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Irreversible inhibitors of c-Src kinase that target a nonconserved cysteine.

ABSTRACT: We have developed the first irreversible inhibitors of wild-type c-Src kinase. We demonstrate that our irreversible inhibitors display improved potency and selectivity relative to that of their reversible counterparts. Our strategy involves modifying a promiscuous kinase inhibitor with an electrophile to generate covalent inhibitors of c-Src. We applied this methodology to two inhibitor scaffolds that exhibit increased cellular efficacy when rendered irreversible. In addition, we have demonstrated the utility of irreversible inhibitors in studying the conformation of an important loop in kinases that can control inhibitor selectivity and cause drug resistance. Together, we have developed a general and robust framework for generating selective irreversible inhibitors from reversible, promiscuous inhibitor scaffolds.

SUBMITTER: Kwarcinski FE

PROVIDER: S-EPMC3500393 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Irreversible inhibitors of c-Src kinase that target a nonconserved cysteine.

Kwarcinski Frank E FE Fox Christel C CC Steffey Michael E ME Soellner Matthew B MB

ACS chemical biology 20120905 11

We have developed the first irreversible inhibitors of wild-type c-Src kinase. We demonstrate that our irreversible inhibitors display improved potency and selectivity relative to that of their reversible counterparts. Our strategy involves modifying a promiscuous kinase inhibitor with an electrophile to generate covalent inhibitors of c-Src. We applied this methodology to two inhibitor scaffolds that exhibit increased cellular efficacy when rendered irreversible. In addition, we have demonstrat ...[more]

PMID: 22928736

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Project description:Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 ?M (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 ?M) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 ?M (Nek2); GI50 (SKBR3) 2.2 ?M] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 ?M concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 ?M and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

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Irreversible inhibitors of c-Src kinase that target a nonconserved cysteine.

Publications

Irreversible inhibitors of c-Src kinase that target a nonconserved cysteine.

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