Kinase conformational

Fig. 2. Conformational free energy of closed, intermediate and open protein kinase conformations. cAPK indicates the unbound form of cAMP-dependent protein kinase, cAPKiATP the binary complex of cAPK with ATP, cAPKiPKP the binary complex of cAPK with the peptide inhibitor PKI(5-24), and cAPK PKI ATP the ternary complex of cAPK with ATP and PKI(5-24). Shown are averaged values for the three crystal structures lATP.pdb, ICDKA.pdb, and ICDKB.pdb. All values have been normalized with respect to the free energy of the closed conformations.

Helms and McCammon 1997] Helms, V., McCammon, J.A. Kinase Conformations A computational study of the effect of ligand binding. Prot. Sci. 6 (1997) 2336-2343... 

Fig. 14.6. Annotations for inhibitors that bind to unactivated kinase conformations. Sorafenib binds to the DGF-out conformation and its core is defined as Bayer like dfg. PD318088 binds to the aC-Glu-out conformation and its template is MEK like 3.

Although lipid modifications are believed to be necessary for bringing soluble, cytoplasmic proteins to their membrane-bound partners, it is not obvious why transmembrane receptors with structural features favouring their anchorage in the lipid bUayer must be acylated (palmitoylated). Therefore, lipid modifications of proteins may have other roles than attachment to membranes. In some cases lipid modification may change the conformation of proteins, just like phosphorylation an example is the Raf kinase, where the membranous environment activates the kinase conformationally (see Chapter 4). 

Naumann and Matter used a set of 26 X-ray structures of eukaryotic protein kinases, which were classified into subfamilies with similar protein-ligand interactions in the ATP binding site. As can be seen in Fig. 3.13, which shows the GPGA score plot, PC 1 separates CDK and MAP/receptor kinases on the left from the family of PKA kinases. The CDK family is represented by two distinct clusters in the target family landscape, formed by two different ATP binding site conformations. They correspond to the activated and inactivated kinase conformations... 

Thus far, no potent ATP competitive inhibitors of ERKl/2 have been reported. Because of the likely cross-reactivity of ATP competitive inhibitors, compounds that inhibit through interactions outside the ATP binding pocket are attractive. Substrates bind to MAPKs on the opposite face from the active site in a region, which are sometimes called the common docking or CD site this site can also influence kinase conformation (21). Small molecule inhibitors that bind to the CD site of ERK2 have been identified recently but have not received extensive testing (23). 

Liu Y, Gray NS. Rational design of inhibitors that bind to inactive kinase conformations. NaLChem Biol 2006 2 358-364. 

In most active protein kinase conformations, the C-helix forms a salt bridge to an invariant Lys-residue within the N-terminal lobe, allowing optimal positioning of... 

Arqule (www.arqule.com) have recently published work on the potential advantages of targeting autoinhibited, inactive kinase conformations. First reported was the discovery of ARQ 197 (14), Figure 2.9, a cMET inhibitor which was shown to bind to an inactive kinase conformation and is currently in Phase III studies.19... 

In some cases, it is important to characterise the effects of compounds on different activation states of the same kinase. Compounds may give prevention of activation by binding to non-activated kinase in addition to inhibition of catalysis by association the activated state. Some compounds show marked changes in affinity according to the kinase activation state. The magnitude of any effect varies considerably between kinases and tends to be similar for inhibitors that bind the same kinase conformation.13,30,37... 

The availabUity of the DFG-out pocket requires the kinase activation loop to adopt a catalytically deficient conformation in which the ATP binding site becomes partially occluded by the Phe side chain of the DFG motif. While the DFG-out conformation is more favorable in the unphosphorylated kinase, phosphorylation of the activation loop shifts conformational equUibria to the more active DFG-in conformation, increases kinase activity, and often reduces the affinity of type 11 and type 111 inhibitors [1]. Although the search for chemical scaffolds which have affinity for the DFG-out pocket is moving to the forefront of kinase inhibitor research, efforts have been constrained by the lack of high-throughput assay technologies which can identify and discriminate for hgands which bind to and stabihze enzymatically inactive kinase conformation. 

Figure 2.3 First HTS for the detection allosteric Src inhibitors. In the absence of ligand, acrylodan-labeled cSrc shows two emission maxima at 475 and 505 nm. Type I ligands induce a robust loss of fluorescence intensity (arrows) at 475 nm, resulting in a red shift in the emission maxima to 510 nm (right panel). Type II and III inhibitors stabilize the inactive kinase conformation and elicit a different response in which the emissions at 475 and 505 nm are equally reduced. The emission signal at 445 nm is less sensitive to ligand binding and serves as an internal reference point, allowing for...

Simard, J.R., Grutter, C., Pawar, V., Aust, B., Wolf, A., Rabiller, M., Wulfert, S., Robubi, A., Milter, S., Ottmann, C., and Rauh, D. (2009) High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38alpha. /. Am. Chem. Soc., 131 (51), 18478-18488. 

Positive Control Turn on the stir bar, inject 0.1-1 pM of a known Type II inhibitor (or any compound which is known to stabilize or bind to the inactive DFG-out kinase conformation). Immediately start the interval scan to obtain a series of changing emission spectra in response to the binding of the chosen ligand (see Note 19). 

To assess protein stability and time-dependence, re-measure the same plates over a period of time and re-plot the data. For slow-binding inhibitors of the DFG-out kinase conformation, a time-dependent leftward shift of the binding curve is often observed. Additionally, re-calculate the Z -factor over time. A sudden decrease in assay window or data point reproducibility often indicates that the protein is no longer stable. 

Simard JR, Grutter C, Pawar V et al. (2009) High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38alpha. J Am Chem Soc 131 18478-18488... 

Simard JR, Getlik M, Grutter C, Schneider R, Wulfert S, Rauh D (2010) Fluorophore labeling of the glycine-rich loop as a method of identifying inhibitors that bind to active and inactive kinase conformations. J Am Chem Soc 132 4152-4160... 

---