The interplay of structural information and functional studies in kinase drug design: insights from BCR-Abl
Introduction
From the perspective of a medicinal chemist engaged in rational drug discovery, an understanding of how biological activity relates to drug-target interactions on a molecular level is of paramount importance. This is particularly well illustrated in the area of cancer drug discovery, where targeting inhibitors of protein kinases is a frequently followed strategy. Cancers comprise a diverse set of over 200 malignancies, all resulting from DNA mutations and other genetic alterations, but with different pathophysiologies. Amongst these DNA mutations, those affecting protein kinases that frequently modify intracellular protein signalling pathways and create a survival advantage for malignant cells are statistically over-represented [1••]. A potential advantage of drugs designed to target a mutated kinase and its associated dysregulated signalling pathways is that they should be much better tolerated with far fewer side-effects compared to cytotoxic chemotherapeutic agents, which usually indiscriminately target rapidly dividing cells. However, in order to realise such an advantage a kinase inhibitor should selectively target that enzyme most relevant to the particular malignancy, since unselective multi-targeted kinase inhibitors [2], can have side-effect profiles which are little better tolerated than those of cytotoxic chemotherapies [3].
Achieving target selectivity for protein kinase inhibitors is not trivial. There are 90 tyrosine and 388 dual specificity, serine–threonine kinases encoded within the human genome [4], all of which usually utilise ATP as substrate for the transfer of phosphate to substrate proteins. Despite the fact that intracellular concentrations of ATP are in the 1–5 mm range and that kinases have a Km for ATP in the micromolar range, the ATP-binding site is druggable, and many chemotypes have been identified that can be engineered to provide highly potent inhibitors [5]. However, the topology of this site is highly conserved with few amino-acid variations, and consequently the design of selective inhibitors is challenging [6, 7].
Staurosporin, first discovered as a protein kinase C inhibitor [8], was one of the first well-characterised ATP-competitive kinase inhibitors [9], but the promiscuity of this and other prototype compounds led many to believe that selectivity within this target class would not be achievable. However, many kinases are rigorously controlled by auto-regulatory mechanisms and in some cases these can be utilised to provide for selectivity. Thus in the case of the MEK MAP kinases, non-ATP competitive inhibitors such as PD184352 stabilise an inactive conformation of the activation loop without disrupting ATP binding [10]. In another example, Gray and co-workers have successfully targeted the myristoyl-binding site on the Abl tyrosine kinase that regulates the rearrangement of the enzyme between catalytically active and catalytically inactive conformations [11]. Another approach also takes advantage of these different conformations, whereby the inhibitor binds to and stabilises this catalytically inactive conformation. The most well-known example of a kinase inhibitor which acts in this manner is imatinib (Gleevec®; Figure 1) [12, 13], a BCR-Abl tyrosine kinase inhibitor that set a new paradigm in anticancer therapy through its ability to control chronic myelogenous leukaemia (CML) [14••, 15].
In order to attempt the rational design of a selective kinase inhibitor, it is crucial to have reliable information regarding the molecular structure of the enzyme bound to either an ATP-analogue or to an inhibitor, and an accurate means of assessing the effects of inhibitors on the catalytic activity and functional efficacy. These two aspects are discussed further below, with particular reference to the design of BCR-Abl inhibitors.
Section snippets
Molecular mechanism of Abl/BCR-Abl inhibition
In the absence of structural information for either the apo Abl kinase or a liganded Abl kinase, imatinib was discovered in 1992 employing empirical medicinal chemistry methods [12, 16]. The activity of imatinib was subsequently rationalised using docking studies with a homology model of the ATP-binding site of Abl, based upon an X-ray crystal structure of the fibroblast growth factor receptor (FGFR) kinase in complex with the pyrido[2,3-d]pyrimidine inhibitor PD173074 [17]. In the resulting
Broader impact of the Abl/imatinib story in kinase inhibitor development
Structural analysis of the Abl-imatinib interaction has had a tremendous influence in the broader area of kinase inhibitor design. Structural biologists have long suggested that targeting the inactive conformation of kinases could lead to more selective inhibitors, because whilst all kinases adopt a highly similar active conformation to carry out phosphotransfer, there is great variety in the manner in which the active site is deconstructed to facilitate catalytic regulation. The DFG-out
Assays for evaluation of kinase inhibition
Reliable quantification of kinase inhibition is crucial for triaging lead-finding hits, developing structure–activity relationships for drug-optimisation, as well as evaluating kinase selectivity. Three common, generally available techniques are competition kinase–ligand binding assays, biochemical kinase transphosphorylation assays and cellular kinase autophosphorylation assays. The binding assays, which utilise kinase constructs fused to a tag or a support, assess the displacement of a ligand
Conclusions
Retrospectively, studies of Abl and its pharmacologic inhibition highlight the potential value of the tight integration of studies of cellular activity with structural biology and in vitro mechanistic characterisation. Although imatinib was discovered, developed and brought to the clinic well before its serendipitous binding mode was elucidated, a detailed structural understanding of its mechanism of action greatly accelerated development of the next generation BCR-Abl inhibitor nilotinib, as
References and recommended reading
Papers of particular interest published within the period of review have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Pascal Furet for providing the coordinates from imatinib-ABL docking studies, Jürgen Mestan and Doriano Fabbro for in vitro kinase data and Daniel Lietha for assistance with Figure 2, Figure 3.
References (49)
- et al.
The conformational plasticity of protein kinases
Cell
(2002) - et al.
Type-II kinase inhibitor docking, screening, and profiling using modified structures of active kinase states
J Med Chem
(2008) - et al.
Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases
Trends Pharm Sci
(1989) - et al.
Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition
Nat Struct Mol Biol
(2004) - et al.
Bcr-Abl inhibition as a modality of CML therapeutics
Biochim Biophys Acta Rev Cancer
(2001) - et al.
Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia
New Engl J Med
(2006) - et al.
Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571)
Cancer Res
(2002) - et al.
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification
Science
(2001) - et al.
Urea-derivatives of STI571 as inhibitors of Bcr-Abl and PDGFR kinases
Bioorg Med Chem Lett
(2004) - et al.
Discovery of N-(2-chloro-6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays
J Med Chem
(2004)
Solution conformations and dynamics of ABL kinase–inhibitor complexes determined by NMR substantiate the different binding modes of Imatinib/Nilotinib and Dasatinib
J Biol Chem
Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF
Cell
Rational design of inhibitors that bind to inactive kinase conformations
Nat Chem Biol
A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells
Cancer Res
Crystal structures of the FAK kinase in complex with TAE 226 and related bis-anilino pyrimidine inhibitors reveal a helical DFG conformation
PLoS One
Molecular pathobiology of gastrointestinal stromal sarcomas
Ann Rev Path Mech Dis
Patterns of somatic mutation in human cancer genomes
Nature
A quantitative analysis of kinase inhibitor selectivity
Nat Biotechnol
Assessing and managing toxicities induced by kinase inhibitors
Curr Opin Drug Discov Dev
The protein kinase complement of the human genome
Science
Features of selective kinase inhibitors
Chem Biol
Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase
Biochem Biophys Res Commun
Allosteric inhibitors of Bcr-abl-dependent cell proliferation
Nat Chem Biol
Potent and selective inhibitors of the ABL-kinase: phenylaminopyrimidine (PAP) derivatives
Bioorg Med Chem Lett
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