLPNPADVLGPMDKELNYFMWMPGFEWRPEPKVGEYDGACESPS-CREGGRPAADEDMQEALEEMVEADEMYARFNARASGGKVST C. hominis MISLLNGTGCVACTSGCKVVVDDYKTTGRIEYGVFVGYYSESEESDYDESDVVTLDKKPSNIQDSSSAEVVSIEDELEEGEADIIKEMFDNASSGGECSL ▼▼ P. falciparum DNASYNARKLGLAPSSIDEKKIKELYGDNLTYEQYLEYLSICVHDKDNVEELIKMFAHFDNNCTGYLTKSQMKNILTTWGDALTDQEAIDALNAFSSEDN P. knowlesi EDASHNARKLGLAPSSTDEKKIRDLYGDSLTYEQYLEYLTMCVHDRDNMEELIKMFSHFDNNSSGFLTKNQMKNILTTWGDALTEQEANDALNAFSSEDR P. vivax EDASQNARKLGLAPSSLDEKKVRDLYGDSLTYEQYLEYLAMCVHDRDNMEELIKMFSHFDNNASGFLTKNQMRNILTTWGDALTEQEANDALNAFSSEDR P. berghei ENASYNARRLGLAPSSKDEEKIRDLYGDNLTYDQYLEYLSMTIHDKDNAEQLVKMFAYFDTNTTGFLTKNQMKNILVTWGDALTEDEAMNALNAFSNDDK P. yoelii ENASHNARRLGLAPSSKDEEKIRELYGDNLTYDQYLEYLSMSIHDKDNAEQLVKMFAYFDTNTTGYLTKNQMKNILITWGDALTEDEAMNALNAFSNDDK T. gondii GDAMILARQLGLAPSYADKQAFEEKSGDNLDYASFQKFVGTSTHPEDNIEDLVEAFAYFDVSKHGYLTRKQMGNILMTYGEPLTTEEFNALAAEYFTSDQ C. hominis DQTCHLAHRMGLAPSKSDLEKLNEETGGKVTYEEFERWIMSITHPEDHIDYMVSYFRKYDLRGNGKISRQQFIWLTSIGGDILTREEAEAILDKLSIGGD P. falciparum IDYKLFCEDILQ---------------------------------------------------------------------------------------- P. knowlesi INYKLFCEDILS---------------------------------------------------------------------------------------- P. vivax IDYKLFCEDILQ---------------------------------------------------------------------------------------- P. berghei IDYKLFCEDILQ---------------------------------------------------------------------------------------- P. yoelii IDYKLFCEDILQ---------------------------------------------------------------------------------------- T. gondii IDYRQFCKAMLERRE------------------------------------------------------------------------------------- C. hominis VYYEDLLRKIMDVEASDKPMVNIGNKKSTPTKMPTTKVLVTQVSTTIRDLADNEAFLPTIDLLMALKATYGNELTEAAAKEATDCCENQDEAAKCLQEFY P. falciparum -------------------------------------------------------------- P. knowlesi -------------------------------------------------------------- P. vivax -------------------------------------------------------------- P. berghei -------------------------------------------------------------- P. yoelii -------------------------------------------------------------- T. gondii -------------------------------------------------------------- C. hominis SEWAQNKGMVSRKIVRGLLMVWKAKLDQVSAEAWITSLCGTDENIDIQSVLNKVECKTNEIN

c.

Ser87Ser86

Ser109

Ser108Ser87Ser86

Ser109

Ser108

Supplementary Figure 6. PfCDPK1 phosphorylates PfMTIP in vitro. (a) Mass spectrometric identification of phosphorylation sites. Mass spectrometric analysis revealed that at least four serine residues (Ser 86, 87, 108 and 109) of PfMTIP can be phosphorylated by PfCDPK1 in vitro. No phosphorylation was observed in PfMTIP incubated without ATP. (b) Amino acid sequence alignment of MTIP orthologues. Some of the phosphorylated serine residues are well conserved not only in Plasmodium but also in Apicomplexa. (c) 3D-mapping of serine phosphorylation sites to the corresponding residues of PkMTIP (pdb code, 2AUC). The phosphorylated Ser 86, 87, 108 and 109 are shown in red.

a.

-11 -10 -9 -8 -7 -6 -50.0

0.5

1.0

-11 -10 -9 -8 -7 -6 -50.0

0.5

1.0

-11 -10 -9 -8 -7 -6 -50.0

0.5

1.0 3D7Dd2FCBHB3W2

3D7Dd2FCBHB3W2

log [Inhibitor], M

Para

site

gro

wth

178

GB3

179169259230EC50 (nM)

W2FCBDd23D7strain

178

GB3

179169259230EC50 (nM)

W2FCBDd23D7strain

b.

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0 2 4 6day after infection

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purfalcamine(compound 1)

Supplementary Figure 7. Purfalcamine as an anti-malaria drug. (a) Inhibitory effect of purfalcamine (1) on five different P. falciparum strains. EC50 values of five different strains of P. falciparum (3D7, Dd2, FCB, HB3, W2) treated with purfalcamine (1) were determined using the SYBR Green assay. Purfalcamine (1) inhibited proliferation of the five strains in nanomolar range, suggesting that the compound is equally effective against drug resistant parasites. (b) in vivo activity of purfalcamine (1) against rodent parasite, P. yoelii. The mice treated with purfalcamine (1) showed a delay in onset of parasitemia when compared with control mice.

a.

PfCDPK1 50 GKIGESYF LG G GEV EK KA K KK NE K LD PN KL F FYLKVRK- S AY LLCR HGHGE I VI SQFDKMKYSITNKIECDDKIHEEIY ISLL S H II FDV EDKKY VTEFY

PfCDPK2 66 GKLEDK IIDEK- Q TY YKGID VTNQLY I EE ---------------DRLKNINRFF IEI K H I YET ENDNY IMELC Y LG G GCV K A K KK QE MK LD PN VKL Y IYL

b. CDPK1 CDPK2 CDPK3 CDPK4 CDPK5

identical residues 29.2% 30.9% 30.7% 30.5% 30.8%

conserved residues 34.7% 36.6% 40.5% 36.8% 38.0%

total homology 63.9% 67.6% 71.2% 67.3% 68.8%

Supplementary Figure 8. Amino acid sequence analysis of the ATP binding pocket residues in PfCDPKs. (a) An amino acid sequence alignment of the P. falciparum family of CDPK1-5 was performed to analyze the amino acid homology between their kinase domains, with a particular emphasis on residues predicted to form the ATP binding pocket (denoted by triangles). Amino acid positions with 100% identity are highlighted in yellow whereas residues that show >50% identity between CDPKs are highlighted in blue. Conserved amino acid changes are highlighted in green. The ATP pocket residues were predicted and labeled with color-coded triangles to distinguish between their location (green, buried region; blue, adenine region; pink, sugar region; purple, phosphate region; red, solvent accessible region) and contribution (solid triangle, side chain interaction; open, main chain interaction). A quartet of amino acids unique to PfCDPK1 (denoted by a green asterisk) comprise a majority of the buried region residues of the ATP binding pocket, and are expected to generate a pocket that has a distinctly different volume and shape complementarity from the other CDPKs. An additional amino acid difference in the adenine-binding region of the PfCDPK1 ATP-binding pocket (denoted by a blue asterisk), is expected to contribute to the widening of the pocket, which would accommodate 2, 6, 9-trisubstituted purines with bulky R1 groups. (b) The percentage of identical and conserved residues from the alignment of the CDPKs in part A are given in table format.

PfCDPK3 111 GNLEDF NLSKEP K TY YKATD LLKIS VV ---------------KKLKNIPRFR IDI N H V LET EDSNQ VMELC Y LG G GCV K RAVK SK QE MK LD PN VKL F IYLPfCDPK4 64 VVFNEQ GIKI- K SF ILSRD HTGHEY I VI ----------KHVKRKTDK--ESLL VEL M I YEF EDNNY VSDVY YK LG G GEV K A K SK RE LK LDHIN MKL F YYLPfCDPK5 118 DELSDV EIDRYK K SY VKAVS RTGQQR I II ---------------KKIHNIERLK ILI Q H I YEV EDNEK VLELC

Y LG G GNV K A K EK RE MK MD PN IKL Y LYL

PfCDPK1 149 E G INRHK CD A M L GI L K IV I LENKHS-LLNI IV LSSFFSKD-NKLRDR Y E RK YNE G ELFEQI FDE A NI KQI S CY H HN HRDIKPEN L K DFG LGTAY IAP VL KPfCDPK2 150 S DS IENG KN A M F AI L S IV L F FQSENK-DSLL I LSKNLGTG-EFTTTK PQ DG YDK GRELF I SFTE A TI KQI S FY H LN HRD KPEN L KI DFG AGTPYYVA VL KPfCDPK3 196 T G DK VKKG TF SF M F VL L I I F FYDMTP-ESLI I LASYFTHNNYEMKTK PQ TG YNY G ELF I CFVE A I KQI S NY H RN CHRDIKPEN L KI DFG AGTPYYVA VL SPfCDPK4 151 T G DE ISRKR ID A L GI K VV L I LETKNKEDMII I LSTHFEYS-KKMKDK Y D HG YDE G ELF I FYE A RIIKQI S TYMH NN HRD KPEN L KI DFG IGTAY IAP VL TPfCDPK5 203 D G DK VKYG YE YK M F AL S I L I YVDNTE-DSPI I FASKCMNN-HNLKSV Y E RG YDK

G ELF I SFSE A I KQI S YYCH KN MHRD KPEN L QI DWG VGTPY IAP IL K

PfCDPK1 247 K C I I C Y QNDQDIIKK EK KY DFNDWKN E KL TYDYN ITAKE NS WIK CDVWS GV LY LL G PPFGG V G YF IS EAKELI ML KR AL KPfCDPK2 248 K S IM T C Y DTDNEVLK KK EF YENDWGS S N TK TYNPN CTIEE NH WIT CDIWS GV Y LL G PPFYG KV G CF IS DAK LI LL ER AL PPfCDPK3 295 K S LF I C Y ESDHEILS KK KY KGKEWNN E D KR TMDAD ICASE QH WFK CDMWS GV Y LL G PPFFG MV G QF IS EAK LI CL KR AL PPfCDPK4 250 I C I I S C SNEYDILKK EA KY DLPQFKK D D KK MYTSA ISARD EH WIK KCD WS GV LY LL G PPFNG V G TF IS KAK LI ML VR AL EPf CD WS GV MY LL G PPFNG V G VF VS DAK LI CL ER VL R

CDPK5 301 R I S I I C Y KNNDEILKK EK EF DSNYWAR D D CQ NYNYK IDVEQ KH WFK

***

*

*

PfCDPK1 50 GKIGES KVRK- S AY LLCR HGHGE I VI SQFDKMKYSITNKIECDDKIHEEIY ISLL S H II FDV EDKKY VTEFY YF LG G GEV EK KA K KK NE K LD PN KL F FYLPfCDPK2 66 GKLEDK IIDEK- Q TY YKGID VTNQLY I EE ---------------DRLKNINRFF IEI K H I YET ENDNY IMELC Y LG G GCV K A K KK QE MK LD PN VKL Y IYLPfCDPK3 111 GNLEDF NLSKEP K TY YKATD LLKIS VV ---------------KKLKNIPRFR IDI N H V LET EDSNQ VMELC Y LG G GCV K RAVK SK QE MK LD PN VKL F IYLPfCDPK4 64 VVFNEQ GIKI- K SF ILSRD HTGHEY I VI ----------KHVKRKTDK--ESLL VEL M I YEF EDNNY VSDVY YK LG G GEV K A K SK RE LK LDHIN MKL F YYLPfCDPK5 118 DELSDV EIDRYK K SY VKAVS RTGQQR I II ---------------KKIHNIERLK ILI Q H I YEV EDNEK VLELC

Y LG G GNV K A K EK RE MK MD PN IKL Y LYL

PfCDPK1 149 E G INRHK CD A M L GI L K IV I LENKHS-LLNI IV LSSFFSKD-NKLRDR Y E RK YNE G ELFEQI FDE A NI KQI S CY H HN HRDIKPEN L K DFG LGTAY IAP VL KPfCDPK2 150 S DS IENG KN A M F AI L S IV L F FQSENK-DSLL I LSKNLGTG-EFTTTK PQ DG YDK GRELF I SFTE A TI KQI S FY H LN HRD KPEN L KI DFG AGTPYYVA VL KPfCDPK3 196 T G DK VKKG TF SF M F VL L I I F FYDMTP-ESLI I LASYFTHNNYEMKTK PQ TG YNY G ELF I CFVE A I KQI S NY H RN CHRDIKPEN L KI DFG AGTPYYVA VL SPfCDPK4 151 T G DE ISRKR ID A L GI K VV L I LETKNKEDMII I LSTHFEYS-KKMKDK Y D HG YDE G ELF I FYE A RIIKQI S TYMH NN HRD KPEN L KI DFG IGTAY IAP VL TPfCDPK5 203 D G DK VKYG YE YK M F AL S I L I YVDNTE-DSPI I FASKCMNN-HNLKSV Y E RG YDK

G ELF I SFSE A I KQI S YYCH KN MHRD KPEN L QI DWG VGTPY IAP IL K

***

*

*

PfCDPK1 247 K C I I C Y QNDQDIIKK EK KY DFNDWKN E KL TYDYN ITAKE NS WIK CDVWS GV LY LL G PPFGG V G YF IS EAKELI ML KR AL KPfCDPK2 248 K S IM T C Y DTDNEVLK KK EF YENDWGS S N TK TYNPN CTIEE NH WIT CDIWS GV Y LL G PPFYG KV G CF IS DAK LI LL ER AL PPfCDPK3 295 K S LF I C Y ESDHEILS KK KY KGKEWNN E D KR TMDAD ICASE QH WFK CDMWS GV Y LL G PPFFG MV G QF IS EAK LI CL KR AL PPfCDPK4 250 I C I I S C SNEYDILKK EA KY DLPQFKK D D KK MYTSA ISARD EH WIK KCD WS GV LY LL G PPFNG V G TF IS KAK LI ML VR AL EPf CD WS GV MY LL G PPFNG V G VF VS DAK LI CL ER VL R

CDPK5 301 R I S I I C Y KNNDEILKK EK EF DSNYWAR D D CQ NYNYK IDVEQ KH WFK

# of tryptic peptides

P. falciparum protein names accession numbers positive competition

hsp60 PF10_0153 21 29 Heat shock protein PFI0875w 21 22 heat shock 70 kDa protein PF08_0054 17 11 polyadenylate-binding protein, putative PFL1170w 17 3 chaperonin cpn60 PFL1545c 16 11 heat shock protein hsp70 homologue PF11_0351 12 16 hypothetical protein PF13_0116 10 0 pyruvate kinase, putative PF10_0363 9 8 hypothetical protein, conserved PF14_0359 9 5 FAD-dependent glycerol-3-phosphate dehydrogenase, putative

PFC0275w 8 0

Heat Shock 70 KDa Protein, (HSP70) MAL7P1.228 7 6 structure specific recognition protein, putative PF14_0393 7 2 hypothetical protein, conserved PFF0785w 7 0 rhoptry-associated protein 1, RAP1 PF14_0102 6 12 hypothetical protein PF11_0037 6 0 calcium-dependent protein kinase 1, (PfCDPK1)

PFB0815w 6 0

merozoite capping protein 1 PF10_0268 5 0 nucleosome assembly protein 1, putative PFL0185c 5 0 hypothetical protein PF14_0186 4 5 V-type H(+)-translocating pyrophosphatase, putative

PF14_0541 4 0

hypothetical protein PFC0720w 4 0 dihydroorotate dehydrogenase, mitochondrial precursor

PFF0160c 4 0

leucine aminopeptidase, putative PF14_0439 4 0 hypothetical protein PF14_0096 3 0

elongation factor 1 alpha PF13_0304 PF13_0305

3 0

replication factor A-related protein, putative PFI0235w 3 0 mitochondrial processing peptidase alpha subunit, putative

PFE1155c 3 0

plasmepsin, putative PF14_0075 3 0 tryptophan/threonine-rich antigen PF08_0003 3 0 hypothetical protein PF11_0191 3 0 peptidase, putative PF14_0517 2 9 alpha tubulin PFI0180w 2 0 conserved protein, putative PF13_0238 2 0 hypothetical protein PFI1680w 2 0 Supplementary Table 1. List of P. falciparum proteins identified by mass spectrometry. Purfalcamine immobilized to agarose resin was incubated with parasite lysate (Positive) and proteins retained after washing were subjected to SDS-PAGE. Entire lanes were excised, subjected to tryptic digestion, and analyzed by LC/MS/MS. Separately, free purfalcamine (2.5 mM) was added to parasite lysate, incubated with the purfalcamine immobilized to agarose resin, and processed identically (Competition). Only proteins

identified by more than three unique tryptic peptides are listed. The database searching conditions employed are specified in Supplementary Methods.

Experiment 100µM 50µM 25µM 12.5µM 6.25µM 3.1µM 1.56µM

1 I (S) I (S) I (S) x I (W/M) E (W) no 2 I (S) I (S) I (M) x no no no 3 I (S) I (S) I (S) 4 I (S) I (S) I (S) 5 I (S) I (S) I (S) I (W) no x E (W) 6 I (S) I (S) I (S) I (M) I (W) I (W/M) I (W) 7 I (S) I (S) I (S) I (M) I (M) I (M) I (M) 8 I (S) I (S) I (S) I (M) I (M) I (S) no 9 I (S) I (S) I (S) I (M) no no no 10 I (S) I (S) I (S) I (M) I (M) I (W) no 11 I (S) I (S) I (S) I (W) no I (W) no 12 I (S) I (S) I (S/M) I (W/M) no I (M) no 13 I (S) I (S) I (S/M) I (M) no I (M) no 14 I (S) I (S/M) I (S) I (S/M) I (W) I (S) no 15 I (S) I (S) I (S) I (S) I (W) no I (W)

average I (S) I (S) I (S) I (M) I (W/M) I (W) no Supplementary Table 2. Effect of purfalcamine on the invasion of BS-C-1 cells by T. gondii. T. gondii tachyzoites were incubated with host cells in the presence of various concentrations of purfalcamine (1) as described in Methods, and scored (relative to DMSO) on a scale of 0 (no effect); I[W] (weak inhibition, ~60-80% invasion compared to control); I[M] (medium inhibition, ~20-60% invasion compared to control); I[S] (strong inhibition; 0-20% invasion compared to control); and E[W] (weak enhancer of invasion). Each experiment was run in duplicate and scored blind. The experiment was repeated seven times; the average result at each concentration of purfalcamine (1) is shown in the bottom row.

SUPPLEMENTARY METHODS for

Gene expression signatures and small molecule compounds link a protein kinase to

Plasmodium falciparum motility Nobutaka Kato1,3, Tomoyo Sakata3, Ghislain Breton1, Karine G. Le Roch3, Advait Nagle3, Carsten Andersen3, Badry Bursulaya3, Adele Godfrey-Certner4, Kerstin Henson1,3, Jeffrey Johnson1, Kota Arun Kumar5, Felix Marr1, Daniel Mason3, Case McNamara3, David Plouffe3, Vandana Ramachandran1, Muriel Spooner3, Tove Tuntland3, Yingyao Zhou3, Eric Peters3, Arnab Chatterjee3, Peter G. Schultz1, 3, Gary E. Ward4, Nathanael Gray3, Jeffrey Harper2, Elizabeth A. Winzeler1, 3

1Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, ICND202 La Jolla, CA 92037, USA. 2Biochemistry Department, University of Nevada, Reno, NV 89557, USA. 3Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA. 4 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA. 5Michael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, New York, NY 10016, USA.

Corresponding Author: Elizabeth A. Winzeler The Scripps Research Institute 10550 North Torrey Pines Road. ICND 202 La Jolla, CA 92037 Tel. 858-784-9468 Fax. 858-784-9860 Email: [email protected]

Running title PfCDPK1 as an anti-malaria drug target

mailto:[email protected]

Supplementary Methods

Disruption of pfCDPK1 and pfCDPK4. The pfcdpk1 and pfcdpk4 disruption vectors

were constructed by amplifying the 5’ (~500 bp) and 3’ (~1,500 bp) non-coding regions

outside of the pfcdpk1 and pfcdpk4 open reading frames by PCR using the following

primers and cloning them into insertion sites of the pHHT-TK vector, respectively.

pfcdpk1 5’ TCCccgcggGAACGTGTAACATGAAATGGGGTG and

GGAggatccGGTTTAATATCTCGATGTACAATATTATG

pfcdpk1 3’ CCCcctaggGCATTCAATTTGTTTGATACTGATAAAAG and

CCCggcgccGTGTATGTGTATTATTTATATGTTTTATTTTTTTTATTTATC

pfcdpk4 5’ TCCccgcggCATTCGTTCATATGTTTTATATTCTATC and

GGAggatccCATATAATTTCATTATATTAATATGATC

pfcdpk4 3’ CCCcctaggGAGTGGATAAAAATGATGACAAGCAAAG and

CCCggcgccGGAAGGATTTAAATTAATATATATGTACAC

SacII, BglII, AvrII and SfoI sites are indicated in small letters.

For transfection, 100 µg of plasmid DNA was used to preload erythrocytes with DNA1.

Parasites were then allowed to invade transfected erythrocytes. The sample was washed

with complete media and transferred to a culturing flask. Blood was added so that the

final hematocrit was 5% and final parasitemia was 3%. WR99210 (2) was added starting

2 days after the transfection for a final concentration of 10 nM. WR99210 (2) was added

for 2 weeks until the parasitemia reached 5%. Ganciclover (3) was added at a final

concentration of 4 µM for 9 days. No drugs were added for 2 weeks. Once parasites

were detected, WR99210 (2) was added again. Gene disruption was verified by

microarray analysis2 and PCR using the following primers.

pfcdpk1 5’ GTAACCGAATTTTATGAAGGTGGGGAATTATTTG and

pfcdpk1 3’ CTCCTCACTGAAAAGGATTTGCTTATCCATAC

pfcdpk4 5’ GTTTGAAAGGGGGTAATGAGAGACATGAAATGAAAG and

pfcdpk4 3’ CAAGATTTGATCAACTTCATATTCGATAGCAGC

hdhfr 5’ CGCTAAACTGCATCGTCGCTGTGTCCCAGAAC and

hdhfr 3’ CGTGTCACTTTCAAAGTCTTGCATGATCCTTG

Expression and purification of recombinant PfCDPK1. The entire coding region of

pfcdpk1 (PFB0815w) was inserted into the expression vector (pET41-b, Novagen,

Madison, WI). Sequence analysis was performed to confirm that the fusion protein was

in frame with the N-terminus glutathione-S-transferase (GST) tag and C-terminus 6X His

tag. Escherichia coli strain (Rosetta 2 pLyse, Novagen) was transformed with the

expression vector and grown to A600 = 0.5. Expression of the recombinant protein was

induced by the addition of IPTG to a final concentration of 0.5 mM and further incubated

for 3 hours at 37oC.

The cultured cells were harvested and resuspended in lysis buffer (20 mM Tris

HCl pH 8.0, 10 mM imidazole, 500 mM NaCl, 10% glycerol, 0.4% Triton X-100, 1 mM

PMSF) and lysed by sonication on ice. The lysate was centrifuged at 14,000 x g for 20

minutes at 4oC. The recombinant protein in the crude supernatant was first purified on a

Ni-NTA agarose column (Novagen) and further purified by GST affinity column (GE

Healthcare, Piscataway, NJ). Glycerol was added to the purified recombinant protein to a

final concentration of 10% and stored at -80oC until used.

Compound library. The approximately 20,000 small molecules used for screening were

a part of kinase-directed heterocyclic combinatorial libraries3. The library consisted of

diverse planar heterocycles (including purines, pyrimidines, quinazolines, pyrazines,

phthalazines, pyridazines, and quinoxalines) bearing hydrogen bond acceptor as well as

hydrogen bond donor moieties which are the hallmarks of an ATP competitive kinase

inhibitor. The general synthetic strategy contains aromatic substitution of

dichloroheterocyclic scaffolds on solid support with amines, or anilines, boronic acids,

and phenols, which therefore generate discrete and highly diverse heterocyclic small

molecules3.

Compound synthesis and characterization.

Abbreviations.

EtOH (ethanol), EtOAc (Ethylacetate), DCM (CH2Cl2),

DHP (3,4-dihydro-2H-pyran), DIEA (diisopropylethylamine),

DMSO (dimethylsulfoxide), DME (dimethoxyethane),

DMF (dimethylformamide), Fmoc-OSu (Fmoc N-hydroxysuccinimide ester),

MeOH (methanol), MS (molecular sieves),

Pd2(dba)3 (tris(dibenzylidenacetone)dipalladium (0)), TEA (triethylamine),

TFA (trifluoroacetic acid), THF (tetrahydrofurane),

p-TSA (p-toluenesulfonic acid monohydrate), r.t. (room temperature)

General synthetic methods.

Unless otherwise noted, materials were obtained from commercial suppliers and were

used without purification. Removal of solvent under reduced pressure refers to

distillation using Büchi rotary evaporator attached to a vacuum pump (~3 mmHg).

Products obtained as solids or high boiling oils were dried under vacuum (~1 mmHg).

Purification of compounds by reverse phase HPLC was achieved using a Waters 2487

series with SunFire Prep C18 OBD column (10 μm, 19 x 50 mm) with 10-90% CH3CN

with 0.035% TFA in H2O with 0.1% TFA. For large scale purification, Waters XTerra

Prep MS C18 OBD column (10 μm, 50 x 150 mm) was used. Flash column

chromatography was performed by CombiFlash (Separation system Sg. 100c, ISCO)

using eluant gradient solvent systems as described. 1H NMR spectra were recorded on

Bruker XWIN-NMR (400 MHz). Proton resonances are reported in parts per million

(ppm) down field from tetramethylsilane (TMS). For spectra obtained in CDCl3, DMSO-

d6, CD3OD, the residual protons (7.27, 2.50 and 3.31 ppm respectively) were used as the

reference. 1H NMR data are reported as chemical shifts (δ) in ppm, multiplicity (s:

singlet; d: doublet; t: triplet; q: quartet; dd: doublet of doublets; ddd: doublet of dd; m:

multiplet; bs: broad singlet), number of protons and coupling constant (J) in hertz (Hz).

13C NMR data are reported as chemical shifts (δ) in ppm and coupling constant (J) in

hertz (Hz) for C-F coupling. Analytical HPLC was conducted on an Agilent 1100 series

liquid chromatograph/mass selective detector to monitor the progress of reactions and

check the purity of products using 254 nm and 220 nm wavelengths, and electrospray

ionization (ESI) positive mode. Mass spectra were obtained in ESI positive mode.

Synthesis of purine compounds mentioned in this article.

N

N NH

N

Cl

F

N

N N

N

Cl

FTHP

(26) (27)

DHP, p-TSA

DCM

6-chloro-2-fluoro-9-(tetrahydro-pyran-2-yl)-9H-purine (27):

A mixture of 6-chloro-2-fluoro-9H-purine (26) (10.0 g, 58 mmol), DHP (7.4 ml,

87 mmol), p-TSA (1.1 g, 5.8 mmol) and DCM (80 ml) was stirred overnight at r.t. After

filtration, it was washed with Sat. Na2CO3 and brine and dried with MgSO4. Evaporation

of the solvent followed by titration with EtOAc and hexanes induced precipitate.

Filterarion yielded white crystal 27 (8.9 g, 60%). 1H NMR (400 MHz, CD3OD): δ 8.70

(s, 1H), 5.75 (dd, J = 2.3, 10.7 Hz, 1H), 4.13 (dd, J = 2.7, 10.6 Hz, 1H), 3.80 (ddd, J = 2.6,

11.6, 11.6 Hz, 1H), 2.24 (m, 1H), 2.09 (m, 2H), 1.89-1.70 (m, 2H), 1.67 (m, 1H); 13C

NMR (400 MHz, CD3OD): δ 159.7, 157.5 154.7, 154.5, 154.4, 153.1, 152.9, 146.6, 146.9,

131.3, 84.1, 69.8, 31.8, 26.0, 23.7; MS (m/z): 173.0 [M-THP+2H]+.

H2N

N

O

O2N

Cl

O

H2Pd/C

EtOH(28) (30)

HN

(29)

DCM

(4-amino-phenyl)-piperidin-1-yl-methanone (30):

To a DCM solution of piperidin (29) (11.6 ml, 117 mmol) was added 4-nitro-

benzoyl chloride (28) (10.9 g, 58.7 mmol) at 0°C. After stirring for 30 minutes at r.t., the

reaction mixture was washed with 1% HCl, and the combined aqueous layer was then

basified and extracted with DCM. The organic layer was dried with MgSO4 and

concentrated in vacuo to yield yellow oil. This product was dissolved in MeOH (100 ml)

and stirred with Pd/C under 1 atm H2 overnight. The reaction mixture was filtered over

celite and concentrated in vacuo to yield 30 as a yellow oil, which was used for the next

reaction without further purification: MS m/z: 205.1 [M+H]+.

N

N N

N

Cl

FTHP

(27) (32)

N

N NH

N

HN

HN

N

O

NH2

H2N

N

O

DIEA, EtOH80ºC

NH2

NH2

TFA

MeOH

(30) (31)

DIEA, EtOH95ºC

2-(4-amino-cyclohexylamino)-6-[4-(piperidine-1-carbonyl)-phenylamino]-9H-purine

(32):

The mixture of 27 (320 mg, 1.25 mmol), 30 (204 mg, 1 mmol), DIEA (260 μl, 1.5

mmol) in EtOH (3 ml) were heated at 80°C for 12 hours. Then trans-cyclohexane-1, 4-

diamine (31) (395 mg, 3.5 mmol) and DIEA (210 μl, 1.2 mmol) were added, and the

mixture was heated at 95°C overnight. After removal of the solvent in vacuo, the

obtained oily residue was dissolved in MeOH (4 ml), and TFA (2 ml) was added at 0oC.

After stirring at r.t. for 4 hours, the solvent was removed in vacuo. The oily residue was

purified by HPLC to yield TFA salt of 32 as a white solid (312 mg, 47% in 3 steps): 1H

NMR (400 MHz, DMSO-d6): δ 8.28 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.88 (bs, 3H), 7.40

(d, J = 8.0 Hz, 2H), 3.69 (m, 1H), 3.65-3.20 (m, 4H), 3.04 (m, 1H), 2.09-1.95 (m, 4H),

1.62 (m, 2H), 1.55-1.34 (m, 8H); 13C NMR (100 MHz, DMSO-d6): δ 168.5, 153.0, 149.9,

149.3, 141.8, 139.2, 131.9, 127.5, 120.5, 106.5, 50.3, 48.4, 42.5, 29.6, 29.1, 25.5, 24.1;

MS (m/z): 435.2 [M+H]+.

(1), (5) - (10)

CuIN,N'-dimethyl-

ethylenediamine

K3PO4, DMF90ºC

N

N NH

N

HN

NH

N

O

H2NN

N N

N

HN

NH

N

O

H2N

(33) - (39)

I

X

(32) X

N

N N

N

HN

NH

N

O

H2N

F

Purfalcamine (1)

Purfalcamine (1):

A reaction vial containing mixture of 32 (400 mg, 0.92 mmol), CuI (190 mg, 1.00

mmol) and K3PO4 (780 mg, 3.67 mmol) was degassed and refilled with dry N2. N,N’-

dimethylethylenediamine (217 μl, 2.0 mmol) and 1-fluoro-3-iodo-benzene (33) (138 μl,

1.17 mmol) in DMF (3 ml) were added, and the mixture was stirred at 90°C overnight.

After filteration, the reaction mixture was purified with HPLC to yield TFA salt of

purfalcamine (1) as a white solid (203 mg, 32%): 1H NMR (400 MHz, CD3OD) δ 8.26 (s,

1H), 7.99 (d, J = 8.6 Hz, 2H), 7.85 (m, 1H), 7.66 (dd, J = 0.9, 8.2 Hz, 1H), 7.58 (ddd, J =

6.2, 8.2, 8.2 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.19 (ddd, J = 2.4, 8.2, 8.2 Hz, 1H), 3.83

(m, 1H), 3.71 (m, 2H), 3.50 (m, 2H), 3.15 (m, 1H), 2.28 (m, 2H), 2.12 (m, 2H), 1.75-1.40

(m, 10H); 13C NMR (100 MHz, CD3OD): δ 172.3, 164.4 (d, J = 246 Hz), 159.5, 153.4,

152.0, 142.3, 138.5, 138.0 (d, J = 10 Hz), 132.3 (d, J = 9 Hz), 131.6, 129.0, 121.3, 119.5,

115.5 (d, J = 21 Hz), 115.0, 111.6 (d, J = 26 Hz), 51.1, 50.9, 50.3, 44.6, 31.4, 30.9, 27.7,

26.9, 25.6; MS m/z 529.3 [M+H]+1.

N

N N

N

HN

NH

N

O

H2N

OBn

(5)

{4-[2-trans-(4-amino-cyclohexylamino)-9-(3-benzyloxy-phenyl)-9H-purin-6-ylamino]-

phenyl}-piperidin-1-yl-methanone (5):

Compound 5 was synthesized from 32 (26 mg, 50 μmol) and 1-benzyloxy-3-iodo-

benzene (34) (16 mg, 52 μmol) in the same way described for 1. HPLC purification

yielded TFA salt of 5 as pale yellow solid (6.9 mg, 19%): 1H NMR (400 MHz, CD3OD) δ

8.20 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.48-7.42 (m, 4H), 7.44 (d, J = 8.4 Hz, 2H), 7.38

(dd, J = 7.2, 7.2 Hz, 2H), 7.33 (m, 2H), 7.10 (dd, J = 2.4, 8.4 Hz, 2H), 5.16 (s, 2H), 3.81

(m, 1H), 3.70 (m, 2H), 3.49 (m, 2H), 3.12 (m, 1H), 2.27 (m, 2H), 2.24 (m, 2H), 1.76-1.36

(m, 10H); 13C NMR (100 MHz, CD3OD): δ172.3, 161.1, 159.6, 153.5, 152.0, 142.5,

138.9, 138.3, 137.4, 131.6, 131.4, 129.6, 129.1, 128.9, 128.7, 121.1, 116.7, 115.4, 114.9,

111.5, 71.4, 51.0, 50.9, 50.3, 44.7, 31.5, 30.9, 27.7, 26.9, 25.5; MS m/z 617.4 [M+H]+1.

N

N N

N

HN

NH

N

O

H2N

O

O

(6)

{4-[2-trans-(4-amino-cyclohexylamino)-9-benzo[1,3]dioxol-5-yl-9H-purin-6-ylamino]-


Compound 6 was synthesized from 32 (30 mg, 56 μmol) and 5-iodo-

benzo[1,3]dioxole (35) (7.3 μl, 56 μmol) in the same way described for 1. HPLC

purification yielded TFA salt of 6 as white solid (13.7 mg, 22%): 1H NMR (400 MHz,

CD3OD) δ 8.14 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.28 (bs, 1H),

7.15 (dd, J = 1.6, 8.3 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.07 (s, 2H), 3.81 (m, 1H), 3.71

(m, 2H), 3.48 (m, 2H), 3.15 (m, 1H), 2.25 (m, 2H), 2.12 (m, 2H), 1.75-1.40 (m, 10H); 13C

NMR (100 MHz, CD3OD): δ 172.3, 159.7, 153.5, 152.1, 149.9, 148.9, 142.6, 139.3,

131.3, 130.2, 128.9, 121.0, 118.3, 115.0, 109.5, 106.4, 103.5, 51.0, 50.9, 50.3, 44.6, 31.5,

30.9, 27.7, 26.8, 25.6; MS m/z 555.4 [M+H]+1.

N

N N

N

HN

NH

N

O

H2N

CO2H

(7)

3-{2-trans-(4-amino-cyclohexylamino)-6-[4-(piperidine-1-carbonyl)-phenylamino]-

purin-9-yl}-benzoic acid (7):

Compound 7 was synthesized from 32 (30 mg, 56 μmol) and 3-iodo-benzoic acid

(36) (14 mg, 56 μmol) in the same way described for 1. HPLC purification yielded a

TFA salt of 7 as white solid (10.0 mg, 16%): 1H NMR (400 MHz, CD3OD) δ 8.57 (bs,

1H), 8.29 (bs, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.5 Hz,

2H), 7.67 (dd, J = 7.9 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 3.82 (m, 1H), 3.71 (m, 2H),

3.49 (m, 2H), 3.13 (m, 1H), 2.28 (m, 2H), 2.11 (m, 2H), 1.78-1.35 (m, 10H); 13C NMR

(100 MHz, DMSO-d6): δ 168.9, 166.5, 158.5, 151.9, 141.3, 141.2, 137.2, 135.6, 131.9,

129.7, 129.3, 127.7, 127.5, 127.3, 123.6, 119.0, 113.8, 49.7, 48.8, 48.4, 42.5, 29.9, 29.3,

25.6, 24.0; MS m/z 555.6 [M+H]+1.

N

N N

N

HN

NH

N

O

H2N

CO2H(8)

4-{2- trans-(4-amino-cyclohexylamino)-6-[4-(piperidine-1-carbonyl)-phenylamino]-

purin-9-yl}-benzoic acid (8):

Compound 8 was synthesized from 32 (30 mg, 56 μmol) and 4-iodo-benzoic acid

(37) (14 mg, 56 μmol) in the same way described for 1. HPLC purification yielded TFA

salt of 8 as white solid (12.5 mg, 20%): 1H NMR (400 MHz,CD3OD) δ 8.30 (s, 1H), 8.19

(d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H) , 7.41 (d, J = 8.8

Hz, 2H), 3.82 (m, 1H), 3.71 (m, 2H), 3.49 (m, 2H), 3.14 (m, 1H), 2.27 (m, 2H), 2.12 (m,

2H), 1.75-1.40 (m, 10H); 13C NMR (100 MHz, CD3OD): δ 172.4, 168.8, 160.4, 153.6,

152.8, 142.8, 140.6, 138.1, 132.1, 131.0, 130.7, 128.9, 123.4, 120.7, 115.4, 51.0, 50.9,

50.3, 44.6, 31.5, 31.0, 27.7, 26.8, 25.6; MS m/z 555.4 [M+H]+1.

N

N N

N

HN

NH

H2N

N

O

OEt(9)

{4-[2- trans-(4-amino-cyclohexylamino)-9-(4-ethoxy-phenyl)-9H-purin-6-ylamino]-


Compound 9 was synthesized from 32 (26 mg, 50 μmol) and 1-ethoxy-4-iodo-

benzene (38) (13 mg, 52 μmol) in the same way described for 1. HPLC purification

yielded TFA salt of 9 as pale yellow solid (8.1 mg, 25%): 1H NMR (400 MHz, CD3OD) δ

8.16 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H),

7.07 (d, J = 8.4 Hz, 2H), 4.10 (q, J = 7.0 Hz, 2H), 3.81 (m, 1H), 3.71 (m, 2H), 3.48 (m,

2H), 3.13 (m, 1H), 2.23 (m, 2H), 2.11 (m, 2H), 1.77-1.41 (m, 10H), 1.42 (t, J = 7.0 Hz,

3H); 13C NMR (100 MHz, CD3OD): δ 172.3, 160.2, 159.8, 153.5, 152.3, 142.6, 139.4,

131.3, 128.9, 126.4, 121.0, 116.3, 114.7, 65.0, 51.0, 50.9, 50.3, 44.6, 31.5, 30.9, 27.7,

26.8, 25.5, 15.1; MS m/z 555.4 [M+H]+1.

N

N N

N

HN

NH

N

O

H2N

t-Bu(10)

{4-[2- trans-(4-amino-cyclohexylamino)-9-(4-tert-butyl-phenyl)-9H-purin-6-ylamino]-


Compound 10 was synthesized from 32 (30 mg, 57 μmol) and 1-tert-butyl-4-

iodo-benzene (39) (10 μl, 56 μmol) in the same way described for 1. HPLC purification

yielded TFA salt of 10 as white solid (11.8 mg, 20%): 1H NMR (400 MHz, CD3OD) δ

8.22 (bs, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.63 (m, 4H), 7.44 (d, J = 8.4 Hz, 2H), 3.52 (m,

1H), 3.71 (m, 2H), 3.48 (m, 2H), 3.13 (m, 1H), 2.24 (m, 2H), 2.11 (m, 2H), 1.76-1.36 (m,

10H), 1.38 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 172.3, 159.5, 153.5, 152.6, 151.8,

142.5, 139.1, 133.7, 131.4, 128.9, 127.6, 124.4, 121.1, 114.9, 51.0, 50.9, 50.3, 44.6, 35.7,

31.7, 31.5, 30.9, 27.7, 26.8, 25.5; MS m/z 567.4 [M+H]+1.

N

N NH

N

Cl

F

Cu(OAc)2

TEAMS (4 Å)Dioxane

B(OH)2

N

N N

N

Cl

F

(41)(26)

(40)

6-chloro-2-fluoro-9-phenyl-9H-purine (41):

A mixture of activated MS (4 Å, 12.0 g), 6-chloro-2-fluoro-9H-purin (26),

phenylboronic acid (40) (8.3 g, 70 mmol), copper acetate (9.0 g, 53 mmol) and TEA (19

ml, 140 mmol) in dry dioxane (100 ml) was stirred at r.t. for 2 days with a drying tube

attached. The reaction mixture was diluted in DCM, filtered through a Celite and washed

with DCM. The combined organic phase was concentrated, and the oily residue was

purified flash column chromatography (SiO2, 10-30% EtOAc/hexanes) to yield 41 as

light yellow solid (2.1 g, 24%): 1H NMR (400 MHz, CDCl3): δ 8.38 (s, 1H), 7.66 (d, J =

7.5 Hz, 2H), 7.60 (dd, J = 7.5, 7.5 Hz, 2H), 7.52 (dd, J = 7.5, 7.5 Hz, 1H); 13C NMR (100

MHz, CDCl3): δ 157.6 (d, J = 220 Hz), 153.3 (d, J = 18 Hz), 153.0 (d, J = 17 Hz), 144.8,

133.3, 130.1, 129.2, 123.4; MS (m/z): 249.0 [M+H]+.

H2N

N

ONH2

H2N

N

N N

N

HN

N

O

HN

NH2(11)(41)

(42) (31)

DIEA, EtOH80ºC

DIEA, EtOH95ºC

N

N N

N

Cl

F

N2-(4-amino-cyclohexyl)-N6-(4-morpholin-4-ylmethyl-phenyl)-9-phenyl-9H-purine-2,6-

diamine (11):

The mixture of 41 (24 mg, 69 μmol), 4-morpholin-4-ylmethyl-phenylamine (42)

(13 mg, 68 μmol) and DIEA (17.4 μl, 100 μmol) in EtOH (1 ml) was heated at 80°C for

12 hours. Then 31 (13 mg, 68 μmol) and DIEA (14.4 μl, 83 μmol) were added, and the

mixture was heated at 95°C overnight. After cooling to r.t., the reaction mixture was

concentrated in vaccuo, and the obtained oily residue was purified by HPLC yielding

TFA salt of 11 as white solid (34.6 mg, 84% in 2 steps): 1H NMR (400 MHz, CD3OD) δ

8.28 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.5 Hz, 2H), 7.58 (dd, J = 7.5, 7.5 Hz,

2H), 7.55 (d, J = 8.4 Hz, 2H), 7.49 (dd, J = 7.5, 7.5 Hz, 1H), 4.36 (s, 2H), 4.05 (m, 2H),

3.80 (m, 3H), 3.40 (m, 2H), 3.25-3.10 (m, 3H), 2.2 (m, 2H), 2.12 (m, 2H), 1.58 (m, 2H),

1.43 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 158.7, 153.1, 151.2, 142.2, 139.1, 136.0,

133.2, 130.8, 129.5, 124.8, 124.5, 122.1, 114.2, 65.0, 61.5, 52.7, 51.0, 50.8, 31.3, 30.8 ;

MS m/z 499.3 [M+H]+1.

N

N

O

H2N

O

O2NCl

H2Pd/C

EtOH

HN

N

(43) (45)

(44)

DCM

(3-amino-phenyl)-(4-methyl-piperazin-1-yl)-methanone (45):

The compound 45 was synthesized by amide formation of 3-nitro-benzoyl

chloride (43) (93 mg, 0.50 mmol) and 1-methyl-piperazine (44) (56 μl, 0.50 mmol)

followed by hydrogenation in the same way described for 30, which was also used for the

next reaction without purification: MS (m/z): 220.1 [M+H]+.

NH2

H2N

N

N N

N

HN

HN

NH2

N

N

O

H2N N

N

O

(12)

(45) (31)

(41)

DIEA, EtOH80ºC

DIEA, EtOH95ºC

N

N N

N

Cl

F

{3-[2-trans-(4-amino-cyclohexylamino)-9-phenyl-9H-purin-6-ylamino]-phenyl}-(4-

methyl-piperazin-1-yl)-methanone (12):

Compound 12 was synthesized by coupling 41 (24 mg, 69 μmol) with 45 (15 mg,

68 μmol) and 31 (31 mg, 271 μmol) sequentially in the same way described for 11.

HPLC purification gave TFA salt of 12 as pale yellow solid (14.0 mg, 33% in 2 steps):

1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 7.97 (m, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.58

(dd, J = 7.8, 7.8 Hz, 2H), 7.50 (m, 2H), 7.25 (d, J = 7.5 Hz, 1H), 3.82 (m, 2H), 3.80-3.20

(m, 4H), 3.12 (m, 2H), 2.96 (s, 3H), 2.2 (m, 2H), 2.09 (m, 2H), 1.55 (m, 2H), 1.42 (m,

2H); 13C NMR (100 MHz, CD3OD): δ 172.4, 159.8, 153.5, 152.3, 141.5, 138.8, 136.5,

136.0, 130.7, 130.4, 129.0, 124.5, 123.6, 122.6, 120.2, 114.9, 54.2, 51.0, 50.7, 43.7, 31.6,

30.9; MS m/z 526.3 [M+H]+1.

N

N N

NH2N

O

O

N

NH2

O

N

HN

HN

N

O

(13)(41)

(30) (46)N

N N

N

Cl

F DIEA, EtOH80ºC

DIEA, EtOH95ºC

{4-[2-(3-morpholin-4-yl-propylamino)-9-phenyl-9H-purin-6-ylamino]-phenyl}-

piperidin-1-yl-methanone (13):

The compound 13 was synthesized by coupling 41 (24 mg, 69 μmol) with 30 (14

mg, 68 μmol) and 3-morpholin-4-yl-propylamine (46) (39 mg, 270 μmol) sequentially in

the same way described for 11. HPLC purification yielded TFA salt of 12 as white solid

(23.2 mg, 53% in 2 steps): 1H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H), 7.92 (d, J = 8.6

Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H), 7.60 (dd, J = 7.5, 7.5 Hz, 2H), 7.50 (d, J = 7.5 Hz, 1H),

7.45 (d, J = 8.6 Hz, 2H), 3.97 (m, 2H), 3.67 (m, 4H), 3.53 (t, J = 6.4 Hz, 2H), 3.45 (m,

2H), 3.40 (m, 2H), 3.20 (m, 2H), 3.04 (m, 2H), 2.07 (m, 2H), 1.78-1.52 (m, 6H); 13C

NMR (100 MHz, CD3OD): δ 172.2, 159.8, 153.2, 151.4, 141.9, 139.1, 136.1, 132.0,

130.9, 129.5, 129.0, 124.8, 121.6, 114.5, 65.0, 56.6, 53.2, 50.3, 44.6, 39.8, 27.7, 26.8,

25.5, 25.0; MS m/z 541.3 [M+H]+1.

TFA

MeOH

(49)

N

N NH

N

I

HN

N

O

N

N N

N

Cl

I

(47) (48)

H2N

N

O

DIEA, EtOH80ºC

(30) N

N N

N

I

HN

N

O

THPTHP

N

N N

N

HN

N

O

N

HN

Pd2(dba)32-(di-tert-butylphosphino)

-biphenyl

K3PO4, DME85ºC

N

NH2

(14)

N

N N

N

HN

N

O

I

Cu(OAc)2

PyridineMS (4 Å)Dioxane

(HO)2B

(40)

(51)

(50)

{4-[2-iodo-9-(tetrahydro-pyran-2-yl)-9H-purin-6-ylamino]-phenyl}-piperidin-1-yl-

methanone (48):

6-Chloro-2-iodo-9-(tetrahydro-pyran-2-yl)-9H-purine (47) was prepared

according to Brun et al4. An EtOH solution (12 ml) of 47 (1.09 g, 3.00 mmol), 30 (0.405

g, 3.00 mmol) and DIEA (0.8 ml, 6.7 mmol) was refluxed for 48 hours. After removal of

the solvent, the resulting yellow foam was purified on flash chromatography (SiO2, 40-

60% EtOAc/DCM) yielding 48 as pale yellow solid (0.60 g, 43%): 1H NMR (400 MHz,

DMSO-d6): δ 8.51 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 5.63 (dd, J

= 1.6, 10.9 Hz, 1H), 4.01 (m, 1H), 3.72 (m, 1H), 3.60-3.33 (m, 4H), 2.24 (m, 1H), 1.97

(m, 2H), 1.76 (m, 1H), 1.61-1.45 (m, 8H); 13C NMR (100 MHz, DMSO-d6): δ 168.6,

150.9, 149.7, 139.9, 139.8, 130.8, 127.4, 119.9, 119.8, 119.6, 80.7, 67.6, 48.1, 42.5, 29.9,

25.6, 24.4, 24.1, 22.2; MS (m/z): 533.1 [M+H]+.

[4-(2-iodo-9H-purin-6-ylamino)-phenyl]-piperidin-1-yl-methanone (49):

To a MeOH solution (1 ml) of 48 (73 mg, 0.14 mmol) TFA (0.5 ml) was added,

and the mixture was stirred at r.t. for 5 hours. The precipitate was collected and washed

with small amount of cold MeOH to yield 49 (60 mg, ~96%) as pale yellow solid, which

was used for the next reaction without further purification: 1H NMR (400 MHz, DMSO-

d6): δ 8.27 (s, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 3.45 (m, 4H), 1.61

(m, 2H), 1.51 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 168.7, 152.1, 150.4, 140.5,

140.0, 130.6, 127.4, 119.6, 119.1, 118.5, 48.5, 42.3, 25.5, 24.1; MS (m/z): 449.0 [M+H]+.

[4-(2-iodo-9-phenyl-9H-purin-6-ylamino)-phenyl]-piperidin-1-yl-methanone (50):

To a suspension of activated MS (4 Å, 60 mg) and 49 (60 mg, 0.13 mmol) in dry

dioxane (0.5 ml) were added 40 (34 mg, 0.28 mmol), Cu(OAc)2 (38 mg, 0.21 mmol) and

pyridine (45 μl, 0.56 mmol). The mixture was heated at 40°C for 36 hours. The mixture

was cooled down to r.t., diluted with DCM, filtered through Celite and washed with

DCM. The filterate was concentrated and the residue was purified by flash column

chromatography (SiO2, 1-5% MeOH/DCM) to yield 50 (28.5 mg, 41%): 1H NMR (400

MHz, CDCl3): δ 8.52 (bs, 1H), 7.97 (s, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 7.6 Hz,

2H), 7.50 (dd, J = 7.6, 7.6 Hz, 2H), 7.40 (dd, J = 7.6, 7.6 Hz, 1H), 7.39 (d, J = 8.5 Hz,

2H), 3.62 (m, 2H), 3.39 (m, 2H), 1.70-1.40 (m, 6H); 13C NMR (100 MHz, CDCl3): δ

175.1, 170.0, 150.9, 139.3, 134.1, 131.3, 129.9, 128.6, 128.1, 123.4, 120.3, 119.9, 119.4,

50.7, 48.8, 43.1, 26.5, 25.6, 24.6, 21.0; MS (m/z): 525.1 [M+H]+.

{4-[9-phenyl-2-(quinolin-3-ylamino)-9H-purin-6-ylamino]-phenyl}-piperidin-1-yl-

methanone (14):

To a suspension of 50 (20 mg, 38 μmol), Pd2(dba)3 (2.7 mg, 2.9 μmol), 2-(di-tert-

butylphosphino)biphenyl (3.4 mg, 11.4 μmol), K3PO4 (37.9 mg, 179 μmol) in dry DME

(1.5 ml) was added 3-aminoquinoline (8.3 mg, 58 μmol). After sealing, the reaction tube

was degassed and filled with N2. The reaction mixture was then stirred at 85°C overnight.

The resulting suspension was cooled down to r.t., filtered and purified by HPLC to yield

yellow solid of 14 as TFA salt (8.0 mg, 33%): 1H NMR (400 MHz, CD3OD) δ 9.43 (bs,

1H), 9.22 (s, 1H), 8.36 (s, 1H), 8.03 (m, 3H), 7.91 (m, 3H), 7.81 (dd, J = 7.0, 7.0 Hz, 1H),

7.75 (dd, J = 7.0, 7.0 Hz, 1H), 7.68 (dd, J = 7.5, 7.5 Hz, 2H), 7.55 (dd, J = 7.5, 7.5 Hz,

1H), 7.45 (d, J = 8.6 Hz, 2H), 3.74 (m, 2H), 3.52 (m, 2H), 1.80-1.50 (m, 6H); 13C NMR

(100 MHz, CD3OD): δ172.3, 156.9, 153.6, 152.8, 151.6, 142.4, 140.7, 140.1, 137.6,

137.4, 136.5, 131.4, 130.8, 130.6, 130.3, 129.1, 129.0, 128.9, 128.0, 124.4, 123.6, 121.3,

117.3, 50.3, 44.6, 27.8, 26.9, 25.5; MS m/z 541.2 [M+H]+1.

N

N N

N

Cl

FTHP

(53)

N

N NH

N

HN

HN

N

O

OH

H2N

N

O

NH2

HO

TFA

MeOH

(52)

(27)

(30)

DIEA, EtOH80ºC

DIEA, EtOH95ºC

{4-[2-trans-(4-hydroxy-cyclohexylamino)-9H-purin-6-ylamino]-phenyl}-piperidin-1-yl-

methanone (53):

Compound 53 was synthesized in the same way described for 32 from 27 (60 mg,

0.23 mmol) using 30 (35 mg, 0.17 mmol) and trans-4-amino-cylohexanol (52) (90 mg,

0.60 mmol). HPLC purification yielded a TFA salt of 53 as white solid (33 mg, 27% in 3

steps): 1H NMR (400 MHz, DMSO-d6): δ 8.29 (s, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.39 (d,

J = 8.3 Hz, 2H), 3.75 – 3.30 (m, 6H), 1.95 (m, 2H), 1.87 (m, 2H), 1.62 (m, 2H), 1.52 (m,

4H), 1.30 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 168.4, 153.0, 150.0, 148.9, 141.4,

139.0, 131.7, 120.7, 106.8, 68.1, 50.6, 48.1, 42.4, 34.0, 29.9, 25.6, 24.0; MS (m/z): 436.2

[M+H]+.

N

N NH

N

HN

HN

N

O

OH (15)

N

N N

N

HN

HN

N

O

OH

F

CuIN,N'-dimethyl-

ethylenediamine

K3PO4, DMF90ºC

I F(33)

(53)

{4-[9-(3-fluoro-phenyl)-2-trans-(4-hydroxy-cyclohexylamino)-9H-purin-6-ylamino]-


Compound 15 was synthesized from 53 (15 mg, 28 μmol) and 33 (3.3 μl, 28

μmol) in the same way described for 1. HPLC purification yielded a TFA salt of 15 as a

white solid (9.1 mg, 28%): 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 7.90 (d, J = 8.4

Hz, 2H), 7.78 (d, J = 9.9 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.57 (ddd, J = 6.2, 8.2, 8.2 Hz,

1H), 7.44 (d, J = 8.6 Hz, 2H), 7.21 (dd, J = 8.2, 9.8 Hz, 1H), 3.79-3.48 (m, 6H), 2.14 (m,

2H), 2.00 (m, 2H), 1.78-1.52 (m, 6H), 1.38 (m, 4H); 13C NMR (100 MHz, CD3OD): δ

172.2, 164.4 (d, J = 246 Hz), 159.1, 153.2, 151.7, 142.0, 138.5, 137.9 (d, J = 11 Hz),

132.3 (d, J = 9 Hz), 131.8, 129.0, 121.4, 119.5, 115.5 (d, J = 22 Hz), 114.9, 111.5 (d, J =

27 Hz), 70.7, 51.8, 50.4, 44.6, 35.1, 31.6, 27.7, 26.9, 25.6; MS m/z 530.3 ([M+H]+1).

NH2

HN

N

N

N N

N

Cl

FTHP

N

N NH

N

HN

N

O

H2N

N

O

HN

NHN

(27)

(30)

(55)

(54) TFA

MeOHDIEA, EtOH80ºC

DIEA, EtOH95ºC

(4-{2-[2-(3H-imidazol-4-yl)-ethylamino]-9H-purin-6-ylamino}-phenyl)-piperidin-1-yl-

methanone (55):

The compound 55 was synthesized in the same way described for 32 from 27 (60

mg, 0.23 mmol) using 30 (35 mg, 0.17 mmol) and 2-(3H-imidazole-4-yl)-ethyamine (54)

(74 mg, 0.67 mmol). HPLC purification yielded TFA salt of 55 as white solid (55 mg,

45% in 3 steps): 1H NMR (400 MHz, CD3OD): δ 8.74 (s, 1H), 8.20 (s, 1H), 7.86 (d, J =

8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.30 (s, 1H), 3.78 (t, J = 6.5 Hz, 2H), 3.72 (m, 2H),

3.45 (m, 2H), 3.07 (t, J = 6.5 Hz, 2H), 1.75-1.57 (m, 6H); 13C NMR (100 MHz, CD3OD):

δ 171.8, 152.0, 150.1, 142.4, 140.5, 134.8, 133.6, 132.8, 128.9, 122.8, 117.7, 108.7, 50.2,

44.5, 41.9, 27.7, 26.8, 25.5; MS (m/z): 432.2 [M+H]+.

N

N NH

N

HN

N

O

HN

NHN

(16)

N

N N

N

HN

N

O

HN

NHN

Cl

CuIN,N'-dimethyl-

ethylenediamine

K3PO4, DMF90ºC

I Cl

(55)

(56)

(4-{9-(3-chloro-phenyl)-2-[2-(3H-imidazol-4-yl)-ethylamino]-9H-purin-6-ylamino}-

phenyl)-piperidin-1-yl-methanone (16):

Compound 16 was synthesized from 55 (26 mg, 50 μmol) and 1-chloro-3-iodo-

benzene (56) (61 μl, 49 μmol) in the same way described for 1. HPLC purification

yielded TFA salt of 16 as white solid (9.7 mg, 30%): 1H NMR (400 MHz, CD3OD) δ

8.69 (d, J = 1.2 Hz, 1H), 8.25 (s, 1H), 8.04 (bs, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.77 (dd, J

= 0.9, 8.1 Hz, 1H), 7.55 (dd, J = 8.1, 8.1 Hz, 1H), 7.44 (ddd, J = 0.9, 2.0, 8.1 Hz, 1H),

7.40 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 1.2 Hz, 2H), 3.75 (t, J = 6.5 Hz, 2H), 3.72 (m, 2H),

3.48 (m, 2H), 3.08 (t, J = 6.5 Hz, 2H), 1.76-1.55 (m, 6H); 13C NMR (100 MHz, CD3OD):

δ 172.3, 160.8, 153.5, 152.5, 142.6, 138.4, 137.9, 136.1, 134.5, 133.7, 132.1, 131.2, 128.9,

128.7, 124.2, 122.1, 120.9, 117.4, 115.3, 50.3, 44.6, 41.7, 27.7, 26.9, 26.0, 25.6; MS m/z

542.3 [M+H]+1.

Synthesis of Fmoc-aminoethylpurfalcamine (34)

H2N

N

O

NH

Boc

HN

NH

Boc

O2N

O

Cl

DIEA, DCM

Pd/C, H2

EtOH

(57)

(28) (58)

{2-[1-(4-amino-benzoyl)-piperidin-4-yl]-ethyl}-carbamic acid tert-butyl ester (58):

To a DCM solution of (2-piperidin-4-yl-ethyl)-carbamic acid tert-butyl ester (57)

(1 g, 4.4 mmol) and DIEA (1.9 ml, 11 mmol) was added 4-nitro-benzoyl chloride (28)

(812 mg, 4.38 mmol) at 0°C. After stirred for 4.5 hours at r.t., the reaction mixture was

washed with water, and the organic layer was dried with MgSO4 and concentrated in

vacuo to yield yellow oil. This product was dissolved in EtOH and stirred with Pd/C

under 1 atm of H2 for 3 hours. The reaction mixture was filtered over celite and

concentrated in vacuo to yield 58 as yellow oil, which was used for the next reaction

without further purification. MS m/z: 348.2 [M+H]+.

N

N N

N

Cl

FTHP

N

N NH

N

HN

HN

O

NH2

6% TFA

MeOH

HN

Boc

(27)

(58)

(59)

NH2

H2N

(31)

DIEA, EtOH80ºC

DIEA, EtOH95ºC

H2N

N

O

HN

Boc

[2-(1-{4-[2-trans-(4-amino-cyclohexylamino)-9H-purin-6-ylamino]-benzoyl}-piperidin-

4-yl)-ethyl]-carbamic acid tert-butyl ester (59):

An EtOH solution (3 ml) of 58 (406 mg, 1.58 mmol), 28 (500 mg, 1.44 mmol)

and DIEA (300 μl, 1.72 mmol) was sealed and stirred at 80°C overnight. To this reaction

mixture was added an EtOH solution (2 ml) of 31 (541 mg, 4.74 mmol) and DIEA (300

μl, 1.72 mmol), and the mixture was sealed and stirred at 95°C overnight. After filtration

and removal of the solvent in vacuo, the obtained oily residue was dissolved in MeOH

(12 ml). To this solution was added TFA (800 μl) at 0°C, and the mixture was stirred at

r.t. overnight. The reaction mixture was neutralized with DIEA, concentrated in vacuo

and purified with HPLC to yield 59 as TFA salt (394 mg, 3 steps 37%); 1H NMR (400

MHz, CD3OD) δ 8.20 (s, 1H), 7.90 (d, 2H, J = 8.4 Hz), 7.49 (d, 2H, J = 8.4 Hz), 4.63 (m,

1H), 3.83 (m, 2H), 3.10 (t, 2H, J = 7.11 Hz), 3.17-3.09 (m, 2H), 2.86 (m, 2H), 2.25-2.20

(m, 2H), 2.17-2.11 (m, 2H), 1.88 (m, 1H), 1.80-1.62 (m, 3H), 1.60-1.40 (m, 5H), 1.44 (s,

9H), 1.30-1.10 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 171.7, 158.5, 154.1, 152.1,

149.5, 142.6, 140.5, 133.4, 128.8, 122.9, 108.5, 79.9, 51.4, 50.5, 43.8, 38.3, 34.9, 33.6,

32.7, 31.1, 30.9, 30.6, 28.8; MS m/z: 578.3 [M+H]+.

N

N NH

N

HN

HN

O

NH2

HN

Boc

CuIN,N'-dimethyl-

ethylenediamine

K3PO4, DMF90ºC

F

N

N N

N

HN

N

O

H2N

HN

NHFmoc

i) Fmoc-OSu,TEA, DCM

ii) 83% TFAMeOH

(59) (60)

I

(33)

F

Fmoc-aminoethylpurfalcamine (60):

A DMF solution (5 ml) of 59 (100 mg, 148 μmol), 33 (17.4 μl, 148 μmol), CuI

(31.0 mg, 163 μmol), dimethylethylendiamine (31.9 μl, 296 μmol), K3PO4 (126 mg, 594

μmol) was sealed, filled with N2 and heated at 90°C overnight. The reaction mixture was

filtered over celite and washed with DCM. To the filterate, saturated NaHCO3 was added,

and the product was extracted with DCM, dried with MgSO4 and concentrated. The oily

residue was then dissolved in DCM (10 ml), and Fmoc-OSu (55.0 mg, 160 μmol) and

TEA (88.0 μl, 630 μmol) were added to the solution and stirred at r.t. overnight. After

removal of the solvent in vacuo, the residue was dissolved in MeOH (200 μl) and TFA (1

ml), and the mixutre was stirred at r.t. overnight and at 40°C for 2 hours. After

concentration in vacuo, the obtained oily residue was purified with HPLC to yield TFA

salt of 60 as white solid (48.3 mg, 3 steps 37%); 1H NMR (400 MHz, CD3OD) δ 8.69 (d,

J = 1.2 Hz, 1H), 8.25 (s, 1H), 8.04 (bs, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.77 (dd, J = 0.9,

8.1 Hz, 1H), 7.55 (dd, J = 8.1, 8.1 Hz, 1H), 7.44 (ddd, J = 0.9, 2.0, 8.1 Hz, 1H), 7.40 (d, J

= 8.6 Hz, 2H), 7.30 (d, J = 1.2 Hz, 2H), 3.75 (t, J = 6.5 Hz, 2H), 3.72 (m, 2H), 3.48 (m,

2H), 3.08 (t, J = 6.5 Hz, 2H), 1.76-1.55 (m, 6H); 13C NMR (100 MHz, CD3OD): δ 172.3,

160.8, 153.5, 152.5, 142.6, 138.4, 137.9, 136.1, 134.5, 133.7, 132.1, 131.2, 128.9, 128.7,

124.2, 122.1, 120.9, 117.4, 115.3, 50.3, 44.6, 41.7, 27.7, 26.9, 26.0, 25.6; MS m/z 542.3

[M+H]+1.

Preparation of agarose-immobilized purfalcamine

N

N N

N

HN

N

O

NH2

HN

NHFmoc

F

piperidine

DMF

affi-gel 10

DIEA

N

N N

N

HN

HN

NH2

F

N

O

NH

O

HN

HN

O

O

O

(60)

One hundred μl bed volume of Affi-gel 10 (Bio-Rad Laboratories, N-hydroxysuccinimide

esters of a derivatized cross-linked agarose gel bead, 15 μmol/ml in 2-propanol) was

transferred to a Micro Bio-Spin column (Bio-Rad Laboratories) and washed with dry

DMSO (3 x 1 ml). After the column tip was plugged, the resin was resuspended in dry

DMSO (200 μl) to which 10 mM DMSO solution of 60 (150 μl, 1.5 μmol) and 0.1 M

DMSO solution of DIEA (60 μl, 6 μmol) were added. The agarose gel beads were then

gently rotated for 17 hours at room temperature and washed with DMSO (2 x 1 ml). The

beads were incubated with 3 M ethanolamine (5 μl, 15 μmol) in DMSO (1 ml) for

overnight to cap unreacted N-hydroxysuccinimide esters. After the solvent was drained,

the beads were washed with DMSO (2 x 1 ml) and resuspended in 2% piperidine-DMF

solution (0.5 ml) and rotated for 30 minutes. The solution was drained, and the

absorbance at 300 nm was measured. Using molar extinction coefficient (7,800 M-1cm-1)

of deprotection product (1-(9H-fluoren-9-ylmethyl)-piperidine), the attached

purfalcamine concentration was calculated as 0.85 μmol (57% coupling yield). The

resulting agarose-immobilized purfalcamine was further washed with DMF (1 ml),

DMSO (3 x 1 ml) and PBS (3 x 1 ml), and stored in PBS with 0.02% NaN3 at 4°C.

Target confirmation by affinity chromatography and mass spectrometry. Parasites

were harvested and extracted as described5. Parasite pellets (~1 ml) were sonicated in 4

ml of lysis buffer (20 mM Tris, 10 mM MgCl2, 250 μM CaCl2, 200 μM EGTA, 250 mM

NaCl, 0.1% TritonX-100, 1 mM DTT, phosphatase inhibitor cocktail I and II (Sigma, St.

Louis, MO), protease inhibitor (Sigma)) on ice. The resulting homogenate was

centrifuged (16,000 x g) at 4°C for 30 minuets, and the supernatant was recovered.

Protein concentration of the lysate was measured as 1.8 mg/ml using RC DC protein

assay kit (Bio-Rad laboratories, Hercules, CA). The prepared lysate was split in two, and

each portion was immediately used for affinity chromatography experiments with or

without free Purfalcamine (1) (2.5 mM of final concentration). Purfalcamine-resin (200

μl) was transferred to a centrifuge tube (2 ml) and washed with the lysis buffer (3 x 1.5

ml). Lysate with or without free purfalcamine (1) was added to resin and incubated for 1

hour at 4°C with gentle rotation. The resulting resins were then washed with lysis buffer

(3 x 1 ml), and the bound proteins were subsequently eluted by submerging the resin in

200 μl of eluting solution (8 M urea, 0.5 M NaCl, 0.1% Triton X-100) for 2 hours at

room temperature. After brief centrifugation, the supernatants were recovered, and the

resins were rinsed with the eluting solution (100 μl). The combined supernatant and rinse

for each resin were then boiled in the presence of Laemli sample buffer (75 μl) for 10

minutes. The protein samples were loaded onto an SDS-PAGE gel (10% Tris/HCl, Bio-

Rad Laboratories). The corresponding gel lanes were excised and cut horizontally into

eight pieces, which were then subjected to mass spectrometry analysis.

Excised gel bands were subjected to in-gel digestion using a Micromass

MassPREP Station in conjunction with the manufacturer specified protocols. Briefly, gel

pieces were rinsed (2 x with 50% acetonitrile/50 mM ammonium bicarbonate),

dehydrated (acetonitrile), reduced (DTT), alkylated (iodoacetamide), and digested with

sequencing grade trypsin overnight. The resulting peptides were extracted with 30 μl of

1% formic acid. Automated nanoscale LC/MS/MS was performed using a

ThermoFinnigan Surveyor HPLC and LTQ XL ion trap mass spectrometer along with a

variation of the “vented column” approach6. Ten μl of the tryptic digest extracts was

loaded onto a 5 cm long x 75 μm i.d. precolumn packed with 5 μm C-18 silica (Monitor

100Å) retained by a Kaisel frit. After thorough washing, the vent was closed and the

sample was transferred to a 12 cm long x 75 μm i.d. column with a pulled 5 μm tip

packed with the same material by starting the reversed-phase run. The chromatographic

profile was from 100% solvent A (0.1% acetic acid) to 50% solvent B (0.1% acetic acid

in acetonitrile) in 30 minutes at approximately 200 nl/min (manual split from 300 μl/min)

as well as appropriate times for column washing and reequilibration. An angiotensin I

standard (1.25 pmol) was run between each sample to eliminate carryover as well as

monitor overall system performance. The LTQ acquisition method involved one MS

precursor ion scan followed by five data-dependant MS/MS scans. Dynamic exclusion

was enabled for a repeat count of 1 second, repeat duration of 15 seconds, and exclusion

duration of 30 seconds.

Tandem MS data was used to search a P. falciparum specific database

(PlasmoDB 5.3, released 06-19-2007) using SEQUEST, to which a reversed copy of the

protein database was appended. Searches were performed allowing for the fixed

modification of cysteine (carbaminomethyl), the variable modifications of Met

(oxidation), and full trypsin cleavage. Proteins were listed as possible identification only

if three unique peptides were assigned, each of which passed a 95% peptide certainty

criterion based on a reversed database searching strategy7. All potential protein

identifications of interest were also manually verified.

Expression analysis of drug treated samples. Purfalcamine (1) was added to a mixed

population of infected human erythrocytes (4% parasitemia) with a final concentration of

560 nM. The parasites were harvested 24 hours after incubation at 37oC. Total RNA was

extracted and hybridized onto a scrMalaria array8. Gene expression levels were

determined using the MOID algorithm9.

Two-way probe-level Mack-Skillings test were applied to control and treatment

pairs at 24 and 48 hour time points, respectively10. Genes were sorted so that those with

higher expression in the treatment sample occurred first, followed by genes that were

absent in both (expression level below noise), then by genes expressed higher in the

control. Genes were sorted by ascending p-value and descending fold change within the

first group; genes were sorted by ascending p-value and descending fold change within

the second group; genes were sorted by descending p-value and ascending fold change

within the third group. In the resultant sorted list, genes ranked closer to the top are more

likely to show up-regulation in the treatment sample, while genes ranked closer to the

bottom are more likely to show down-regulation in the treatment sample.

There are a total of N genes on the array that are associated with any piece of GO

annotation. For up-regulation, we analyzed the subset of n genes belonging to the top of

the sorted list. If m out of a given GO group of size M also occurs in this top subset, the

enrichment of the particular GO group can be statistically characterized by an

accumulative hypergeometric p-value:

∑=

⎟⎟⎠

⎞⎜⎜⎝

⎛

⎟⎟⎠

⎞⎜⎜⎝

⎛−−

⎟⎟⎠

⎞⎜⎜⎝

⎛

=),min(

),,,(nM

mi

nN

inMN

iM

mMnNP

To avoid an ad hoc selection of n, n is chosen so that the above P function was

minimized. The optimal p-value was identified through an iterative parameter

optimization routine introduced before as OPI11:

),,,(minarg100

mMnNPpn≤

=

Notice that m above is actually determined by the selection of n, and we limit n to be no

larger that 100 in order to focus on the most up-regulated genes in our analysis. We then

carried out 1,000 permutation simulations where gene labels were shuffled and the above

OPI process was reapplied. An original p-value was rejected, if the same or better

number could be reproduced by more than 5% of the permutations.

The same analysis was then repeated on both 24 hour and 48 hour time points,

and the gene lists were also reversed for enrichment analysis in the down-regulated genes.

We then took each of the 15 k-means gene clusters obtained previously as customized

GO groups for similar enrichment analysis of temporally-expressed genes8.

Microscopic analysis. Purfalcamine (1) was added to highly synchronized, infected

human erythrocytes starting at the ring stage with 3% parasitemia. Sample smears were

taken every 8 hours for 48 hours. Slides were then fixed with methanol and stained with

10% Giemsa. Images were captured using the Olympus AX70 upright brightfield

microscope with a UPLanApo 100 x 1.35 oil immersion objective.

In vitro phosphorylation of PfMTIP by PfCDPK1. The entire coding region of pfmtip

(PFL2225w) was inserted into the expression vector (pET28-b, Novagen, Madison, WI).

Sequence analysis was performed to confirm that the fusion protein was in frame with the

C-terminus 6X His tag. Escherichia coli strain (BL21-CodonPlus (DE3) RIL, Stratagene,

La Jolla, CA) was transformed with the expression vector and grown to A600 = 0.5.

Expression of the recombinant protein was induced by the addition of IPTG to a final

concentration of 0.5 mM and further incubated for 10 hours at 24oC.

The cultured cells were harvested and resuspended in lysis buffer (20 mM Tris

HCl pH 8.0, 10 mM imidazole, 500 mM NaCl, 10% glycerol, 0.4% Triton X-100, 1 mM

PMSF) and lysed by sonication on ice. The lysate was centrifuged at 14,000 x g for 20

minutes at 4oC. The recombinant protein in the crude supernatant was purified on a Ni-

NTA agarose column (Novagen).

Purified PfCDPK1 (approximately 50 ng) and PfMTIP (approximately 1 μg) were

incubated in a buffer (20 mM Tris HCl, pH 7.5, MgCl2 10 mM, EGTA 1 mM, CaCl2 1.1

mM, 50 μM ATP) for 3 hours at 37oC. Then, the proteins were separated by 12% SDS

PAGE gel and a band corresponding PfMTIP was excised. The gel band was reduced

with DTT, cysteines alkylated with iodoacetamide (IAM) and digested with trypsin

overnight at 37oC. The digested peptides were recovered and dried to a minimal volume

and reconstituted with 20 mL 0.1 M HOAc. Ten ml from the in-gel digestions was

analyzed by nano-LC/MS on an LTQ XL (Thermo Fisher Scientific) as follows. The

samples was loaded and rinsed on a 360 x 100 mm precolumn packed with 2 cm of

Monitor C18 (Column Engineering, Ontario, CA) and then placed inline with a 360 x 75

mm column (10 cm of Monitor C18) with integrated electrospray tip. Peptides were

eluted with a linear gradient of 0B to 40B in 60 minutes (B, 0.1 M acetic acid,

acetonitrile). The LTQ was set to acquire 1 full scan MS followed by data dependent

acquisition of the 5 most abundant ions from the full scan. Dynamic exclusion was

enabled for a repeat count of 1 second, repeat duration of 15 seconds, and exclusion

duration of 30 seconds.

The data were searched against the NCBI nr database (downloaded on 16 Feb

2007) using Mascot (2.2.03), allowing for the fixed modification of cysteine

(carbaminomethyl), the variable modifications of Ser and Thr (phosphorylation) and Met

(oxidation), and trypsin cleavage. The search results were compiled in Scaffold (01-07-

00) and phosphorylation sites were manually verified.

Efficacy studies. In vivo efficacy studies were carried out on two groups of six mice

each. Male BALB/c mice (Jackson Labs), 7 weeks of age, were housed in microisolator

caging, maintained in a 12:12 hour light/dark schedule and fed regular rodent chow (5001

Lab Diets Inc, Richmond, IN). Starting one day prior to infecting mice with the malaria

parasite, mice were dosed by oral gavage BID with compound 1 at 10 mg/kg (n = 6) or

vehicle control (n = 6) at dosing volume 8 ml/kg. The formulation was a 1.25 mg/ml

solution of 50% Captisol (0.6 g/l), 0.3% HCL and 49.7% sodium phosphate buffer at pH

7.4. The mice were injected via intraperitoneal injection with 100 μl of freshly infected

blood of P. yoelii of titer 10 /ml. Blood samples (5-10 μl) were obtained daily from the

tip of the tail, and parasitemia was monitored by flow cytometry analysis.

7

Dosing

continued for up to 6 days or until the mice were euthanized for humane reasons. Animal

studies described in this report were performed according to the Animal Welfare Act and

the Guide for the Care and Use of Laboratory Animals and approved by the Institutional

Animal Care and Use Committee. All experiments were conducted in a USDA-certified

vivarium.

Homology modeling and virtual ligand screening. In the absence of an experimentally

determined atomic structure of PfCDPK1, a homology model was built with Prime 1.6

(Schrödinger, Inc., San Diego, CA) using death-associated kinase coordinates (pdb code,

1JKK) as a template. The homology model was used for flexible ligand docking with

Glide 4.5 (Schrödinger, Inc.). The binding model of purfalcamine (1) was further refined

using mixed Monte Carlo Multiple Minimum (MCMM)/LLMOD (Large Scale Low

Mode) conformational search strategy available in MacroModel 9.5 (Schrodinger, Inc.).

Comparison of ligand binding was performed using PyMOL (DeLano Scientific, Palo

Alto, CA) by superimposing (RMSD 1.48 Å) the Cα traces of the PfCDPK1 model

docked with purfalcamine (1) (PfCDPK1 residues 53-220) and PfPK5 co-crystallized

with purvalanol B (23) (PDB ID code 1V0P; residues 4-151).

References

1. Deitsch, K., Driskill, C. & Wellems, T. Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res 29, 850-3 (2001).

2. Kidgell, C. et al. A systematic map of genetic variation in Plasmodium falciparum. PLoS Pathog 2, e57 (2006).

3. Ding, S., Gray, N. S., Wu, X., Ding, Q. & Schultz, P. G. A combinatorial scaffold approach toward kinase-directed heterocycle libraries. J Am Chem Soc 124, 1594-6 (2002).

4. Brun, V., Legraverend, M. & Grierson, D. S. Traceless solid-phase synthesis of 2,6,9-trisubstituted purines from resin bound 6-thiopurines. Tetrahedron 58, 7911-7923 (2002).

5. Barik, S., Taylor, R. E. & Chakrabarti, D. Identification, cloning, and mutational analysis of the casein kinase 1 cDNA of the malaria parasite, Plasmodium falciparum. Stage-specific expression of the gene. J Biol Chem 272, 26132-8 (1997).

6. Licklider, L. J., Thoreen, C. C., Peng, J. & Gygi, S. P. Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column. Anal Chem 74, 3076-83 (2002).

7. Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J. & Gygi, S. P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res 2, 43-50 (2003).

8. Le Roch, K. G. et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-8 (2003).

9. Zhou, Y. & Abagyan, R. Match-only integral distribution (MOID) algorithm for high-density oligonucleotide array analysis. BMC Bioinformatics 3, 3 (2002).

10. Barrera, L., Benner, C., Tao, Y. C., Winzeler, E. & Zhou, Y. Leveraging two-way probe-level block design for identifying differential gene expression with high-density oligonucleotide arrays. BMC Bioinformatics 5, 42 (2004).

11. Zhou, Y. et al. In silico gene function prediction using ontology-based pattern identification. Bioinformatics 21, 1237-45 (2005).

NMR spectra of compounds used in this study

tsakata

Typewritten Text

Supplementary Figures and Tables - media.nature.com · 1.5 Kb 2.0 Kb 1.0 Kb M wt sampleA sampleB pfcdpk1 pfcdpk4 hdhfr b Supplementary Figure 1. Disruption of pfcdpk1 and pfcdpk4.

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