Sequence-based Design of Kinase Inhibitors Applicable for Therapeutics and Target Identification

Abstract

A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce TM , utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, threedimensional structure, and involvement in proteinprotein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.Protein kinases are important drug targets for oncologic, immunologic, and metabolic disorders. The development of selective protein kinase inhibitors is widely considered a promising approach to drug development. The common strategy, which has led to the development of drugs, such as Glivec and Iressa (1), is to target the ATP binding site (1-3). Other approaches include substrate mimicking inhibitors (4 -9), bi-substrate analogs that target both the ATP and the acceptor binding sites (10), and molecules that target the Src homology 2 domain (11). The KinAce TM (12) approach presented in this article is based upon deriving short peptides from specific regions in the catalytic domain of the kinase that are implicated in kinase-substrate interactions. The KinAce TM peptides mimic regions of the kinase and therefore compete with the kinase for binding to the substrate (or to other modulators of the kinase), and subsequently abrogate the kinase-dependent signaling.Several other groups have used peptides to disrupt proteinprotein interactions and thus modulate kinase signaling. In contrast to our technology, which derives the peptides from the catalytic domain of the kinase, others have derived inhibitory peptides from the substrates (7), pseudosubstrate (13), regulators interacting with the kinase (14 -17), or from non-catalytic domains of the kinase participating in substrate binding (11,18). One of the unique characteristics of the KinAce TM technology is that the regions from which the inhibitory peptides are derived share conserved structural patterns in all kinases. Evidence from the literature supports a potential role for these regions in substrate binding (19 -22). Our technology suggests a general recipe for generating inhibitors of kinase-dependent signaling, applicable to any kinase. To enable permeation of peptides into cells researchers conjugated peptides to membrane penetrat...

Antidiabetic effect of novel modulating peptides of G-protein-coupled kinase in experimental models of diabetes

“…A method for modulating the activity of a protein kinase is to use peptides that contain sequences that are identical or complementary to specific regions within the target protein [38,39,40]. The presumed mechanism of action of these sequence-based peptides is to interfere with the interaction between the kinase and its specific substrate [19]. In the case of GRK2, relatively long peptides complementary to the C terminus of the kinase have been successfully introduced into myocytes using viral vectors containing DNA sequences encoding for the peptide.…”

“…This peptide inhibits the endogenous activity of GRK2 and reverses GRK2-mediated heart failure [41]. To avoid the need for a relatively long peptide or the requirement of transfection, we developed an alternative approach of coupling fatty acid moieties to short synthetic peptides derived from the HJ-loop of GRK2/3, a kinasesubstrate interaction site that has been identified in protein kinases [19].…”

“…Utilising a peptide corresponding to the C-terminus of GRK2, several groups have shown that the activities of the β-adrenergic and parathyroid hormone receptors are increased due to endogenous GRK2 inhibition [17,18]. In previous studies, we have found that small sequence-based peptides taken from a loop (hereafter the HJ loop) that encompasses parts of α helix F and α helix G (subdomains IX-X located in the catalytic domain of the kinase) interfere with kinase-substrate binding among several protein kinases, leading to a decrease in substrate phosphorylation [19].…”

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases

“…The wide use of acylation in general for peptide delivery (Adachi et al, 1999b;Boehning et al, 2005) and of myristoylation in particular (Eichholtz et al, 1993;Kelemen et al, 2002;Niv et al, 2004) made it likely that also the present peptides entered into the cytoplasm. To confirm this assumption, 100 mM of fluorescence-labelled derivatives of peptide 18AD and its non-myristoylated equivalent were added to suspensions of 7TD1 cells.…”

“…Thus, the full apoptosis induction in 7TD1 cells by the IL-6-antagonistic peptide RGKEELD was achieved by concentrations above 30 mM (Manfredini et al, 2003). For myristoylated peptides derived from the catalytic domains of different protein kinases and used at a concentration of 10 mM, the threshold for a proof of efficacy against cancer cell lines was set at 40% growth inhibition (Niv et al, 2004). The inhibition of Erk phosphorylation by membranepenetrating MEK1-derived peptides exhibited IC 50 -values of 10-30 mM (Kelemen et al, 2002).…”