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

Date Published: August 1, 2012

Publisher: International Union of Crystallography

Author(s): Alexander Pflug, Kenneth A. Johnson, Richard A. Engh.


The anomalous dispersion signal of the bromine-containing kinase inhibitor H-89 was used to characterize discrete binding modes of the compound when complexed with the catalytic subunit of protein kinase A.

Partial Text

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 ▶).

The approach of incorporating bromine into small-molecule ligands in order to quickly screen for binding and to subsequently efficiently determine the binding geometry has been employed as a drug-discovery business model (Antonysamy et al., 2008 ▶; Blaney et al., 2006 ▶; Wolf et al., 2002 ▶). Details of its application and utility are sparse in the scientific literature. However, this would be one approach to address the problem of evaluating ligand flexibility in drug design (Seddon et al., 2012 ▶). Here, we show the successful use of this approach for a very specific application, namely the characterization of the apparently heterogeneous binding mode of a kinase inhibitor, which was not possible using electron-density maps (2mFo − DFc) alone.




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