preparation of nek2 and nek8 sensors by solid phase peptide synthesis

8/6/2019 Preparation of NeK2 and Nek8 Sensors by Solid Phase Peptide Synthesis

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Preparation of NeK2 and Nek8Preparation of NeK2 and Nek8Sensors by Solid Phase PeptideSensors by Solid Phase Peptide

SynthesisSynthesis

Syed Ali and Hugo JhunDr. Sanjai KumarChemistry & Biochemistry

DepartmentQueens College CUNY


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Outline Introduction

Relevance to human cancer

Research goals

Procedure-Solid phase synthesis-Purification-Characterization-Enzymatic/Radioactive Assay

Conclusion


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IntroductionIntroduction Nek2 is a Serine/Threonine centrosomalNek2 is a Serine/Threonine centrosomal

kinase that tightly regulates centrosomekinase that tightly regulates centrosomeorganization during mitosis.organization during mitosis.

NeK2 KinaseNeK2 Kinase

Protein substrate + ATP ADP + PhosphorylatedProtein substrate + ATP ADP + Phosphorylated

protein Substrateprotein Substrate

Any aberrant role ofcentrosome can leadAny aberrant role ofcentrosome can leadto chromosome instability, which can leadto chromosome instability, which can leadto aneuploidy.to aneuploidy.


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IntroductionIntroduction Nek8 is also a serine/threonine kinase,

but its functions are poorly understoodas of now

Figure 1: A phylogenetic analysis of the Nek family.


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Nek2s Relevance toNek2s Relevance to

Human CancerHuman Cancer Nek2 enzyme has been found to be abnormallyNek2 enzyme has been found to be abnormally

expressed in many cancer cells.expressed in many cancer cells.

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


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Nek2 roles in Mitosis cellNek2 roles in Mitosis cell

divisiondivision

Fig. 3 : Different stages of Mitosis cell divisionFig. 3 : Different stages of Mitosis cell division


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Overexpression of Nek8 occurs in

Murine and feline juvenile polycystic kidney

disease

Human breast tumors

Implicated in nephronphtisis

Most likely mechanism is through mutationdisrupting wild-type actin regulation

Nek8 and Mammalian CancerNek8 and Mammalian Cancer


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Research GoalsResearch Goals

Many biological functions of NeK2/8 are stillunknown.

Design and synthesize specific activity-basedsensors for monitoring Nek2/8 activity in vitro.

Determine optimal sensor monitoring in vivo.

Develop specific inhibitors of Nek2/8 enzyme


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Working Principle of NeK2/8Working Principle of NeK2/8

Sensors without QuencherSensors without Quencher

Figure 2. 14-3-3 proteins molecular anvils that mediate conformational changes or steric hindrance with

functional consequences (Hermeking, 2003).


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Working Principle of NeK2/8Working Principle of NeK2/8

Sensors with External QuencherSensors with External Quencher

Figure 3. nhanced Sensing of Protein Kinase Activity via a Deeply Quenched Kinase Peptide Substrate (Sharma et al., 2007).


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Target Sequences of NeK2 SensorsTarget Sequences of NeK2 Sensors

H


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CH C

CH

O

CH 3CH 2

CH 3

HN C HC

CH2

O

CH2

CH2

NH

C

NH2

NH

HN C HC

CH2

O

CH2

CH2

NH

C

NH2

NH

HN C HC

CH2

O

CHCH3

CH3

HN C HC

CH2

O

OH

HN C HC

CH2

O

OH

HN C HC

CH2

O

CH2

CH2

NH

C

NH2

NH

HN C HC

CH2

O

CH2

CH2

NH

C

NH2

NH

HN C HC

CH2

NH 2

O

CH2

CH2

NH

C

NH2

NH

HN

O

PLM-7

CH C

CH

O

CH 3

CH 2CH 3

HN C HC

CH 2

O

CH 2CH 2

NH

C

NH 2

NH

HN C HC

CH 2

O

CH 2CH 2

NH

C

NH 2

NH

HN C HC

CH 2

O

CHCH3CH 3

HN C HC

CH 2

O

SH

HN CH C

CH 2

O

OH

HN C HC

CH 2

O

CH 2CH 2

NH

C

NH 2

NH

HN C HC

CH 2

O

CH 2CH 2

NH

C

NH 2

NH

HN C HC

CH 2

NH 2

O

CH 2CH 2

NH

C

NH 2

NH

HN

O

PLM-8


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NH

C H C

C H 3

OHN C H C

C H 2

O

C H 2

C

N H 2

O

HN C H C

C H

O

O H

C H 3

HN C H C

C H 2

O

C H 2

C

N H 2

O

HN C H C

C H 2

O

O H

HN C H C

C H 2

O

C H C H 3

C H 3

HN C H C

C H

O

C H 3

C H 3

HN C H C

C H 2

O

O H

N

C N H 2

O

O

N EK 8-P YR -161

P Y R -A LA -G LU -T H R -SE R -LE U -V A L-TY R -P R O

OO

ONH

OHN CHC

CH3

OHN CHC

CH2

O

CH2

C

NH2

O

HN CHC

CH

O

OH

CH3

HN CHC

CH2

O

CH2

C

NH2

O

HN CHC

CH2

O

OH

HN CHC

CH2

O

CHCH3

CH3

HN CHC

CH

O

CH3

CH3

HN CHC

CH2

O

OH

N

C

ONEK8-PYR-162

NH2

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


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OO

O

NH

H

N CH C

CH3

OH

N CHC

CH2

O

CH2

C

NH2

O

H

N CH C

CH

O

OH

CH3

H

N CH C

CH2

O

CH2

C

NH2

O

H

N CHC

CH2

O

OH

H

N CH C

CH2

O

CHCH3

CH3

H

N CHC

CH

O

CH3

CH3

H

N CH C

CH2

O

OH

N

C

ONEK8- Y - 3

NH2O

OO

OH

N

PY - i iPEG- i iPEG- -GLU-THR- ER-LEU- L-TYR-PRO


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Experimental ProcedureExperimental Procedure

To prepare the peptide sensor, we use theTo prepare the peptide sensor, we use thesolid phase peptide method (SPPS).solid phase peptide method (SPPS).

The general principle of SPPS is one ofThe general principle of SPPS is one ofrepeated cycles ofcoupling and Fmocrepeated cycles ofcoupling and Fmoc

deprotection.deprotection.


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Solid Phase Synthesis SchemeSolid Phase Synthesis Scheme

Fig. 4: Systematic approach of solid phase synthesisFig. 4: Systematic approach of solid phase synthesis


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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 CO2and a free NH2 group on the resin, which will serve as the

nucleophile in the reaction with carboxy group of amino acid.


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Ninhydrin Test for Primary AmineNinhydrin Test for Primary Amine

We used three different reagents to doWe used three different reagents to dothis test.this test.

For deprotection, the resin should be blueFor deprotection, the resin should be blueand the solution should be dark blue.and the solution should be dark blue.(positive)(positive)

For coupling, the resin should be clear andFor coupling, the resin should be clear andthe solution should be light blue.the solution should be light blue.(negative)(negative)


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Cleavage of NeK2 Sensors fromCleavage of NeK2 Sensors from

ResinResinAfter the final coupling of 1After the final coupling of 1--

Pyrenecarboxylic acid or acetyl group, wePyrenecarboxylic acid or acetyl group, we

were ready for resin cleavage.were ready for resin cleavage.

We addedWe added TrifluoroaceticTrifluoroacetic acid (TFA),acid (TFA),Water andWater and ofofTriisopropysilaneTriisopropysilane (TIS)(TIS)

(95:2.5:2.5) and gently shake the mixture(95:2.5:2.5) and gently shake the mixturefor 3.5 hours.for 3.5 hours.


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Purification by Reverse Phase HPLCPurification by Reverse Phase HPLC

Final purification was achieved by usingFinal purification was achieved by usingAcetonitrile and water containing. BothAcetonitrile and water containing. Both

contains 0.1% TFA.contains 0.1% TFA.

2998 at 254.00 - 2998 (210-800)nm

0.00

0.50

1.00

2998 at 335.00 - 2998 (210-800)nm

0.00

1.00

2.00

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

HPLC Graph for SA-28


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2998 at 254.00 - 2998 (210-800)nm

0.00

2.00

4.00

2998 at 335.00 - 2998 (210-800)nm

0.00

2.00

4.00

Minutes

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

2998 at 254.00 - 2998 (210-800)nm

0.00

1.00

2.00

2998 at 300.00 - 2998 (210-800)nm

0.00

0.20

0.40

0.60

Minutes

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




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Fig. 4. UV detector indicates time whenFig. 4. UV detector indicates time whenthe peptide was collectedthe peptide was collected

We used the Lyophilizer to remove theWe used the Lyophilizer to remove theaqueous solution.aqueous solution.

2998 at 254.00 - 2998 (210-800)nm

0.00

1.00

2.00

2998 at 350.00 - 2998 (210-800)nm

0.00

1.00

2.00

3.00

Minutes

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



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AU

0.00

1.00

2.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00

AU

0.00

0.50

1.00

1.50

2.00

2.50

Minutes

0.00 10.00 20.00 30.00 40.00 50.00

HPLC Graph for PLM-7

HPLC Graph for PLM-8


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Principle of Mass Spectrometry

Characterize product based on mass tocharge ratio (M/Z)

Fig.5.- Basic principle of Mass spectrometer


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SA-27syed-pep27-tune_090616112943 #1 RT: 0.00 AV: 1 NL: 3.10E1

T: ITMS + p ESI Full ms [200.00-1000.00]

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbundance

436.50

569.42

518.67

872.17

444.67

291.42502.67

298.58 354.83

428.58

985.75343.42 370.50

592.50 716.08322.58476.50

535.58392.58 605.33 907.92281.00 625.42 685.50

229.00 780.58 953.08744.17

843.50794.83

M/2 +1

M + 1M/3 + 1


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SA-28SA28-1 #30 RT: 0.10 AV: 1 NL: 5.64E2


200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbundance

858.58

430.17

880.50

702.75

838.75

971.92

994.00566.83441.17

229.25 287.17 355.42 509.00 576.92421.67 712.58 780.50452.08 644.75 938.50413.67 469.92

258.50

M/2 +1

M+ 1

M + 23

M/3 + 1


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SA-29SA29-1 #30 RT: 0.08 AV: 1 NL: 7.89E3T: ITMS + p ESI Full ms [300.00-1200.00]

300 400 500 600 700 800 900 1000 1100 1200

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbundance

971.75

486.92

1085.17

619.67 815.92

325.00

914.67

804.83729.75

663.67 901.83929.33 1051.25408.58

861.58 993.75458.58372.67 1147.17685.75564.92503.33 758.75592.83

430.33

M + 1M/2 +1

M/3 + 1

M + 23


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SA-30-1i #30 RT: 0.11 AV: 1 NL: 2.74E3


200 300 400 500 600 700 800 900 1000 1100

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

R

elativeAbundance

493.75

985.67

829.67

379.67 533.58

1065.42

330.08229.33 415.83 974.58702.83 838.67

943.08 1007.50371.08

431.25 906.67 1042.58770.67610.58566.83 671.50315.50

M/2 +1

M+ 1

M/3 + 1

SA-30

M + 23


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PLMPLM--77

PLM7-1 #30 RT: 0.09 AV: 1 NL: 6.01E4


200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbundance

414.58

621.08

311.58

677.58452.25

734.50385.67 543.00

249.42 791.33464.92 870.42 1197.83928.33 1084.50 1267.251041.67

M/3 + 1

M/2 +1

M+ 1

Chemical Formula: C50H97N25O12

Exact Mass: 1239.7749


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PLMPLM--88

PLM8-1 #31 RT: 0.09 AV: 1 NL: 4.37E4


200 300 400 500 600 700 800 900 1000 1100 1200 1300

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbundance

420.00

629.08

315.67

685.67457.67

742.50

551.00368.25

252.67 799.334 95.58 607.67 859.25 972.83896.75 1213.921100.421016.92 1257.42

M/3 + 1

M/2 +1

Chemical Formula: C50H97N25O11S

Exact Mass: 1255.752


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FluoremetricFluoremetricAssayAssay

1) The activity of our bio1) The activity of our bio--senosrsenosr will bewill bejustified byjustified by fluorometerfluorometer..

2) We will take our bio2) We will take our bio--sensor, ATP andsensor, ATP andobserve fluorescence activity.observe fluorescence activity.

3) 143) 14--33--3 domain will be added to the3 domain will be added to the

solution and observe the fluorescence .solution and observe the fluorescence .


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Radioactive AssayRadioactive Assay

1) Sensors and enzymesare added to solution

2) 32P-ATP is added to initiate kinaseactivity

3) Kinase phosphorylates sensor

4) Sensor is isolated and radioactiveemission is recorded


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ConclusionConclusion

We have successfully prepared nineWe have successfully prepared ninepeptides.peptides.

Right now, we are preparing seven moreRight now, we are preparing seven morepeptides.peptides.

After monitoring the activity of NeK2/8,After monitoring the activity of NeK2/8,

we will observe the activity of NeK2/8 inwe will observe the activity of NeK2/8 inliving cells.living cells.


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AcknowledgementAcknowledgement

Dr.Dr. SanjaiSanjai Kumar for his greatmentorshipKumar for his greatmentorshipand guidance.and guidance.

Special thanks to Dr. RaviSpecial thanks to Dr. Ravi LankalapalliLankalapalliwho modify our peptide protocol, createwho modify our peptide protocol, createnew method for HPLC ,improve thenew method for HPLC ,improve the

lyophilizationlyophilization procedure.procedure.


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ReferenceNek2 kinase in chromosome instability and cancer

Cancer Letters, Volume 237, Issue 2, 18 June 2006, Pages 155-166Daniel G. Hayward, Andrew M. Fry

Synthesis and Biological Evaluation of a Series of NovelInhibitor of Nek2/Hec1 AnaloguesXiao-LongQiu, GuidengLi, GuikaiWu, JiewenZhu,LongenZhou,Phang-LangChen, A. RichardChamberlin, Wen-HwaLee

JournalofMedicinalChemistry200952 (6), 1757-1767

Coordinate Regulation of the Mother Centriole Component Nlp byNek2 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


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Reference

Liu S, LuW, Obara T, Kuida S, Lehoczky J, Dewar K,Drummond IA Beier DR: A defect in a novel Nek-familykinase causes cystic kidney disease in the mouse and in

zebrafish. Development 129: 58395846, 2002 Bowers AJ, Boylan JF: Nek8, a NIMA family kinase

member, is overexpressed in primary human breasttumors. Gene 328: 135142, 2004

Otto, E, Trapp, ML, Schultheiss, UT, Helou, J,Quarmby,LM, Hildebrandt, F. J Am Soc Nephrol 19: 587-592, 2008

preparation of nek2 and nek8 sensors by solid phase peptide synthesis

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