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Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.

Hussein D, Starr A, Heikal L, McNeill E, Channon KM, Brown PR, Sutton BJ, McDonnell JM, Nandi M - Br. J. Pharmacol. (2015)

Bottom Line: Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential.We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo.In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King's College London, London, UK.

No MeSH data available.


Related in: MedlinePlus

Surface plasmon resonance sensorgrams and tabulated data for His-tag captured native/full length GCH1 (F-GCH1) or truncated GCH1 (T-GCH1) interacting with GCH1 feedback regulatory protein (GFRP) analyte in the absence and presence of L-phenylalanin (L-phe). (A) Representative sensorgrams comparing F-GCH1–GFRP binding curves (left) and T-GCH1–GFRP binding curves (right). GTP-cyclohydrolase 1 (GCH1) is captured at a surface density of ∼1300 RU via His-capture, and GFRP is introduced in varying concentrations (12.5–400 nM) with tabulated first-order dissociation constants (KD) and on- and off-rate constants (n = 4). (B) Representative sensorgrams for GFRP binding to F-GCH1 in the presence of L-phe. GCH1 immobilized at a surface density of ∼1500 RU via His-capture, and GFRP and L-phe are introduced in varying concentrations (12.5–400 nM), with tabulated first-order dissociation constants (KD) and on- and off-rate constants are tabulated (n = 4). (C) Comparison of binding kinetics; binding profiles for T-GCH1 and F-GCH1 in the presence and absence of ligands with 400 nM GFRP; F-GCH1 + GFRP; T-GCH1 + GFRP; F-GCH1 + GFRP + L-phe.
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fig02: Surface plasmon resonance sensorgrams and tabulated data for His-tag captured native/full length GCH1 (F-GCH1) or truncated GCH1 (T-GCH1) interacting with GCH1 feedback regulatory protein (GFRP) analyte in the absence and presence of L-phenylalanin (L-phe). (A) Representative sensorgrams comparing F-GCH1–GFRP binding curves (left) and T-GCH1–GFRP binding curves (right). GTP-cyclohydrolase 1 (GCH1) is captured at a surface density of ∼1300 RU via His-capture, and GFRP is introduced in varying concentrations (12.5–400 nM) with tabulated first-order dissociation constants (KD) and on- and off-rate constants (n = 4). (B) Representative sensorgrams for GFRP binding to F-GCH1 in the presence of L-phe. GCH1 immobilized at a surface density of ∼1500 RU via His-capture, and GFRP and L-phe are introduced in varying concentrations (12.5–400 nM), with tabulated first-order dissociation constants (KD) and on- and off-rate constants are tabulated (n = 4). (C) Comparison of binding kinetics; binding profiles for T-GCH1 and F-GCH1 in the presence and absence of ligands with 400 nM GFRP; F-GCH1 + GFRP; T-GCH1 + GFRP; F-GCH1 + GFRP + L-phe.

Mentions: SPR data were analysed using the curve fitting software Origin 7.0 (OriginLab Corporation, Northampton, MA, USA) and Biaevaluation software to determine the kon and koff rate constants and binding parameters, using both first and second order kinetic models. Bmax calculations were normalized for surface density when this differed between experiments. A global fitting approach using the Biaevaluation software was not adequate to fully describe and fit the binding curves. Therefore, individual curve fitting was conducted in order to calculate binding parameters and rate constants (Supporting Information Fig. S2). The representative data shown in the results (Figure 2) were best described using first-order kinetics; hence the values were determined using monophasic fits.


Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.

Hussein D, Starr A, Heikal L, McNeill E, Channon KM, Brown PR, Sutton BJ, McDonnell JM, Nandi M - Br. J. Pharmacol. (2015)

Surface plasmon resonance sensorgrams and tabulated data for His-tag captured native/full length GCH1 (F-GCH1) or truncated GCH1 (T-GCH1) interacting with GCH1 feedback regulatory protein (GFRP) analyte in the absence and presence of L-phenylalanin (L-phe). (A) Representative sensorgrams comparing F-GCH1–GFRP binding curves (left) and T-GCH1–GFRP binding curves (right). GTP-cyclohydrolase 1 (GCH1) is captured at a surface density of ∼1300 RU via His-capture, and GFRP is introduced in varying concentrations (12.5–400 nM) with tabulated first-order dissociation constants (KD) and on- and off-rate constants (n = 4). (B) Representative sensorgrams for GFRP binding to F-GCH1 in the presence of L-phe. GCH1 immobilized at a surface density of ∼1500 RU via His-capture, and GFRP and L-phe are introduced in varying concentrations (12.5–400 nM), with tabulated first-order dissociation constants (KD) and on- and off-rate constants are tabulated (n = 4). (C) Comparison of binding kinetics; binding profiles for T-GCH1 and F-GCH1 in the presence and absence of ligands with 400 nM GFRP; F-GCH1 + GFRP; T-GCH1 + GFRP; F-GCH1 + GFRP + L-phe.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543619&req=5

fig02: Surface plasmon resonance sensorgrams and tabulated data for His-tag captured native/full length GCH1 (F-GCH1) or truncated GCH1 (T-GCH1) interacting with GCH1 feedback regulatory protein (GFRP) analyte in the absence and presence of L-phenylalanin (L-phe). (A) Representative sensorgrams comparing F-GCH1–GFRP binding curves (left) and T-GCH1–GFRP binding curves (right). GTP-cyclohydrolase 1 (GCH1) is captured at a surface density of ∼1300 RU via His-capture, and GFRP is introduced in varying concentrations (12.5–400 nM) with tabulated first-order dissociation constants (KD) and on- and off-rate constants (n = 4). (B) Representative sensorgrams for GFRP binding to F-GCH1 in the presence of L-phe. GCH1 immobilized at a surface density of ∼1500 RU via His-capture, and GFRP and L-phe are introduced in varying concentrations (12.5–400 nM), with tabulated first-order dissociation constants (KD) and on- and off-rate constants are tabulated (n = 4). (C) Comparison of binding kinetics; binding profiles for T-GCH1 and F-GCH1 in the presence and absence of ligands with 400 nM GFRP; F-GCH1 + GFRP; T-GCH1 + GFRP; F-GCH1 + GFRP + L-phe.
Mentions: SPR data were analysed using the curve fitting software Origin 7.0 (OriginLab Corporation, Northampton, MA, USA) and Biaevaluation software to determine the kon and koff rate constants and binding parameters, using both first and second order kinetic models. Bmax calculations were normalized for surface density when this differed between experiments. A global fitting approach using the Biaevaluation software was not adequate to fully describe and fit the binding curves. Therefore, individual curve fitting was conducted in order to calculate binding parameters and rate constants (Supporting Information Fig. S2). The representative data shown in the results (Figure 2) were best described using first-order kinetics; hence the values were determined using monophasic fits.

Bottom Line: Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential.We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo.In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King's College London, London, UK.

No MeSH data available.


Related in: MedlinePlus