Design, synthesis, biological activity and structural analysis of cyclic peptide inhibitors targeting the substrate recruitment site of cyclin-dependent kinase complexes

Abstract

Inhibition of cyclin A- and cyclin E-associated cyclin-dependent kinase-2 (CDK2) activities is an effective way of selective induction of apoptotic cell death via the E2F pathway in tumour cells. The cyclin groove recognition motif (CRM) in the natural CDK-inhibitory (CDKI) tumour suppressor protein p27KIP1 was used as the basis for the design and synthesis of a series of cyclic peptides whose biological activity and structural characterisation by NMR and X-ray crystallography is reported. Whereas linear p27KIP1 sequence peptides were comparatively ineffective, introduction of side chain-to-tail constraints was found to be productive. An optimal macrocyclic ring size for the conformational constraint was determined, mimicking the intramolecular H-bonding system of p27. Molecular dynamics calculations of various macrocycles suggested a close correlation between ring flexibility and biological activity. Truncated inhibitor peptide analogues also confirmed the hypothesis that introduction of a cyclic conformational constraint is favourable in terms of affinity and potency. The structural basis for the potency increase in cyclic versus linear peptides was demonstrated through the determination and interpretation of X-ray crystal structures of complexes between CDK2/cylin A (CDK2A) and a constrained pentapeptide.

Identification of an Hexapeptide That Binds to a Surface Pocket in Cyclin A and Inhibits the Catalytic Activity of the Complex Cyclin-dependent Kinase 2-Cyclin A

“…A number of groups have reported the identification of inhibitors of cdk2-cyclin A through the design of molecules that bind to the CRM (20,32,33). As stated above the goal of this study was to identify inhibitors of cdk2-cyclin A complexes but not targeting the ATP binding site nor the CRM.…”

“…To this aim we screened combinatorial libraries in the appropriate experimental conditions, thus we adjusted the ATP concentration to be high enough to difficult ATP competition while minimizing the use of [␥-32 P]ATP. As substrate we used histone H1 that is phosphorylated directly and without the required binding to the cyclin A CRM that have other substrates as pRb and E2F protein families (32).…”

Protein Based Drug Discovery

“…The cyclin groove recognition motif (CRM) in the natural CDKinhibitory (CDKI) tumour suppressor protein p27KIP1 was used as the basis for the design and synthesis of a series of cyclic peptides whose biological activity and structural characterisation by NMR and X-ray crystallography is reported. The structural basis for the potency increase in cyclic versus linear peptides was demonstrated through the determination and interpretation of X-ray crystal structures of complexes between CDK2/cylin A (CDK2A) and a constrained pentapeptide [50].…”

Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27KIP1-derived peptidomimetic inhibitors

“…Therefore, interruption of protein-protein interaction (PPI) between CDK2 complex and regulator proteins leads to the structural modification and affects the enzyme association range. Disrupting the conserved cyclin-binding groove (CBG) of cyclin A is one of the possible ways to specifically inactivate the G1 to S phase transition of CDK2 complex [2]. Designing of peptide-based inhibitors that target the CBG site is a revolutionary approach to block the malignant cellular proliferation [9,26,27].…”

“…The most conserved cyclin groove recognition motif (CRM) of p27 has "Leu-Phe-Gly" (LFG) residues that involve the primary anchoring at Ile213, Leu214, Trp217, Arg250, Leu253, and Gln254 residues of cyclin A [18]. The studies on CDK2 enzyme inhibition illustrated the CRM region-derived acetyl-capped Arg-Lys-Leu-Phe-Gly (penta peptide, RKLFG) sequence which significantly restricts the CDK2 complex activity even at low micromolar range [2]. But, the peptide inhibitors have two main obstacles: (i) the intracellular protein-degrading enzymes easily degrade the peptides before target binding and (ii) they slow down the lipid bilayer penetration of the cell membrane [15].…”