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Development of paramagnetic probes for molecular recognition studies in protein kinases.

ABSTRACT: We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.

SUBMITTER: Vazquez J

PROVIDER: S-EPMC2825083 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Development of paramagnetic probes for molecular recognition studies in protein kinases.

Vazquez Jesus J De Surya K SK Chen Li-Hsing LH Riel-Mehan Megan M Emdadi Aras A Cellitti Jason J Stebbins John L JL Rega Michele F MF Pellecchia Maurizio M

Journal of medicinal chemistry 20080601 12

We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the re ...[more]

PMID: 18494454

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Project description:Protein kinases are key enzymes that catalyze the covalent phosphorylation of substrates via the transfer of the γ-phosphate of ATP, playing a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. Due to their pivotal cellular role, the aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Decades of intense development of protein kinase inhibitors (PKIs) resulted in 71 FDA-approved PKI drugs that target dozens of protein kinases for the treatment of various diseases. How do FDA-approved protein kinase inhibitor PKI drugs compete with ATP in their own binding pocket? This is the central question we attempt to address in this work. Based on modes of non-bonded interactions and their calculated interaction strengths by means of the advanced double hybrid DFT method B2PLYP, the molecular recognition of PKI drugs in the ATP-binding pockets was systematically analyzed. It was found that (1) all the FDA-approved PKI drugs studied here form one or more hydrogen bond(s) with the backbone amide N, O atoms in the hinge region of the ATP binding site, mimicking the adenine base; (2) all the FDA-approved PKI drugs feature two or more aromatic rings. The latter reach far and deep into the hydrophobic regions I and II, forming multiple CH-π interactions with aliphatic residues L(3), V(11), A(15), V(36), G(51), L(77) and π-π stacking interactions with aromatic residues F(47) and F(82), but ATP itself does not utilize these regions extensively; (3) all FDA-approved PKI drugs studied here have one thing in common, i.e., they frequently formed non-bonded interactions with a total of 12 residues L(3),V(11), A(15), K(17), E(24),V(36),T(45), F(47), G(51), L(77), D(81) and F(82) in the ATP binding. Many of those 12 commonly involved residues are highly conserved residues with important structural and catalytic functional roles. K(17) and E(24) are the two highly conserved residues crucial for the catalytic function of kinases. D(81) and F(82) belong to the DFG motif; T(45) was dubbed the gate keeper residue. F(47) is located on the hinge region and G(51) sits on the linker that connects the hinge to the αD-helix. It is this targeting of highly conserved residues in protein kinases that led to promiscuous PKI drugs that lack selectivity. Although the formation of hydrogen bond(s) with the backbone of the hinge gives PKI drugs the added binding affinity and the much-needed directionality, selectivity is sacrificed. That is why so many FDA-approved PKI drugs are known to have multiple targets. Moreover, off-target-mediated toxicity caused by a lack of selectivity was one of the major challenges facing the PKI drug discovery community. This work suggests a road map for future PKI drug design, i.e., targeting non-conserved residues in the ATP binding pocket to gain better selectivity so as to avoid off-target-mediated toxicity.

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Development of paramagnetic probes for molecular recognition studies in protein kinases.

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Development of paramagnetic probes for molecular recognition studies in protein kinases.

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