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Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89.

Pflug A, Johnson KA, Engh RA - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Bottom Line: Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented.H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket.Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Structural Biology Centre, Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.

ABSTRACT
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

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Chemical structures of adenosine-5′-triphosphate (ATP), Fasudil and H-89 in similar orientations with respect to the ATP pocket (hinge region at the left). The isoquinoline portion of H-89 is highlighted in blue, the sulfonamide portion in green and the bromobenzene moiety in red. The numbering of the H-89 molecule refers to C3 and N4, consistent with the designation of the atoms in the structure 3vqh. ‘PKA’ refers to cAMP-dependent protein kinase catalytic subunit α isoform 1 and ‘ROCK2’ to Rho kinase α. Affinity values were taken from the literature: *, Rajagopalan et al. (2010 ▶); †, Gassel et al. (2003 ▶); ‡, Yano et al. (2008 ▶); §, Engh et al. (1996 ▶).
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fig1: Chemical structures of adenosine-5′-triphosphate (ATP), Fasudil and H-89 in similar orientations with respect to the ATP pocket (hinge region at the left). The isoquinoline portion of H-89 is highlighted in blue, the sulfonamide portion in green and the bromobenzene moiety in red. The numbering of the H-89 molecule refers to C3 and N4, consistent with the designation of the atoms in the structure 3vqh. ‘PKA’ refers to cAMP-dependent protein kinase catalytic subunit α isoform 1 and ‘ROCK2’ to Rho kinase α. Affinity values were taken from the literature: *, Rajagopalan et al. (2010 ▶); †, Gassel et al. (2003 ▶); ‡, Yano et al. (2008 ▶); §, Engh et al. (1996 ▶).

Mentions: Apart from the approved drug Fasudil (HA-1077), H-89 is one of the most prominent representatives of the ‘H-series’ of kinase inhibitors, a set of ATP-competitive isoquinoline sulfonamides (Chijiwa et al., 1990 ▶; Hidaka et al., 1984 ▶; Ono-Saito et al., 1999 ▶; Fig. 1 ▶). H-89 was developed and reported to be selective towards the catalytic subunit of cAMP-dependent protein kinase, also known as protein kinase A (PKA). Despite its misregulation in certain types of cancer, PKA is usually considered to be an ‘antitarget’ in drug development owing to the ubiquitous and essential nature of the cellular processes that it regulates. Hence, the use of H-89 has largely remained confined to academic research. In contrast, the Rho kinase-targeting inhibitor Fasudil was approved in Japan in 1995 for the prevention of cerebral vasospasm in patients with subarachnoid haemorrhage and was found to potentially be useful to enhance the memory and improve the prognosis of Alzheimers patients (Huentelman et al., 2009 ▶). However, H-89 became particularly popular for in vitro studies requiring the absence of PKA activity or on the regulatory role of PKA itself. It is still used frequently, but now in the context of recent studies that have shown H-89 to be a rather general AGC kinase inhibitor (Davies et al., 2000 ▶; Lochner & Moolman, 2006 ▶). While one barrier to the development of H-series compounds as drugs may be the inhibition of PKA, H-89 has also proven to be useful in drug-design projects. The H-­89 scaffold has provided the basis for the design of new compounds with selectivity towards protein kinase B (PKB/Akt; Caldwell et al., 2008 ▶; Collins et al., 2006 ▶; Reuveni et al., 2002 ▶), which is structurally similar to PKA (Gassel et al., 2003 ▶) and remains an important drug target (Cheng et al., 2005 ▶; Wu & Hu, 2010 ▶).


Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89.

Pflug A, Johnson KA, Engh RA - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Chemical structures of adenosine-5′-triphosphate (ATP), Fasudil and H-89 in similar orientations with respect to the ATP pocket (hinge region at the left). The isoquinoline portion of H-89 is highlighted in blue, the sulfonamide portion in green and the bromobenzene moiety in red. The numbering of the H-89 molecule refers to C3 and N4, consistent with the designation of the atoms in the structure 3vqh. ‘PKA’ refers to cAMP-dependent protein kinase catalytic subunit α isoform 1 and ‘ROCK2’ to Rho kinase α. Affinity values were taken from the literature: *, Rajagopalan et al. (2010 ▶); †, Gassel et al. (2003 ▶); ‡, Yano et al. (2008 ▶); §, Engh et al. (1996 ▶).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412763&req=5

fig1: Chemical structures of adenosine-5′-triphosphate (ATP), Fasudil and H-89 in similar orientations with respect to the ATP pocket (hinge region at the left). The isoquinoline portion of H-89 is highlighted in blue, the sulfonamide portion in green and the bromobenzene moiety in red. The numbering of the H-89 molecule refers to C3 and N4, consistent with the designation of the atoms in the structure 3vqh. ‘PKA’ refers to cAMP-dependent protein kinase catalytic subunit α isoform 1 and ‘ROCK2’ to Rho kinase α. Affinity values were taken from the literature: *, Rajagopalan et al. (2010 ▶); †, Gassel et al. (2003 ▶); ‡, Yano et al. (2008 ▶); §, Engh et al. (1996 ▶).
Mentions: Apart from the approved drug Fasudil (HA-1077), H-89 is one of the most prominent representatives of the ‘H-series’ of kinase inhibitors, a set of ATP-competitive isoquinoline sulfonamides (Chijiwa et al., 1990 ▶; Hidaka et al., 1984 ▶; Ono-Saito et al., 1999 ▶; Fig. 1 ▶). H-89 was developed and reported to be selective towards the catalytic subunit of cAMP-dependent protein kinase, also known as protein kinase A (PKA). Despite its misregulation in certain types of cancer, PKA is usually considered to be an ‘antitarget’ in drug development owing to the ubiquitous and essential nature of the cellular processes that it regulates. Hence, the use of H-89 has largely remained confined to academic research. In contrast, the Rho kinase-targeting inhibitor Fasudil was approved in Japan in 1995 for the prevention of cerebral vasospasm in patients with subarachnoid haemorrhage and was found to potentially be useful to enhance the memory and improve the prognosis of Alzheimers patients (Huentelman et al., 2009 ▶). However, H-89 became particularly popular for in vitro studies requiring the absence of PKA activity or on the regulatory role of PKA itself. It is still used frequently, but now in the context of recent studies that have shown H-89 to be a rather general AGC kinase inhibitor (Davies et al., 2000 ▶; Lochner & Moolman, 2006 ▶). While one barrier to the development of H-series compounds as drugs may be the inhibition of PKA, H-89 has also proven to be useful in drug-design projects. The H-­89 scaffold has provided the basis for the design of new compounds with selectivity towards protein kinase B (PKB/Akt; Caldwell et al., 2008 ▶; Collins et al., 2006 ▶; Reuveni et al., 2002 ▶), which is structurally similar to PKA (Gassel et al., 2003 ▶) and remains an important drug target (Cheng et al., 2005 ▶; Wu & Hu, 2010 ▶).

Bottom Line: Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented.H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket.Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Structural Biology Centre, Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.

ABSTRACT
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

Show MeSH
Related in: MedlinePlus