Identification of 4-(4-Aminopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidines as Selective Inhibitors of Protein Kinase B through Fragment Elaboration

Abstract

Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3- d]pyrimidine inhibitors of PKBbeta with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.

Measuring and interpreting the selectivity of protein kinase inhibitors

“…[ATP] in cells is typically estimated at 1-5 mM the kinase binding site in cells should be achieved with a given inhibitor [6,27]. However, it is important to note that this assumes no limits to cell permeability of the compound, which may further reduce the potency achieved in cellular assays.…”

“…Moreover, although the occupancy of the active site may reflect engagement of the inhibitor with the target enzyme as measured, for example, in a binding assay or a direct measurement of an immediate downstream phosphorylation by the kinase, it does not guarantee the same response in a broader phenotypic assay, e.g. growth inhibition, which depends also on the sensitivity to perturbation of the signalling network in which the kinase participates [27]. Not all kinases can be used in biochemical assays in their full-length form and may require removal of transmembrane or regulatory domains.…”

“…For example, a single amino acid difference was found to be the major determinant of selectivity for Akt versus PKA (www.cellsignal.com). With sponsorship by Cell Signaling Technology and Sugen, the figure was originally presented as a poster in Science to accompany the first analysis of the complete human kinome [100] in two series of Akt inhibitors [27,45]. A library of fragments has been screened against 10 kinases to investigate if these very low molecular weight compounds show intrinsic specificity [1].…”

Diversity of Bisubstrate Binding Modes of Adenosine Analogue–Oligoarginine Conjugates in Protein Kinase A and Implications for Protein Substrate Interactions

“…The structure of ARC-670 is based on ARC-902 (Table 1) and distinct from that of ARC-1012 and ARC-1039, regarding both the substratemimicking moiety and the linker. The adenosine analogue in the ATP competitive head group of ARC-902 was replaced by a purine-piperazine pair, 19 and an octanedioic acid moiety links the piperazine to six D-arginine residues ( Fig. 2 and Table 1) instead of 6-aminohexanoic acid in ARC-902.…”

Design, synthesis and biological characterization of selective LIMK inhibitors

“…Kinase hinge-binding motifs were introduced to the piperidine derivatives through S N Ar reaction of aryl chloride which occurred selectively on the piperidine nitrogen atom. 21 When the aryl groups were not introduced at the last step of the synthesis, isopropanol was replaced by DMSO/water as solvent, providing better solubility of the starting materials, allowing higher temperature and thereby accelerating the reaction. Moreover, hydrolysis of the methyl ester occurred in situ under these conditions, reducing the number of steps for some of the compounds.…”