Preparation of NeK2 and Nek8 Sensors by Solid Phase Peptide Synthesis

Syed Ali and Hugo Jhun Dr. Sanjai Kumar Chemistry & Biochemistry Department Queens College CUNY

Outline


Introduction Relevance to human cancer Research goals Procedure -Solid phase synthesis -Purification -Characterization -Enzymatic/Radioactive Assay Conclusion

Introduction


Nek2 is a Serine/Threonine centrosomal kinase that tightly regulates centrosome organization during mitosis.
NeK2 Kinase

Protein substrate + ATP



ADP + Phosphorylated protein Substrate

Any aberrant role of centrosome can lead to chromosome instability, which can lead to aneuploidy.

Introduction


Nek8 is also a serine/threonine kinase, but it s functions are poorly understood as of now

Figure 1: A phylogenetic analysis of the Nek family.

Nek2 s Relevance to Human Cancer



Nek2 enzyme has been found to be abnormally expressed in many cancer cells.

Fig. 1. Increased Nek2 protein expression in primary breast tumours. Nek2 expression is substantially increased in the carcinoma cells (arrows) compared to the surrounding stromal cells with both the nucleus and cytoplasm staining for Nek2. Scale bars: (A) 100 (B) 30 m.

Nek2 roles in Mitosis cell division

Fig. 3 : Different stages of Mitosis cell division

Nek8 and Mammalian Cancer



 

Overexpression of Nek8 occurs in  Murine and feline juvenile polycystic kidney disease  Human breast tumors Implicated in nephronphtisis Most likely mechanism is through mutation disrupting wild-type actin regulation

Research Goals


 

Many biological functions of NeK2/8 are still unknown. Design and synthesize specific activity-based sensors for monitoring Nek2/8 activity in vitro. Determine optimal sensor monitoring in vivo. Develop specific inhibitors of Nek2/8 enzyme

Working Principle of NeK2/8 Sensors without Quencher

Figure 2. 14-3-3 proteins molecular anvils that mediate conformational changes or steric hindrance with functional consequences (Hermeking, 2003).

Working Principle of NeK2/8 Sensors with External Quencher

Figure 3.

nhanced Sensing of Protein Kinase Activity via a Deeply Quenched Kinase Peptide Substrate (Sharma et al., 2007).

Target Sequences of NeK2 Sensors

H

Target Sequences of NeK2 Sensors

Target Sequences of NeK2 Sensors

P LM -7 O H H N CH C N CH CH 3 O CH 2 CH 3 O H CHC N CH2 CH2 CH2 NH C NH NH2 O H CHC N CH2 CH2 CH2 NH C NH NH2 O O O O H H H H C H C N C HC N C HC N C H C N CH2 CH2 CH2 CH2 C H CH 3 O H OH CH2 CH3 CH2 NH C NH NH2 O H CHC N CH2 CH2 CH2 NH C NH NH2 O C HC NH 2 CH2 CH2 CH2 NH C NH NH2

PLM-8 O H HN CHC N CH CH 3 O CH 2 CH 3 O H CHC N CH 2 CH 2 CH 2 NH C NH NH 2 O H CHC N CH 2 CH 2 CH 2 NH C NH NH 2 O O O O H H H H CHC N CHC N CHC N CHC N CH 2 CH 2 CH 2 CH 2 CHCH 3 SH OH CH 2 CH 3 CH 2 NH C NH NH 2 O H CHC N CH 2 CH 2 CH 2 NH C NH NH 2 O CHC NH 2 CH 2 CH 2 CH 2 NH C NH NH 2

Target Sequences of NeK8 Sensors

N E K 8 -P Y R -1 6 1 O N H O CHC CH3 H N O CHC CH2 CH2 C O NH2 H N O H CHC N CHOH CH3 O CHC CH2 CH2 C O NH2 H N O CHC CH2 OH H N O H CHC N CH2 CHCH3 CH3 O H CHC N CHCH3 CH3 O CHC CH2 O C N NH2

OH P Y R -A L A -G L U -T H R -S E R -L E U -V A L -T Y R -P R O

NEK8-PYR-162 O N H O O O O O H H H H N CHC N CHC N CHC N CH3 CH2 CHOH O CH2 CH3 C O NH2 O O O O O H H H H CHC N CHC N CHC N CHC N CHC N CH2 CH2 CH2 CHCH3 CH2 CH2 OH CHCH3 CH3 C O CH3 NH2 OH

O C NH2

PYR-miniPEG-ALA-GLU-THR-SER-LEU-VAL-TYR-PRO

Target Sequences of NeK8 Sensors

NEK8- Y O 3 O O O N H O O O O O H H H H N CHC N CHC N CHC N CH3 CH2 CHOH O CH2 CH3 C O NH2 O O O O O H H H H CHC N CHC N CHC N CHC N CHC N CH2 CH2 CH2 CHCH3 CH2 CH2 OH CHCH3 CH3 C O CH3 NH2 OH O C NH2

H N

PY - i iPEG- i iPEG-

-GLU-THR- ER-LEU-

L-TYR-PRO

Experimental Procedure


To prepare the peptide sensor, we use the solid phase peptide method (SPPS). The general principle of SPPS is one of repeated cycles of coupling and Fmoc deprotection.

Solid Phase Synthesis Scheme

Fig. 4: Systematic approach of solid phase synthesis

Deprotection Mechanism

Figure (a)

Figure (b) Fig. 5 (a) : Piperidine base attacks and removes proton from Fmoc. Fig. 5(b) : This next step is decarboxylation The result is CO2 and a free NH2 group on the resin, which will serve as the nucleophile in the reaction with carboxy group of amino acid.

Ninhydrin Test for Primary Amine



We used three different reagents to do this test. For deprotection, the resin should be blue and the solution should be dark blue. (positive) For coupling, the resin should be clear and the solution should be light blue. (negative)

Cleavage of NeK2 Sensors from Resin



After the final coupling of 11Pyrenecarboxylic acid or acetyl group, we were ready for resin cleavage. We added Trifluoroacetic acid (TFA), Water and of Triisopropysilane (TIS) (95:2.5:2.5) and gently shake the mixture for 3.5 hours.

Purification by Reverse Phase HPLC



Final purification was achieved by using Acetonitrile and water containing. Both contains 0.1% TFA.
HPLC Graph for SA-28
2998 at 254.00 - 2998 (210-800)nm 1.00 0.50 0.00 2.00 1.00 0.00 0.00 2998 at 335.00 - 2998 (210-800)nm

5.00

10.00

15.00

20.00

25.00

30.00 Minutes

35.00

40.00

45.00

50.00

55.00

60.00

HPLC Graph for SA-28

2998 at 254.00 - 2998 (210-800)nm 4.00 2.00 0.00 4.00 2998 at 335.00 - 2998 (210-800)nm

2.00

0.00 0.00

2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 Minutes

HPLC Graph for SA-29

2.00 2998 at 254.00 - 2998 (210-800)nm

1.00

0.00 0.60 0.40 0.20 0.00 0.00 2998 at 300.00 - 2998 (210-800)nm

2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 Minutes

HPLC Graph for SA-30

2998 at 254.00 - 2998 (210-800)nm 2.00 1.00 0.00 3.00 2.00 1.00 0.00 0.00 2998 at 350.00 - 2998 (210-800)nm

2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 Minutes

Fig. 4. UV detector indicates time when the peptide was collected We used the Lyophilizer to remove the aqueous solution.

HPLC Graph for PLM-7

2.00 AU

1.00

0.00 0.00 10.00 20.00 Minutes 30.00 40.00 50.00

HPLC Graph for PLM-8

2.50 2.00 AU 1.50 1.00 0.50 0.00 0.00 10.00 20.00 Minutes 30.00 40.00 50.00

Principle of Mass Spectrometry



Characterize product based on mass to charge ratio (M/Z)

Fig.5.- Basic principle of Mass spectrometer

SA-27
s yed-pep27-tune_090616112943 #1 RT: 0.00 T: ITMS + p ESI Full m s [200.00-1000.00] 100 95 90 85 80 75 70 65 60 55 518.67 50 45 40 35 291.42 30 298.58 25 428.58 20 15 10 229.00 5 0 200 343.42 322.58 392.58 281.00 370.50 592.50 476.50 535.58 605.33 625.42 685.50 744.17 780.58 794.83 843.50 907.92 953.08 716.08 985.75 354.83 502.67 569.42 AV: 1 NL: 3.10E1 436.50

M + /2 1

Relative Abundance

M+ 1 M +1 /3
444.67

872.17

250

300

350

400

450

500

550

600 m /z

650

700

750

800

850

900

950

1000

SA-28
M 1 +
SA28-1 #30 RT: 0.10 AV: 1 NL: 5.64E2 T: ITMS + p ESI Full m s [200.00-1000.00] 100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 702.75 40 35 30 971.92 25 20 15 10 229.25 5 258.50 0 200 250 300 350 400 287.17 355.42 421.67 413.67 509.00 452.08 469.92 576.92 644.75 712.58 780.50 938.50 838.75 858.58

M + /2 1
430.17

M + 23
880.50

M/3 + 1

441.17

566.83

994.00

450

500

550

600 m /z

650

700

750

800

850

900

950

1000

SA29-1 #30 RT: 0.08 AV: 1 NL: 7.89E3 T: ITMS + p ESI Full m s [300.00-1200.00] 100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45 40 35 30 25 619.67 20 15 663.67 10 5 0 300 408.58 372.67 458.58 564.92 592.83 685.75 325.00 486.92

SA-29
M/2 +1

971.75

M+1

M + 23 M/3 + 1
815.92 914.67 729.75 804.83 901.83 861.58 758.75 929.33 993.75 1051.25 1147.17 1085.17

503.33

430.33

400

500

600

700 m /z

800

900

1000

1100

1200

SA-30
M + /2 1
493.75

SA-30-1i #30 RT: 0.11 AV: 1 NL: 2.74E3 T: ITMS + p ESI Full m s [200.00-1100.00] 100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 25 20

M + 23 M+1
985.67

M +1 /3
829.67 379.67 533.58 1065.42 229.33 330.08 371.08 315.50 300 400 500 415.83 702.83 431.25 566.83 610.58 600 m /z 671.50 700 770.67 800 838.67 974.58 943.08 906.67 900

15 10 5 0 200

1007.50 1042.58 1000 1100

PLMPLM-7
PLM7-1 #30 RT: 0.09 AV: 1 NL: 6.01E4 T: ITMS + p ESI Full m s [200.00-1300.00] 414.58 100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 621.08 25 311.58 20 15 10 5 249.42 0 200 300 400 385.67 464.92 500 600 700 m /z 452.25 677.58 734.50 543.00 791.33 800 870.42 928.33 1041.67 1000 1084.50 1100 1197.83 1200 1267.25 1300

M +1 /3

Chemical Formula: C50H97N25O12 Exact Mass: 1239.7749

M + /2 1

M 1 +
900

PLMPLM-8
PLM8-1 #31 RT: 0.09 AV: 1 NL: 4.37E4 T: ITMS + p ESI Full m s [200.00-1300.00] 420.00 100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 35 30 629.08 25 315.67 20 685.67 15 10 5 0 200 368.25 495.58 300 400 500 551.00 607.67 600 700 m /z 799.33 859.25 896.75 900 972.83 1016.92 1100.42 1100 1213.92 1200 1257.42 1300 457.67

Chemical Formula: C50H97N25O11S Exact Mass: 1255.752

M +1 /3

M + /2 1

742.50 252.67

800

1000

Fluoremetric Assay
1) The activity of our bio-senosr will be biojustified by fluorometer. fluorometer. 2) We will take our bio-sensor, ATP and bioobserve fluorescence activity. 3) 14-3-3 domain will be added to the 14solution and observe the fluorescence .

Radioactive Assay
1) 2) 3) 4) Sensors and enzymes are added to solution 32P-ATP is added to initiate kinase activity Kinase phosphorylates sensor Sensor is isolated and radioactive emission is recorded

Conclusion


We have successfully prepared nine peptides. Right now, we are preparing seven more peptides. After monitoring the activity of NeK2/8, we will observe the activity of NeK2/8 in living cells.

Acknowledgement


Dr. Sanjai Kumar for his great mentorship and guidance. Special thanks to Dr. Ravi Lankalapalli who modify our peptide protocol, create new method for HPLC ,improve the lyophilization procedure.

Reference
Nek2 kinase in chromosome instability and cancer Cancer Letters, Volume 237, Issue 2, 18 June 2006, Pages 155-166 Daniel G. Hayward, Andrew M. Fry


Synthesis and Biological Evaluation of a Series of Novel Inhibitor of Nek2/Hec1 Analogues Xiao-Long Qiu, Guideng Li, Guikai Wu, Jiewen Zhu, Longen Zhou, Phang-Lang Chen, A. Richard Chamberlin, Wen-Hwa Lee Journal of Medicinal Chemistry 2009 52 (6), 1757-1767 Coordinate Regulation of the Mother Centriole Component Nlp by Nek2 and Plk1 Protein Kinases Joseph Rapley,1 Joanne E. Baxter,1 Joelle Blot,1 Samantha L. Wattam,1 Martina Casenghi,2 Patrick Meraldi,2 Erich A. Nigg,2 and Andrew M. Fry1

Reference


Liu S, Lu W, Obara T, Kuida S, Lehoczky J, Dewar K, Drummond IA Beier DR: A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish. Development 129: 5839 5846, 2002 Bowers AJ, Boylan JF: Nek8, a NIMA family kinase member, is overexpressed in primary human breast tumors. Gene 328: 135 142, 2004 Otto, E, Trapp, ML, Schultheiss, UT, Helou, J, Quarmby,LM, Hildebrandt, F. J Am Soc Nephrol 19: 587592, 2008