Structural Analysis of ARC-Type Inhibitor (ARC-1034) Binding to Protein Kinase A Catalytic Subunit and Rational Design of Bisubstrate Analogue Inhibitors of Basophilic Protein Kinases

Abstract

The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

Bisubstrate Inhibitors of Protein Kinases: from Principle to Practical Applications

“…[24,31] This characteristic of ARCs provides further evidence of their bisubstrate character. However, the selectivity data are mostly based on the results of single-point inhibition measurements with wide panels of PKs (up to 80) performed by commercial service suppliers; the results of these assays are hence prone to significant uncertainty and should be considered as semi-quantitative estimations.…”

“…[24] ARCA C H T U N G T R E N N U N G (III) compounds were comprehensively tested against three closely related AGC kinases, PKAc, PKB and ROCK, by measuring IC 50 values in inhibition assays and dissociation constants (K d ) in binding studies. [31] PKAc and ROCK were almost equally inhibited by the ARCA C H T U N G T R E N N U N G (III) compounds, while the affinity of the conjugates towards PKB was at least tenfold weaker. A similar tendency was also revealed for the previous generation of ARCs.…”

“…[25,26] In the case of an enzymebisubstrate inhibitor complex, the crystal structure also provides the most definitive evidence for true bisubstrate binding as it reveals whether the inhibitor occupies both substratebinding sites of the enzyme. To date, bisubstrate inhibitors have been co-crystallized with five PKs, including tyrosine PKs (the core tyrosine kinase domain of the insulin receptor, cIRK; [27] the epidermal growth factor receptor kinase domain, EGFR; [28] and Abelson tyrosine kinase, Abl [29] ), and serine/threonine PKs (CMGC group, cyclin-dependent kinase 2, pCDK2; [30] and AGC group, PKAc [31] ). In all cases, the bisubstrate conjugates incorporated an ATP mimic and a substrate peptide mimic.…”

“…In the co-crystal structure of ARC-1034 (Adc-Ahx-(d-Arg) 2 -NH 2 ) in complex with PKAc (PDB:3BWJ), the peptide substrate-mimicking fragment of the inhibitor does not occupy the presumed binding site, revealing that ARC-1034 is not a bisubstrate inhibitor, but rather a lead scaffold for the longer ARC-type conjugates. [31] In order to position the peptide-mimicking fragment in proximity to the protein/peptide substrate-binding site of the PK, elongation of the linker between the nucleosidic and peptidic moieties was necessary. The co-crystal of PKAc with these ARC-derived inhibitors proved the bisubstrate character of these novel ARC-type…”

“…[24] The only PKs outside the AGC and CAMK groups that are strongly inhibited by ARC-659-1b and ARC-1028 (Adc-Ahx-(d-Lys)-Ahx-(d-Arg) 6 -NH 2 ) are the basophilic PAK kinases from the STE group. [31,59] The isoquinoline-containing ARC-903 (H9-Hex-(d-Arg) 6 -NH 2 ) is a more general inhibitor; the residual activities of 13 out of 52 PKs were less than 10 %. This is probably due to the stronger inter- Figure 6.…”