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
Introduction Property of Daniel Andre Novoa • What is known:
Introduction Property of Daniel Andre Novoa • What is known: • Nek2 is a NIMA-related serine/threonine kinase
Introduction Property of Daniel Andre Novoa • What is known: • Nek2 is a NIMA-related serine/threonine kinase • Regulates centrosome separation & spindle formation
Introduction Property of Daniel Andre Novoa • What is known: • Nek2 is a NIMA-related serine/threonine kinase • Regulates centrosome separation & spindle formation • Greatly over-expressed in many types of cancer
Introduction Property of Daniel Andre Novoa • 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
Introduction Property of Daniel Andre Novoa • 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
Introduction Property of Daniel Andre Novoa • What is needed:
Introduction Property of Daniel Andre Novoa • What is needed: • A small molecule substrate to use in the design of:
Introduction Property of Daniel Andre Novoa • What is needed: • A small molecule substrate to use in the design of: • Biosensors to track the intracellular activity of Nek2
Introduction Property of Daniel Andre Novoa • 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
Introduction Property of Daniel Andre Novoa • What was done:
Introduction Property of Daniel Andre Novoa • What was done: • 2 peptides were synthesized based on the consensus sequence of C-Nap1:
Introduction Property of Daniel Andre Novoa • 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-NH 2
Introduction Property of Daniel Andre Novoa • 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-NH 2 • NAP_4: Ac-Ala-Thr-Gln-Ser-Leu-Thr-Ser-Ala- Gly-Ahx-Arg-Arg-Arg-NH 2
Introduction Property of Daniel Andre Novoa • 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-NH 2 • NAP_4: Ac-Ala-Thr-Gln-Ser-Leu-Thr-Ser-Ala- Gly-Ahx-Arg-Arg-Arg-NH 2 • The two peptides have been purified, characterized and lyophilized
Peptide Design Property of Daniel Andre Novoa
Peptide Design 3 Phosphorylatable Residues Property of Daniel Andre Novoa
Peptide Design Property of Daniel Andre Novoa
Peptide Design 4 Phosphorylatable Residues Property of Daniel Andre Novoa
Property of Daniel Andre Novoa
Property of Daniel Andre Novoa Aminohexanoic Acid Linker
Property of Daniel Andre Novoa 3 Arginine Residues
Methodology Property of Daniel Andre Novoa
Methodology Insoluble Property of Daniel Andre Novoa Resin Bead
Methodology Insoluble Property of Daniel Andre Novoa Resin Bead Resin Linker
Methodology Insoluble Property of FMOC Protection Group Daniel Andre Novoa Resin Bead Resin Linker
Methodology Property of Daniel Andre Novoa
Methodology Free N-Terminus Property of Daniel Andre Novoa
Methodology Property of Daniel Andre Novoa
Methodology Property of Daniel Andre Novoa
Methodology Property of Daniel Andre Novoa
Methodology Property of Daniel Andre Novoa
Methodology Property of Daniel Andre Novoa Desired Peptide
Purification Property of HPLC Chromatogram of NAP_3 Daniel Andre Novoa 4.00 3.50 3.00 2.50 AU 2.00 1.50 1.00 0.50 0.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 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
Purification Property of HPLC Chromatogram of NAP_3 Daniel Andre Novoa 4.00 3.50 NAP_3 at 7 Minutes 3.00 2.50 AU 2.00 1.50 1.00 0.50 0.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 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
Purification Property of HPLC Chromatogram of NAP_4 Daniel Andre Novoa 4.00 3.50 3.00 2.50 2.00 AU 1.50 1.00 0.50 0.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 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
Purification Property of HPLC Chromatogram of NAP_4 Daniel Andre Novoa 4.00 3.50 NAP_4 at 9 Minutes 3.00 2.50 2.00 AU 1.50 1.00 0.50 0.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 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
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] 495.4 100 Property of 95 Daniel Andre Novoa 90 85 80 75 70 65 Mass Spectrum of NAP_3 60 Relative Intensity Molecular Weight = 1484.70 g/mol 55 50 45 40 35 30 25 20 212.2 742.7 15 545.8 583.8 634.5 10 663.2 477.4 452.9 874.5 782.1 433.3 148.8 950.5 5 381.5 1047.6 1211.8 1083.9 1228.1 1289.0 1468.9 1578.7 1680.0 1764.5 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z
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] 495.4 100 Our Product with 3 Charges Property of 95 Daniel Andre Novoa 90 85 80 75 70 65 Mass Spectrum of NAP_3 60 Relative Intensity Molecular Weight = 1484.70 g/mol 55 50 45 40 35 30 25 20 Our Product with 2 Charges 212.2 742.7 15 545.8 583.8 634.5 10 663.2 477.4 452.9 874.5 782.1 433.3 148.8 950.5 5 381.5 1047.6 1211.8 1083.9 1228.1 1289.0 1468.9 1578.7 1680.0 1764.5 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z
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] 486.7 100 Property of 95 Daniel Andre Novoa 90 85 80 75 70 65 Mass Spectrum of NAP_4 60 Relative Intensity Molecular Weight = 1458.62 g/mol 55 50 45 40 35 30 25 20 729.1 15 10 537.0 769.0 375.0 5 574.9 785.6 804.6 365.3 403.4 707.3 861.8 587.6 116.8 166.8 361.2 891.7 969.2 1108.8 1175.3 1252.3 1305.0 1409.2 1456.7 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z
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] 486.7 100 Our Product with 3 Charges Property of 95 Daniel Andre Novoa 90 85 80 75 70 65 Mass Spectrum of NAP_4 60 Relative Intensity Molecular Weight = 1458.62 g/mol 55 50 45 40 35 30 25 20 Our Product with 2 Charges 729.1 15 10 537.0 769.0 375.0 5 574.9 785.6 804.6 365.3 403.4 707.3 861.8 587.6 116.8 166.8 361.2 891.7 969.2 1108.8 1175.3 1252.3 1305.0 1409.2 1456.7 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z
Future Work Property of Daniel Andre Novoa Append Fluorophore Biosensor NAP_3 Enzyme Optimal Kinetics Substrate NAP_4 Enzyme Append Key Inhibitor Residue
Aknowledgements Property of Daniel Andre Novoa • Dr. Sanjai Kumar, Queens College • Queensborough Community College NIH Bridges to Baccalaureate Program, Director: Dr. Patricia Schneider
References Property of Daniel Andre Novoa 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.
Questions? Property of Daniel Andre Novoa
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