AGC kinases

AKT-1 kinase (also called protein kinase B or PKBa) is a serine/threonine kinase belonging to the AGC kinase family [1], AKT was identified from a viral oncogene, v-akt, found in tumor lines established from spontaneous thymomas found in AKR mice [2]. Subsequently, two more AKT isoforms, AKT-2 (or PKB(3) and AKT-3 (or PKBy) have been identified [3]. Reviews exist detailing the structural and cell biology of AKT and the reader is referred to these for further information [4,7,12]. 

The 3-phosphoinositide-dependent protein kinase-1 (PDKl) is a 556-amino acid enzyme composed of three well-differentiated motifs an N-terminal domain, a constitutively activated serine/threonine kinase domain, and a Pleck-strin homology (PH) domain at its C-terminus [87-91]. The attractiveness of PDKl as a potential anticancer target is hnked to its ability to control the activity of a diverse set of AGC kinase members, in particular the three PKB isoforms [92], Full activation of PKB requires phosphorylation at two sites. 

Protein kinase B (PKB), which is also known as Akt, is a serine/threonine kinase that also belongs to the AGC kinase subtype. Three mammalian isoforms—PKBo , /3, and y have been identified. These proteins are broadly expressed and, although isoform-specific patterns of expression exists in some tissues, the three kinases have a similar organizational structure an amino-terminal PH domain, a central serine/threonine catalytic domain, and a short regulatory region at the carboxy terminal end containing the so-called hydrophobic motif [118-120]. 

Mora, A., Komander, D., van Aalten, D. M., and Alessi, D. R. PDKl, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 15 (2004) 161-170. 

Small-molecule inhibitors can induce phosphoiylation priming of AGC kinases. Priming by ATP binding pocket conformation, rather than intrinsic kinase activity, has significant implications for drug discovery and therapeutic efficacy. 

BI-D1870 (Table 7.1) has been reported to be a specific p90RSK inhibitor that lacks activity against other closely related kinases (in the so-called AGC family, which includes the S6 kinases and PKB [Sapkota et al, 2007]). 

The most potent PDKl kinase inhibitor reported to date is UCN-01 (compound 16 Fig. 4 IC50 = 6 to 33 nM) [101], a staurosporine analogue isolated from the culture broth of Streptomyces sp. Originally developed as an inhibitor of calcium-dependent PKC, UCN-01 has the capacity to inhibit a broad spectrum of kinases [102], including other members of the AGC subfamily of kinases (e.g., IC5o = 491nM for PKB) [103]. UCN-Ol-induced PDKl inhibition has also been observed in in vivo murine and human tumor xenografts [101]. 

Other kinases this region is fully accessible, and commonly occupied by inhibitors, so a substantially different binding profile is thus possible for AGC family members, relative to other protein kinases. 

The lipophilic environment created by residues that form the adenine-binding pocket extends sufficiently beyond the opening of the pocket to create an additional lipophilic binding area in front of the adenine. This pocket may not be accessible to inhibitors that bind into tightly closed adenine pockets. Inhibitor accessibility to this pocket may also be restricted for some kinase families. For example, the AGC family kinases (PKA, PKC, AKT) have a C-terminal tail that extends back into N-terminal domain and caps the adenine site with a phenylalanine (see Section 2.2.3.3). Inhibitor-kinase structures that bind an aryl ring in this site include 10 in CDK2 (Fig. 2.5 B) [67], 9 in CDK2 [66], and 17 (Scheme 2.4) in p38... 

Figure 1 Regulation of activation of serine/threonine kinases of the AGC family as modulated conformationally by occupation of the ATP pocket by small-molecule inhibitors or active site mutations. Intrinsic activation refers to a mechanism that does not depend on inhibition of the kinase s activity, whereas extrinsic activation implies a dependence on inhibition of the kinase s catalytic activity and subsequent pathway feedback to result in activation. Surprisingly, wild-ty 3e kinase (wfkinase), an analog-sensitive mutant (askinase—a specific mutant able to bind inhibitors that do not interact with wild-type kinases) and kinase-dead mutants are all intrinsically enabled for activating phosphorylation upon either inhibitor binding or (in the case of certain kinase-dead mutants) conformational priming.

Abbott laboratories reported the ATP-competitive Akt inhibitor A-443654 (in vitro Aktl iCj = 160 pM) °. A-443654 inhibits all three Akt isofonns in FL5.12 cells stably transfected with consti-tutively active myristoylated Aktl, Akt2 and Akt3, and shows moderate selectivity when screened against related kinases in the AGC family, such as PKA and PKC (ref, 20). To obtain a more complete view of A-443654 s cellular targets, we tested it against a... 

---