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Feb 13, 2024 23 likes •497 views

Development of Nek2 Substrates Based on C-Nap1 Using FMOC Solid Property of Phase Peptide Synthesis Daniel Andre Novoa Daniel Andre Novoa Introduction Property of Daniel Andre Novoa What is known What is needed What was done

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SLIDE 1

Development of Nek2 Substrates Based on C-Nap1 Using FMOC Solid Phase Peptide Synthesis

Daniel Andre Novoa

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SLIDE 2

Introduction

  • What is known
  • What is needed
  • What was done

Property of Daniel Andre Novoa

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SLIDE 3

Introduction

  • What is known:

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SLIDE 4

Introduction

  • What is known:
  • Nek2 is a NIMA-related serine/threonine kinase

Property of Daniel Andre Novoa

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SLIDE 5

Introduction

  • What is known:
  • Nek2 is a NIMA-related serine/threonine kinase
  • Regulates centrosome separation & spindle

formation

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SLIDE 6

Introduction

  • What is known:
  • Nek2 is a NIMA-related serine/threonine kinase
  • Regulates centrosome separation & spindle

formation

  • Greatly over-expressed in many types of cancer

Property of Daniel Andre Novoa

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SLIDE 7

Introduction

  • What is known:
  • Nek2 is a NIMA-related serine/threonine kinase
  • Regulates centrosome separation & spindle

formation

  • Greatly over-expressed in many types of cancer
  • Biological functions are largely unknown

Property of Daniel Andre Novoa

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SLIDE 8

Introduction

  • What is known:
  • Nek2 is a NIMA-related serine/threonine kinase
  • Regulates centrosome separation & spindle

formation

  • Greatly over-expressed in many types of cancer
  • Biological functions are largely unknown
  • C-Nap1 is a known in vivo substrate of Nek2

Property of Daniel Andre Novoa

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SLIDE 9

Introduction

  • What is needed:

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SLIDE 10

Introduction

  • What is needed:
  • A small molecule substrate to use in the design of:

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SLIDE 11

Introduction

  • What is needed:
  • A small molecule substrate to use in the design of:
  • Biosensors to track the intracellular activity of

Nek2

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SLIDE 12

Introduction

  • What is needed:
  • A small molecule substrate to use in the design of:
  • Biosensors to track the intracellular activity of

Nek2

  • A competitive enzyme inhibitor for anti-cancer

research

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SLIDE 13

Introduction

  • What was done:

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SLIDE 14

Introduction

  • What was done:
  • 2 peptides were synthesized based on the

consensus sequence of C-Nap1:

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SLIDE 15

Introduction

  • What was done:
  • 2 peptides were synthesized based on the

consensus sequence of C-Nap1:

  • NAP_3: Ac-Leu-Thr-Gln-Ala-Leu-Thr-Ser-Ala-

Gly-Ahx-Arg-Arg-Arg-NH2

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SLIDE 16

Introduction

  • What was done:
  • 2 peptides were synthesized based on the

consensus sequence of C-Nap1:

  • NAP_3: Ac-Leu-Thr-Gln-Ala-Leu-Thr-Ser-Ala-

Gly-Ahx-Arg-Arg-Arg-NH2

  • NAP_4: Ac-Ala-Thr-Gln-Ser-Leu-Thr-Ser-Ala-

Gly-Ahx-Arg-Arg-Arg-NH2

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SLIDE 17

Introduction

  • What was done:
  • 2 peptides were synthesized based on the

consensus sequence of C-Nap1:

  • NAP_3: Ac-Leu-Thr-Gln-Ala-Leu-Thr-Ser-Ala-

Gly-Ahx-Arg-Arg-Arg-NH2

  • NAP_4: Ac-Ala-Thr-Gln-Ser-Leu-Thr-Ser-Ala-

Gly-Ahx-Arg-Arg-Arg-NH2

  • The two peptides have been purified, characterized

and lyophilized

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SLIDE 18

Peptide Design

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SLIDE 19

Peptide Design

3 Phosphorylatable Residues

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SLIDE 20

Peptide Design

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SLIDE 21

Peptide Design

4 Phosphorylatable Residues

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SLIDE 22

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SLIDE 23

Aminohexanoic Acid Linker

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SLIDE 24

3 Arginine Residues

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SLIDE 25

Methodology

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SLIDE 26

Methodology

Insoluble Resin Bead

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SLIDE 27

Methodology

Insoluble Resin Bead Resin Linker

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SLIDE 28

Methodology

Insoluble Resin Bead Resin Linker FMOC Protection Group

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SLIDE 29

Methodology

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SLIDE 30

Methodology

Free N-Terminus

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SLIDE 31

Methodology

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SLIDE 32

Methodology

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SLIDE 33

Methodology

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SLIDE 34

Methodology

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SLIDE 35

Methodology

Desired Peptide

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SLIDE 36

Purification

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 AU 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 Minutes 220.00 240.00 260.00 280.00 300.00 320.00 340.00

HPLC Chromatogram of NAP_3

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SLIDE 37

Purification

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 AU 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 Minutes 220.00 240.00 260.00 280.00 300.00 320.00 340.00

HPLC Chromatogram of NAP_3

NAP_3 at 7 Minutes

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SLIDE 38

Purification

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 AU 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 Minutes 220.00 240.00 260.00 280.00 300.00 320.00 340.00

HPLC Chromatogram of NAP_4

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SLIDE 39

Purification

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 AU 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 Minutes 220.00 240.00 260.00 280.00 300.00 320.00 340.00

HPLC Chromatogram of NAP_4

NAP_4 at 9 Minutes

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SLIDE 40

Results

#81 AV: 10 IT: 11.630 ST: 0.24 uS: 1 NL: 5.33E3 F: ITMS + p ESI Full ms [100.00-1800.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Intensity 495.4 212.2 742.7 545.8 583.8 634.5 663.2 477.4 452.9 874.5 782.1 433.3 148.8 950.5 381.5 1047.6 1211.8 1083.9 1228.1 1289.0 1468.9 1578.7 1680.0 1764.5

Mass Spectrum of NAP_3

Molecular Weight = 1484.70 g/mol

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SLIDE 41

Results

#81 AV: 10 IT: 11.630 ST: 0.24 uS: 1 NL: 5.33E3 F: ITMS + p ESI Full ms [100.00-1800.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Intensity 495.4 212.2 742.7 545.8 583.8 634.5 663.2 477.4 452.9 874.5 782.1 433.3 148.8 950.5 381.5 1047.6 1211.8 1083.9 1228.1 1289.0 1468.9 1578.7 1680.0 1764.5

Mass Spectrum of NAP_3

Molecular Weight = 1484.70 g/mol Our Product with 3 Charges Our Product with 2 Charges

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SLIDE 42

Results

#137 AV: 10 IT: 5.093 ST: 0.21 uS: 1 NL: 5.79E4 F: ITMS + p ESI Full ms [100.00-1500.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Intensity 486.7 729.1 537.0 769.0 375.0 574.9 785.6 804.6 365.3 861.8 707.3 403.4 587.6 116.8 891.7 361.2 166.8 969.2 1108.8 1252.3 1175.3 1305.0 1409.2 1456.7

Mass Spectrum of NAP_4

Molecular Weight = 1458.62 g/mol

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SLIDE 43

Results

#137 AV: 10 IT: 5.093 ST: 0.21 uS: 1 NL: 5.79E4 F: ITMS + p ESI Full ms [100.00-1500.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Intensity 486.7 729.1 537.0 769.0 375.0 574.9 785.6 804.6 365.3 861.8 707.3 403.4 587.6 116.8 891.7 361.2 166.8 969.2 1108.8 1252.3 1175.3 1305.0 1409.2 1456.7

Mass Spectrum of NAP_4

Molecular Weight = 1458.62 g/mol Our Product with 3 Charges Our Product with 2 Charges

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SLIDE 44

Future Work

NAP_3 NAP_4

Enzyme Kinetics

Append Fluorophore Append Key Residue

Optimal Substrate

Biosensor

Enzyme Inhibitor

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SLIDE 45

Aknowledgements

  • Dr. Sanjai Kumar, Queens College
  • Queensborough Community College NIH

Bridges to Baccalaureate Program, Director:

  • Dr. Patricia Schneider

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SLIDE 46

References

1) Toshio Kokuryo, Takeshi Senga, Yukihiro Yokoyama, Masato Nagino, Yuji Nimura, Michinari Hamaguchi. Nek2 as an Effective Target for Inhibition of Tumorigenic Growth and Peritoneal Dissemination of

  • Cholangiocarcinoma. Cancer Res. 2007;15;67(20):9637-42.

2) Nobuyuki Tsunoda, Toshio Kokuryo, Koji Oda, Takeshi Senga, Yukihiro Yokoyama, Masato Nagino, Yuji Nimura, Michinari Hamaguchi. Nek2 as a novel molecular target for the treatment of breast carcinoma. Cancer

  • Sci. 2009;100(1):111-6.

3) Andrew M. Fry, Patrick Meraldi, and Erich A. Nigg. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J. 1998;17(2):470–481. 4) Andrew M Fry. The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene. 2002;21(40):6184-94. 5) Daniel G. Hayward and Andrew M. Fry. Nek2 Kinase in Chromosome Instability and Cancer. Cancer Lett. 2006;8;237(2):155-66.

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SLIDE 47

Questions?

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