1. Funding and Acknowledgement: F A special thanks to our sponsors
Synthetic Contributors: Christopher Ching
ITC Data Contributor: Ayesha Mahmood
The thermodynamic effects of binding a pyrimidine to MUP-11,2,3,4,5
Caroline Cisneros*, Arno Dunstatter*, Kristen Procko! and Stephen F. Martin ‡
*
= Synthesis and Biological Recognition , ! = Research Educator, ‡ = Principal Investigator
Abstract: Mouse major urinary protein-1 (MUP-1) is a lipocalin with a hydrophobic binding pocket that can bind diverse ligands, making it useful to the group’s research into the thermodynamic
contributions to protein-ligand binding. Gibbs free energy of binding and the binding constant (Ka) have been found via isothermal titration calorimetry (ITC) for related ligands, and possible
structural contributions to binding can be elucidated by X-ray crystallography. Understanding binding will make the production of high affinity ligands possible, which can aid in drug design. 4-
Isobutyl-5-methoxypyrimidine was determined to indirectly hydrogen bond to MUP-1 using two water molecules while its closely related analogue 2-isobutyl-3-methoxypyrazine makes a direct bond
to Y-120 in the active site. Through ITC, it was noted that the pyrimidine had a considerably more favorable change in enthalpy and an unfavorable change in entropy with an overall less favorable
binding compared to the pyrazine. Due to the considerable loss of entropy observed in the binding of the original pyrimidine, 5-methoxy-4-phenylpyrmidine was synthesized in an effort to optimize
affinity through increased van der Waals interactions. This ligand is still in a working process for ITC data as of Spring 2014.
1Bingham, R.J.; Findlay, J.B.C.; Hsieh, S-Y.; Kalverda, A.P.; Kjellberg, A.; Perazzolo, C.; Phillips, S.E.V.; Seshadri, K.; Trinh, C.H.; Turnbull, W.B.; Bodenhausen, G.; Homans, S.W. Thermodynamics of Binding of 2-Methoxy-3-isopropylpyrazine and 2-Methoxy-3-isobutylpyrazine to the Major Urinary Protein. J. Am. Chem. Soc. 2004, 126, 1675-1681.
2Pertinhez, T.A.; Ferrari, E.; Casali, E.; Patel, J.A.; Spisni, A.; Smith, L.J. The binding cavity of mouse major urinary protein is optimized for a variety of ligand binding modes. Biochem. Biophys. Res. Commun. 2009, 390, 1266-1271.
3Barratt, E.; Bingham, R. J.; Warner, D. J.; Laughton, C. A.; Phillips, S. E. V.; Homans, S. W. Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc., 2005, 127, 11827-11834.
4Sharrow, S. D.; Vaughn, J. L.; Žídek, L.; Novotny, M.V.; Stone, M. J. Pheromone binding by polymorphic mouse major urinary proteins. Protein Sci. 2002. 11, 2247-2256.
5Timm, D. E.; Baker, L. J.; Mueller, H.; Zidek, L.; Novotny, M. V. Structural basis of pheromone binding to the mouse major urinary protein (MUP-I). Protein Sci., 2001, 10, 997–1004.
6SBRS unpublished data
7Sharrow, S.D.; Novotny, M.V.; Stone, M. J. Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec- butyl-4,5-dihydrothiazole. Biochemistry 2003, 42, 6302-6309.
8Snyder, P. W.; Mecinovic, J.; Moustakas, D.T.; Thomas III, S.W.; Harder, M.; Mack, E.T.; Lockett, M.R.; Héroux, A.; Sherman, W.; Whitesides, G.M. Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17889-17894.
9Gilligan, P. J.; Folmer, B. K.; Hartz, R. A. ; Koch, S.; Nanda, K. K.; Andreuski, S.; Fitzgerald, L.; Miller, K.; Marshall, W. J. Pyrazolo-[1,5-a]-1,3,5-triazine Corticotrophin-Releasing Factor (CRF) Receptor Ligands. Bioorg. Med. Chem. 2003, 11, 4093-4102.
10Ho-Shen Lin; Author, Leo A. Paquette. A Convenient Method for Determining the Concentration of Grignard Reagents. Synth. Commun. 1994, 24, 2503-2506.
11Tyagaranjan, S.; Chakravarty, P. K. Synthesis of pyrimidines from ketones using microwave irradiation. Tetrahedron Lett. 2005, 46, 7889-7891
Binding Modes in Active Site
• water-mediated hydrogen bonding network
• Entropy is increased by having two water molecules in the
hydrophobic binding pocket
• Hydrogen bond interactions between the water molecules,
the ligand, and side residues Phe38, Leu40, and Tyr 120 7
• Numerous van der Waals contacts4
• Altered R group of pyrimidine to increase binding modes
by synthesizing 5-methoxy-4-phenylpyrimidine
• Shows alternate binding modes between
pyrimidine and pyrazine
Isothermal Titration Calorimetry1,6
• Measures the heat energy given off or taken up by the ITC instrument as a function of time
• Integrates the titration curve to find the enthalpy exchange
• Enthalpy exchange (ΔH) and binding affinity (Ka) are found
• Gibb’s free energy equation ΔG=ΔH-TΔS=-RTln(Ka) used to find ΔG and ΔS.
Funding & Acknowledgement:
Isothermal Titration Calorimetry1,6
Mouse Major Urinary Protein (MUP-I)
• Protein found in mouse urine that
protects and transports pheromones
• Can cause physiological changes due to
a pheromone binding 7
• Contains alpha (α) helixes and eight beta
(β) sheets forming its β-barrel
• β-barrel is its hydrophobic binding site5
• Experiences enthalpy driven binding8
Mouse Major Urinary Protein (MUP-I)
4-isobutyl-5-methoxypyrazine4-isobutyl-5-methoxypyrimidine
Binding Modes in Active Site
Synthetic Routes9,10,11Synthetic Routes9,10,11
![Funding and Acknowledgement: F A special thanks to our sponsors
Synthetic Contributors: Christopher Ching
ITC Data Contributor: Ayesha Mahmood
The thermodynamic effects of binding a pyrimidine to MUP-11,2,3,4,5
Caroline Cisneros*, Arno Dunstatter*, Kristen Procko! and Stephen F. Martin ‡
*
= Synthesis and Biological Recognition , ! = Research Educator, ‡ = Principal Investigator
Abstract: Mouse major urinary protein-1 (MUP-1) is a lipocalin with a hydrophobic binding pocket that can bind diverse ligands, making it useful to the group’s research into the thermodynamic
contributions to protein-ligand binding. Gibbs free energy of binding and the binding constant (Ka) have been found via isothermal titration calorimetry (ITC) for related ligands, and possible
structural contributions to binding can be elucidated by X-ray crystallography. Understanding binding will make the production of high affinity ligands possible, which can aid in drug design. 4-
Isobutyl-5-methoxypyrimidine was determined to indirectly hydrogen bond to MUP-1 using two water molecules while its closely related analogue 2-isobutyl-3-methoxypyrazine makes a direct bond
to Y-120 in the active site. Through ITC, it was noted that the pyrimidine had a considerably more favorable change in enthalpy and an unfavorable change in entropy with an overall less favorable
binding compared to the pyrazine. Due to the considerable loss of entropy observed in the binding of the original pyrimidine, 5-methoxy-4-phenylpyrmidine was synthesized in an effort to optimize
affinity through increased van der Waals interactions. This ligand is still in a working process for ITC data as of Spring 2014.
1Bingham, R.J.; Findlay, J.B.C.; Hsieh, S-Y.; Kalverda, A.P.; Kjellberg, A.; Perazzolo, C.; Phillips, S.E.V.; Seshadri, K.; Trinh, C.H.; Turnbull, W.B.; Bodenhausen, G.; Homans, S.W. Thermodynamics of Binding of 2-Methoxy-3-isopropylpyrazine and 2-Methoxy-3-isobutylpyrazine to the Major Urinary Protein. J. Am. Chem. Soc. 2004, 126, 1675-1681.
2Pertinhez, T.A.; Ferrari, E.; Casali, E.; Patel, J.A.; Spisni, A.; Smith, L.J. The binding cavity of mouse major urinary protein is optimized for a variety of ligand binding modes. Biochem. Biophys. Res. Commun. 2009, 390, 1266-1271.
3Barratt, E.; Bingham, R. J.; Warner, D. J.; Laughton, C. A.; Phillips, S. E. V.; Homans, S. W. Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. J. Am. Chem. Soc., 2005, 127, 11827-11834.
4Sharrow, S. D.; Vaughn, J. L.; Žídek, L.; Novotny, M.V.; Stone, M. J. Pheromone binding by polymorphic mouse major urinary proteins. Protein Sci. 2002. 11, 2247-2256.
5Timm, D. E.; Baker, L. J.; Mueller, H.; Zidek, L.; Novotny, M. V. Structural basis of pheromone binding to the mouse major urinary protein (MUP-I). Protein Sci., 2001, 10, 997–1004.
6SBRS unpublished data
7Sharrow, S.D.; Novotny, M.V.; Stone, M. J. Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec- butyl-4,5-dihydrothiazole. Biochemistry 2003, 42, 6302-6309.
8Snyder, P. W.; Mecinovic, J.; Moustakas, D.T.; Thomas III, S.W.; Harder, M.; Mack, E.T.; Lockett, M.R.; Héroux, A.; Sherman, W.; Whitesides, G.M. Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17889-17894.
9Gilligan, P. J.; Folmer, B. K.; Hartz, R. A. ; Koch, S.; Nanda, K. K.; Andreuski, S.; Fitzgerald, L.; Miller, K.; Marshall, W. J. Pyrazolo-[1,5-a]-1,3,5-triazine Corticotrophin-Releasing Factor (CRF) Receptor Ligands. Bioorg. Med. Chem. 2003, 11, 4093-4102.
10Ho-Shen Lin; Author, Leo A. Paquette. A Convenient Method for Determining the Concentration of Grignard Reagents. Synth. Commun. 1994, 24, 2503-2506.
11Tyagaranjan, S.; Chakravarty, P. K. Synthesis of pyrimidines from ketones using microwave irradiation. Tetrahedron Lett. 2005, 46, 7889-7891
Binding Modes in Active Site
• water-mediated hydrogen bonding network
• Entropy is increased by having two water molecules in the
hydrophobic binding pocket
• Hydrogen bond interactions between the water molecules,
the ligand, and side residues Phe38, Leu40, and Tyr 120 7
• Numerous van der Waals contacts4
• Altered R group of pyrimidine to increase binding modes
by synthesizing 5-methoxy-4-phenylpyrimidine
• Shows alternate binding modes between
pyrimidine and pyrazine
Isothermal Titration Calorimetry1,6
• Measures the heat energy given off or taken up by the ITC instrument as a function of time
• Integrates the titration curve to find the enthalpy exchange
• Enthalpy exchange (ΔH) and binding affinity (Ka) are found
• Gibb’s free energy equation ΔG=ΔH-TΔS=-RTln(Ka) used to find ΔG and ΔS.
Funding & Acknowledgement:
Isothermal Titration Calorimetry1,6
Mouse Major Urinary Protein (MUP-I)
• Protein found in mouse urine that
protects and transports pheromones
• Can cause physiological changes due to
a pheromone binding 7
• Contains alpha (α) helixes and eight beta
(β) sheets forming its β-barrel
• β-barrel is its hydrophobic binding site5
• Experiences enthalpy driven binding8
Mouse Major Urinary Protein (MUP-I)
4-isobutyl-5-methoxypyrazine4-isobutyl-5-methoxypyrimidine
Binding Modes in Active Site
Synthetic Routes9,10,11Synthetic Routes9,10,11](https://image.slidesharecdn.com/b1bade69-f6f0-4a01-998c-72c56557fac5-160412144403/85/Poster-Dunstatter-A-and-Cisneros-C-FINAL-1-320.jpg)
