Iterative Conversion of Cyclin Binding Groove Peptides into Druglike CDK Inhibitors with Antitumor Activity

Abstract

The cyclin groove is an important recognition site for substrates of the cell cycle cyclin dependent kinases and provides an opportunity for highly selective inhibition of kinase activity through a non-ATP competitive mechanism. The key peptide residues of the cyclin binding motif have been studied in order to precisely define the structure–activity relationship for CDK kinase inhibition. Through this information, new insights into the interactions of peptide CDK inhibitors with key subsites of the cyclin binding groove provide for the replacement of binding determinants with more druglike functionality through REPLACE, a strategy for the iterative conversion of peptidic blockers of protein–protein interactions into pharmaceutically relevant compounds. As a result, REPLACE is further exemplified in combining optimized peptidic sequences with effective N-terminal capping groups to generate more stable compounds possessing antitumor activity consistent with on-target inhibition of cell cycle CDKs. The compounds described here represent prototypes for a next generation of kinase therapeutics with high efficacy and kinome selectivity, thus avoiding problems observed with first generation CDK inhibitors.

Benzamide capped peptidomimetics as non-ATP competitive inhibitors of CDK2 using the REPLACE strategy

“…Phenyl-(1,2,4)-triazole derived capping groups successfully interface with the hydrophobic subsite however do not make effective contacts with the arginine binding site [12, 14] and benzoic acid derived cyclin groove inhibitors do not interact with the minor hydrophobic pocket [17]. Based on these precedents, the design and synthesis of a unique series of benzamide fragment alternatives was undertaken (Table 1).…”

“…Based on these precedents, the design and synthesis of a unique series of benzamide fragment alternatives was undertaken (Table 1). Since this scaffold has considerable potential to further exploit peptide interacting residues and therefore more completely mimic the peptide-cyclin interface compared to previously investigated N-terminal capping groups[12–14], expansion of this as a core structure was investigated through additional substitutions. In the first instance, a number of piperazine containing functional groups (BA1 scaffold, Table 1, Supplementary information, SI) were installed at the 4 position by reductive amination of 4-formyl methyl benzoate with the appropriate cyclic amine to form the secondary amine.…”

“…After generation of the capping groups, FLIP molecules (Table 1) were synthesized using previously described methods[12] (also see SI) and evaluated in a fluorescence polarization competitive binding assay shown to be a robust measurement of the ability of compounds to bind to the cyclin groove [12] (also see SI) and therefore inhibit CDK activity through blocking of substrate recruitment. Compounds containing the benzamide scaffold (Table 1) were potent mimics of the N-terminal tetrapeptide including 1, possessing a guanidinomethyl group at the 4-position which was the most effective inhibitor (CDK2/cyclin A 0.69 μM and CDK4/cyclin D1 - 15.32 μM).…”

“…Methylation of the piperazine N in compound 6 resulted in a 2 fold increase in binding to CDK2A however did not affect interaction with CDK4D. Subsequent capping group modifications were investigated in either the Arg-βhomoLeu-N-MePhe-NH 2 or Arg-βhomoLeu-3-thienylalanine(3TA)-NH 2 contexts previously utilized to improve proteolytic stability and cellular permeability[12]. These sequences have comparable potency in vitro binding as the native p21 sequence, (RRLIF) to cyclin A even with one less amino acid residue.…”

“…The previously optimized octapeptide inhibitor, HAKRRLIF based on a sequence found in the endogenous CDK inhibitor, p21 waf1 binds potently to the cyclin groove through multiple peptide determinants including ion pairing interactions of the KRR motif and through the hydrophobic side chains with primary and secondary lipophilic subsites present[10, 11]. Previous studies applying the REPLACE strategy[9, 12–16] have identified capping groups replacing the N-terminal tetrapeptide that interact with either the secondary hydrophobic pocket or the critical arginine binding site but not effectively with both[12–14, 16]. From this perspective, an alternative scaffold for presenting binding groups that could interact with both regions was sought and a substituted benzoic acid derivatives were generated and appended to a drug-like peptidomimetic sequence previously identified.…”