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Exploring weak ligand-protein interactions by long-lived NMR states: improved contrast in fragment-based drug screening.

Buratto R, Mammoli D, Chiarparin E, Williams G, Bodenhausen G - Angew. Chem. Int. Ed. Engl. (2014)

Bottom Line: Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy.In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment.This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.

View Article: PubMed Central - PubMed

Affiliation: Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, Batochime (BCH), 1015 Lausanne (Switzerland). roberto.buratto@epfl.ch.

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Identification of a weak binder in a mixture. 1) Weak LLS signals of the spy ligand after sustaining the LLS for Δ=2.5 s in the absence of a competing binder in mixture 1 (spy ligand [II]=500 μm with KD=790 μm protein [Hsp90]=2.5 μm and three nonbinding ligands: 600 μm tyrosine, 600 μm 3,4-difluorobenzylamine, and 600 μm 4-trifluoromethylbenzamidine). 2) Enhanced LLS signals in the presence of a weak binder (mixture 2 contains 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine (KD=2.2 mm), instead of 600 μm of the nonbinding ligand 3,4-difluorobenzylamine). 3) LLS signals observed in the presence of only the binding fragment (mixture 3 contains 500 μm spy ligand [II], 2.5 μm protein [Hsp90], and 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine). 4) Conventional 1H NMR spectrum of mixture 2.
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fig04: Identification of a weak binder in a mixture. 1) Weak LLS signals of the spy ligand after sustaining the LLS for Δ=2.5 s in the absence of a competing binder in mixture 1 (spy ligand [II]=500 μm with KD=790 μm protein [Hsp90]=2.5 μm and three nonbinding ligands: 600 μm tyrosine, 600 μm 3,4-difluorobenzylamine, and 600 μm 4-trifluoromethylbenzamidine). 2) Enhanced LLS signals in the presence of a weak binder (mixture 2 contains 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine (KD=2.2 mm), instead of 600 μm of the nonbinding ligand 3,4-difluorobenzylamine). 3) LLS signals observed in the presence of only the binding fragment (mixture 3 contains 500 μm spy ligand [II], 2.5 μm protein [Hsp90], and 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine). 4) Conventional 1H NMR spectrum of mixture 2.

Mentions: In the absence of competing binders, the interaction between the spy ligand and the protein leads to rapid LLS relaxation and hence to the attenuation of the LLS signal (spectrum 1 in Figure 4); conversely, the presence of a competitor leads to a partial displacement of the spy ligand, hence to slower LLS relaxation and a partial restoration of the LLS signal of the spy (spectrum 2 in Figure 4). This change in LLS signal is due to a mere 13 % change in the amount of bound ligand, which itself is only 0.3 % of the total ligand concentration.


Exploring weak ligand-protein interactions by long-lived NMR states: improved contrast in fragment-based drug screening.

Buratto R, Mammoli D, Chiarparin E, Williams G, Bodenhausen G - Angew. Chem. Int. Ed. Engl. (2014)

Identification of a weak binder in a mixture. 1) Weak LLS signals of the spy ligand after sustaining the LLS for Δ=2.5 s in the absence of a competing binder in mixture 1 (spy ligand [II]=500 μm with KD=790 μm protein [Hsp90]=2.5 μm and three nonbinding ligands: 600 μm tyrosine, 600 μm 3,4-difluorobenzylamine, and 600 μm 4-trifluoromethylbenzamidine). 2) Enhanced LLS signals in the presence of a weak binder (mixture 2 contains 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine (KD=2.2 mm), instead of 600 μm of the nonbinding ligand 3,4-difluorobenzylamine). 3) LLS signals observed in the presence of only the binding fragment (mixture 3 contains 500 μm spy ligand [II], 2.5 μm protein [Hsp90], and 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine). 4) Conventional 1H NMR spectrum of mixture 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4497601&req=5

fig04: Identification of a weak binder in a mixture. 1) Weak LLS signals of the spy ligand after sustaining the LLS for Δ=2.5 s in the absence of a competing binder in mixture 1 (spy ligand [II]=500 μm with KD=790 μm protein [Hsp90]=2.5 μm and three nonbinding ligands: 600 μm tyrosine, 600 μm 3,4-difluorobenzylamine, and 600 μm 4-trifluoromethylbenzamidine). 2) Enhanced LLS signals in the presence of a weak binder (mixture 2 contains 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine (KD=2.2 mm), instead of 600 μm of the nonbinding ligand 3,4-difluorobenzylamine). 3) LLS signals observed in the presence of only the binding fragment (mixture 3 contains 500 μm spy ligand [II], 2.5 μm protein [Hsp90], and 600 μm of the weakly binding ligand [V] 3-bromo-5-methylpyridin-2-ylamine). 4) Conventional 1H NMR spectrum of mixture 2.
Mentions: In the absence of competing binders, the interaction between the spy ligand and the protein leads to rapid LLS relaxation and hence to the attenuation of the LLS signal (spectrum 1 in Figure 4); conversely, the presence of a competitor leads to a partial displacement of the spy ligand, hence to slower LLS relaxation and a partial restoration of the LLS signal of the spy (spectrum 2 in Figure 4). This change in LLS signal is due to a mere 13 % change in the amount of bound ligand, which itself is only 0.3 % of the total ligand concentration.

Bottom Line: Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy.In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment.This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.

View Article: PubMed Central - PubMed

Affiliation: Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, Batochime (BCH), 1015 Lausanne (Switzerland). roberto.buratto@epfl.ch.

Show MeSH
Related in: MedlinePlus