Stepwise combinatorial evolution of Akt bisubstrate inhibitors
Stepwise combinatorial evolution of Akt bisubstrate inhibitors
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DOI：
10.1002/cbic.200700583
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发表时间：
2008-03-03
期刊：
影响因子：
3.2
通讯作者：
Lawrence, David S.
中科院分区：
文献类型：
--
作者：
Lee, Jung Hwan;Kumar, Sanjai;Lawrence, David S.
Protein kinases are key enzymatic participants in the signal transduction pathways that control nearly every aspect of normal cell function. In addition, many of these enzymes are often found to be aberrantly expressed or abnormally active in a diverse array of human ailments. For example, the Akt protein kinase is hyperactive in a variety of cancers, where it promotes tumor cell survival by blocking cell death.[1] Not surprisingly, there is considerable interest in acquiring inhibitors for Akt. Like other members of the protein kinase family, Akt employs ATP as the phosphoryl donor for the phosphorylation of Ser residues in protein substrates. Several ATP analogues and nonphosphorylatable peptides have been reported that block the catalytic activity of Akt.[2] Recently, bisubstrate analogues have been championed as useful structural, mechanistic, and biological probes for protein kinases.[3] These inhibitory agents simultaneously block the binding of both substrates in a two substrate enzyme-catalyzed reaction. We report herein the directed molecular evolution of a potent Akt bisubstrate inhibitor using a stepwise, combinatorial library-based strategy. Bisubstrate inhibitors for protein kinases have been constructed by assembling three component parts into a single unimolecular species. Generally, a tether, which links an ATP surrogate to a peptide-based species, is finely tuned to ensure that both the ATP and the protein binding sites can be comfortably occupied by the inhibitor. However, there is no compelling structural or enzymatic requirement that limits the bisubstrate strategy to previously described components (for example, ATP analogues such as adenosine or H-7). Rather, we felt that it might be possible to use the bisubstrate concept as a means to identify a new functionality that prevents ATP from binding to the kinase under study. We anticipated using a peptide, which targets the protein-binding region, as a framework upon which the ATP-blocking site moiety could be created. Three parent peptides (Ac-Ala-Arg-Arg-Gly-Ala-Leu-Arg-Gln-Ala-HNACHTUNGTRENNUNG (CH2) 2SH; Ac-Ala-Arg-Arg-Gly-Dap (Ac)-Leu-Arg-Gln-Ala-HNACHTUNGTRENNUNG (CH2) 2SH; Ac-Ala-Arg-Arg-Gly-Ala-Leu-Arg-Dap (Ac)-Ala-HNACHTUNGTRENNUNG (CH2) 2SH) were prepared so that, upon subsequent combinatorial expansion, a bisubstrate-like species could be identified by utilizing an appropriate screening protocol. The amino acid ACHTUNGTRENNUNGsequence contained in these parent peptides was based on known Akt preferences.[4] Detailed analysis of one of these peptides (1) revealed competitive behavior versus ATP (Supporting Information; Scheme 1). However, given the absence of aromatic moieties, it is unlikely that 1 significantly encroaches on the ATP binding region (thereby providing a potential opportunity to identify new functional groups that block ATP binding). Although the three-dimensional structure of Akt has been solved [5] it was simply not clear which site on the peptide might be responsible for the observed competitive inhibition pattern versus ATP. Nonetheless, based on known Akt protein binding site recognition preferences, we ruled out certain residues (vide infra) as likely culprits and chose three sites (highlighted with arrows) upon which to focus our attention. Although peptide 1 represented an exciting starting point, it is an extraordinarily poor Akt inhibitor, exhibiting a Ki value of 3.2 Æ1. 1 mM. Consequently, we required a synthetic strategy that would allow us to simultaneously evolve this derivative into a more potent inhibitor while preserving bisubstrate inhibitory behavior.We initially examined whether the N terminus acts …
