Selection of cyclic-peptide inhibitors targeting Aurora kinase A: problems and solutions.

The Aurora family of serine/threonine kinases, which consist of Aurora A, B, and C, play a central role in coordinating cytoskeletal and chromosomal events during mitosis.1 Specifically, Aurora A localizes to the spindle poles and is involved in centrosome maturation and separation, initiation of mitosis, spindle assembly, and cytokinesis.2, 3 On the other hand, Aurora B, an important element of the chromosomal passenger complex,4 functions at the kinetochore to regulate proper alignment of the chromosomes on the mitotic spindle.5, 6 Aurora C, although not as extensively studied, is believed to be complementary in function to Aurora B.7 Both Aurora A and Aurora B are regarded as oncogenes, showing transformative potential when overexpressed in vitro and have been shown to be aberrantly expressed and amplified in several cancers.8–11 As such, both kinases have been extensively targeted for potential cancer therapeutics.8 In general, the development of truly selective protein kinase inhibitors has proven to be extremely challenging, as the structure of the kinase catalytic domain and particularly the ATP-binding region are highly conserved among the greater than 500 members of the human kinome,12 while numerous enzymes also utilize ATP as a substrate. The favored methods of generating kinase inhibitors, namely screening small molecule libraries against the catalytic domain of a chosen kinase, generally result in compounds that bind in the ATP-binding site (so-called Type I inhibitors) and are usually poorly selective across the kinome.13 More recently, a few compounds have been discovered that exploit non-conserved regions of the ATP-binding site, such as a hydrophobic pocket blocked in many kinases by a bulky “gatekeeper” residue or a pocket present in the inactive, or “DFG out” conformation of several kinases.14, 15 This has lead to heightened interest in developing strategies to identify kinase inhibitors that not only do not occupy the ATP-binding site but perhaps target kinases outside the core catalytic domain (true allosteric inhibitors).16 Unexplored regions of the kinase, namely anywhere but the ATP cleft, hold the potential to reveal novel sites for inhibitor development. Owing to the intricate regulation of protein kinases and their conformational flexibility, such allosteric sites may possibly exist. Recently several allosteric kinase inhibitors have been identified through novel screening methods. For example, the inclusion of regulatory domains and the use of differential screening with varying ATP concentration have identified several allosteric ligands of AKT isoforms.17, 18 However, methods for identifying allosteric ligands that target the kinase domain directly have been more elusive. A recent approach combining HTS using MS and NMR has identified MAPK inhibitors (biaryl-tetrazole class) with 11 – 16 μM Kd values for the unactive kinase and prevent activation.19 In another example, differential cytotoxicity screening against BCR-ABL positive cells was utilized and after discarding hits resembling known ATP-competitive compounds, a new class of inhibitors containing a 4,6-pyrimidine core were discovered. These new inhibitors were shown to operate in an allosteric fashion by targeting a distal myristoyl binding pocket of c-ABL.20, 21 Betzi and coworkers in another example of allosteric inhibitor screening combined fluorescent probes and protein crystallography where the probe, 8-anilino-1-naphthalene sulfonate (ANS), bound an allosteric pocket near the ATP site in CDK2 with an apparent Kd of 37 μM.22 Due to the lower affinity of most initial allosteric hits, which are typically greater than 10 μM, many allosteric ligands may be potentially missed during traditional HTS campaigns. However, the potential for selectivity for these new classes of allosteric ligands provides the impetus for redesigning current methodologies to discover such inhibitors. Unlike most small molecule inhibitors, peptides are potentially amenable to targeting the peptide binding site or kinase surface as opposed to binding the ATP-cleft, and hence have the potential advantage of probing less conserved regions. An exciting application of these surface-targeting ligands has been in the generation of selective bivalent inhibitors, which covalently combine surface binding peptide moieties with small molecules that are known to target the ATP-binding site. This combined targeting has been successfully employed against protein kinases to produce inhibitors of enhanced potency and selectivity compared to their starting fragments.23–26 Recently, we have developed an approach to generate bivalent inhibitors utilizing phage displayed peptide libraries, and successfully demonstrated its feasibility in creating a new class of potent and selective inhibitors of a model kinase, cAMP-dependent protein kinase A (PKA).27, 28 In our approach, the ATP-binding site is occupied with a pan-inhibitor, staurosporine, and a phage displayed peptide library is directed to the kinase surface via the non-covalent assembly of two coiled-coils conjugated to each moiety, allowing for their simultaneous binding (Figure 1). After several rounds of in vitro selection, the two ligands (staurosporine and a selected peptide) are covalently linked to generate a potential bivalent inhibitor with greater binding affinity and perhaps an enhanced selectivity profile, due to the targeting of the kinase surface. The initial application of this approach to PKA produced bivalent ligands that are >90-fold more potent than the starting staurosporine derivative alone.27 Importantly, kinetic analysis of the cyclic peptide demonstrated it to be a noncompetitive inhibitor.28 In our efforts to test the generality of this approach and potentially discover noncompetitive inhibitors against therapeutically relevant kinases, we chose to target the most extensively studied kinase of the Aurora family1, Aurora Kinase A (Aurora A). Figure 1 The Bivalent Selection Strategy Applied to Aurora A (AUR2) Our bivalent phage display approach shown in Figure 1 was applied to Aurora A as described previously for PKA;27, 28 however, challenges arose concerning high background binding phage (streptavidin binding) and low potencies of selected sequences for Aurora A. These problems were overcome by appropriate changes in selection conditions. The final selection protocol resulted in the discovery of two peptides with low micromolar IC50 values for Aurora A, which to our knowledge are among the most potent peptides identified to date for Aurora A. One of these peptides was further interrogated by kinetic analysis and showed a noncompetitive mode of inhibition.