Fascaplysin as a specific inhibitor for CDK4: insights from molecular modelling

Abstract

Cyclin-dependent kinases (CDKs) play a key role in the cell cycle and are important anti-cancer drug targets. The natural product fascaplysin inhibits CDK4 with surprising selectivity (IC(50) = 0.4 µM) compared to the close homolog CDK2 (IC(50) = 500 µM). Free energy calculations of the positively charged fascaplysin and an uncharged iso-electronic derivative in the CDK2 and CDK4 inhibitor complexes indicate that the positive charge of fascaplysin is crucial for selectivity. This finding will guide further improvements in the design of fascaplysin-based selective inhibitors for CDK4.

Figure 1. Molecular structures of fascaplysin (FAS) and “carbofascaplysin” (CRB).

Fascaplysin (A) is a natural product from the sponge Fascaplysinopsis Bergquist. Isoelectronic substitution of the positively charged nitrogen with a carbon atom leads to an electrically neutral derivative (B), which for the ease of discussion we refer to as “carbofascaplysin”.

Figure 2. Active site conservation in CDK2, CDK4 and CDK6.

(A) Structural overlay of active site residues for CDK2 (green, PDB-ID: 1FIN), CDK4 (blue, PDB-ID: 2W96) and CDK6 (red, PDB-ID: 1G3N). (B) Corresponding sequence alignment of the active site residues, the colour scheme is as in (A).

Figure 3. Thermodynamic scheme for calculating the contribution of inhibitor charge to the free energy difference in CDK4/fascaplysin and CDK2/fascaplysin complexes.

The energetic contribution of inhibitor charge to specificity is calculated as difference from two independent steps, the transformation of the CDK4/carbofascaplysin complex to CDK4/fascaplysin and the transformation of the CDK2/carbofascaplysin complex to CDK2/fascaplysin.

Figure 4. Predicted binding modes for the fascaplysin/CDK4 and the fascaplysin/CDK2 complexes.

The predicted binding modes for CDK4 (A) and CDK2 (B) show two hydrogen bonds between NH and carbonyl groups of fascaplysin and the backbone carbonyl and NH of Val96 in CDK4 and of Leu83 in CDK2, respectively. In the CDK4/fascaplysin binding mode an additional hydrogen bond between the Nδ-H of the His95^CDK4 imidazole side chain and fascaplysin is possible.

Figure 5. 5 ns MD simulations for CDK4 and CDK2 complexed with fascaplysin and carbofascaplysin.

(A) Comparison of backbone rmsd (relative to the energy minimised starting structure) as a function of time for the CDK4/fascaplysin (red) and CDK4/carbofascaplysin (black) simulations (B) Comparison of rmsd (relative to the energy minimised starting structure) for the CDK2/fascaplysin (red) and CDK2/carbofascaplysin (black) simulations. (C) Cα RMSF values for the simulations shown in (A). The peak for residues 42–48 corresponds to a flexible poly-glycine region not present in CDK2 (D) Cα RMSF values for the in the simulations shown in (C).

Figure 6. Binding of fascaplysin and carbofascaplyin in CDK4 and CDK2.

(A) Distances between the carbonyl oxygen (black), and amide hydrogen (red) of V96^CDK4 and the indoyl hydrogen and carbonyl oxygen of FAS, respectively. (B) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of V96^CDK4 and the indoyl hydrogen and carbonyl oxygen of CRB, respectively. (C) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of L83^CDK2 and the indoyl hydrogen and carbonyl oxygen of FAS, respectively. (D) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of L83^CDK2 and the indoyl hydrogen and carbonyl oxygen of CRB, respectively. (E) Distances between the His95^CDK4 Nδ-H and the carbonyl oxygen of FAS (red) and CRB (black), respectively. (F) Distances between Lys89^CDK2 amine-N and the N+/C of FAS (red) and CRB (black), respectively.

Figure 7. Thermodynamic Integration.

Each point (200 ps window) represents the free energy “cost” of the carbofascaplysin to fascaplysin transformation in the CDK4 and CDK2 complex calculated from 19 values for λ. The difference (ΔΔG⁰) between the two plots quantifies the energetic contribution for selectivity that can be attributed to the positive fascaplysin charge.