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Remarkably Stereospecific Utilization of ATP ?,?-Halomethylene Analogues by Protein Kinases.

ABSTRACT: ATP analogues containing a CXY group in place of the ?,?-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ? Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a d-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of ?,?-CHF-ATP and ?,?-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of ?,?-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to ?,?-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of ?,?-CHF-?-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes.

SUBMITTER: Ni F

PROVIDER: S-EPMC5541949 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases.

Ni Feng F Kung Alvin A Duan Yankun Y Shah Vivek V Amador Carolina D CD Guo Ming M Fan Xuegong X Chen Lin L Chen Yongheng Y McKenna Charles E CE Zhang Chao C

Journal of the American Chemical Society 20170602 23

ATP analogues containing a CXY group in place of the α,β-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a d-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of ...[more]

PMID: 28535041

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Project description:BACKGROUND:(-)-Balanol is an ATP mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is a tumour promoter, PKC isozymes act as tumour promoters or suppressors, depending on the cancer type. In particular, PKC? is frequently implicated in cancer promotion, making it a potential target for anticancer drugs. To improve isozyme selectivity of balanol, exhaustive structural and activity relationship (SAR) studies have been performed in the last two decades, but with limited success. More recently, fluorination on balanol has shown improved selectivity for PKC?, although the fluorine effect is not yet clearly understood. Understanding the origin to this fluorine-based selectivity will be valuable for designing better balanol-based ATP mimicking inhibitors. Computational approaches such as molecular dynamics (MD) simulations can decipher the fluorine effect, provided that correct charges have been assigned to a ligand. Balanol analogues have multiple ionisable functional groups and the effect of fluorine substitutions on the exact charge state of each analogue bound to PKA and to PKC? needs to be thoroughly investigated in order to design highly selective inhibitors for therapeutic applications. RESULTS:We explored the charge states of novel fluorinated balanol analogues using MD simulations. For different potential charge states of these analogues, Molecular Mechanics Generalized Born Surface Area (MMGBSA) binding energy values were computed. This study suggests that balanol and the most potent fluorinated analogue (5S fluorine substitution on the azepane ring), have charges on the azepane ring (N1), and the phenolic (C6''OH) and the carboxylate (C15''O2H) groups on the benzophenone moiety, when bound to PKC? as well as PKA. CONCLUSIONS:To the best our knowledge, this is the first study showing that the phenolate group is charged in balanol and its analogues binding to the ATP site of PKC?. Correct charge assignments of ligands are important to obtain predicted binding energy values from MD simulations that reflect experimental values. Both fluorination and the local enzymatic environment of the ATP site can influence the exact charge states of balanol analogues. Overall, this study is highly valuable for further rational design of potent balanol analogues selective to PKC?.

Dataset Information

Remarkably Stereospecific Utilization of ATP ?,?-Halomethylene Analogues by Protein Kinases.

Publications

Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases.

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