Limits...
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: 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.The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K(D).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

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

License
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.


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

License
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: 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.The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K(D).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