Molecular Basis of Differential Selectivity of Cyclobutyl-Substituted Imidazole Inhibitors against CDKs: Insights for Rational Drug Design

<div><p>Cyclin-dependent kinases (CDKs) belong to the CMGC subfamily of protein kinases and play crucial roles in eukaryotic cell division cycle. At least seven different CDKs have been reported to be implicated in the cell cycle regulation in vertebrates. These CDKs are highly homologous and contain a conserved catalytic core. This makes the design of inhibitors specific for a particular CDK difficult. There is, however, growing need for CDK5 specific inhibitors to treat various neurodegenerative diseases. Recently, cis-substituted cyclobutyl-4-aminoimidazole inhibitors have been identified as potent CDK5 inhibitors that gave up to 30-fold selectivity over CDK2. Available IC₅₀ values also indicate a higher potency of this class of inhibitors over commercially available drugs, such as roscovitine. To understand the molecular basis of higher potency and selectivity of these inhibitors, here, we present molecular dynamics simulation results of CDK5/p25 and CDK2/CyclinE complexed with a series of cyclobutyl-substituted imidazole inhibitors and roscovitine. The atomic details of the stereospecificity and selectivity of these inhibitors are obtained from energetics and binding characteristics to the CDK binding pocket. The study not only complements the experimental findings, but also provides a wealth of detailed information that could help the structure-based drug designing processes.</p></div

DataSheet1_Evaluating the Spike–hACE2 interactions in the wild type and variants of concern of SARS -CoV-2 at different temperatures.docx

The effect of temperature on SARS-CoV-2 is frequently debated upon. There is evidence of temperature sensitivity of the viral proteins; however, how heat influences the protein–protein interaction between a SARS-CoV-2 protein and the human angiotensin-converting enzyme 2 (ACE2) receptor remains to be elucidated. Here, we studied the receptor-binding domain of the surface glycoprotein of SARS-CoV-2 wild type and variants of concern bound to the human ACE2 receptor at different temperatures through atomistic simulations. We found that although there were no major conformation changes in the protein complexes at high temperatures, the dynamics of the proteins significantly increased. There was loss of protein–protein contacts and interaction energies. Thus, the protein–protein interaction was found to be rather strong. This study would be useful for viral protein studies and the design of peptide-based vaccines and therapeutics.</p

Average solvent accessible surface area (SASA) of the substrate binding pocket of CDKs.

<p>SASA is calculated by removing the cis-N-acetyl inhibitor from the pocket and rolling a probe of radius 1.4 Å across the pocket.</p

Interaction energy of CDK5 with cis-N-acetyl (red) and roscovitine (blue).

<p>Residue-level decomposition of the total energy is also included.</p

Superimposed structures of cis-N-acetyl and roscovitine bound CDK complexes:

<p>(A) CDK2 (B) CDK5. In roscovitine-CDK complexes, the drug and protein residues are shown in pink and grey, respectively. Remaining color scheme is similar to Fig. 3.</p

Interaction energies between CDKs and cis-OH/cis-N-acetyl inhibitors.

<p>(A) CDK2 bound with cis-OH (green) and cis-N-acetyl (red); (B) similar CDK5 complexes. Residue-level decomposition of the total energy is also included.</p

Why Are the Truncated Cyclin Es More Effective CDK2 Activators than the Full-Length Isoforms?

Cell cycle regulating enzymes, CDKs, become activated upon association with their regulatory proteins, cyclins. The G1 cyclin, cyclin E, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. <i>In vivo</i> and <i>in vitro</i> studies have shown that these LMW isoforms of cyclin E hyperactivate CDK2 and accelerate the G1-S phase of cell division. The molecular basis of CDK2 hyperactivation due to LMW cyclin E isoforms in cancer cells is, however, unknown. Here, we employ a computational approach, combining homology modeling, bioinformatics analyses, molecular dynamics (MD) simulations, and principal component analyses to unravel the key structural features of CDK2-bound full-length and LMW isoforms of cyclin E1 and correlate those features to their differential activity. Results suggest that the missing N- and C-terminal regions of the cyclin E LMW isoforms constitute the Nuclear Localization Sequence (NLS) and PEST domains and are intrinsically disordered. These regions, when present in the full-length cyclin E/CDK2 complex, weaken the cyclin-CDK interface packing due to the loss of a large number of key interface interactions. Such weakening is manifested in the decreased contact area and increased solvent accessibility at the interface and also by the absence of concerted motions between the two partner proteins in the full-length complex. More effective packing and interactions between CDK2 and LMW cyclin E isoforms, however, produce more efficient protein–protein complexes that accelerate the cell division processes in cancer cells, where these cyclin E isoforms are overexpressed

Free energy of binding of cis-OH and cis-N-acetyl inhibitors to CDKs from MMPBSA calculations.

<p>All energy values are in kcal/mol and ΔΔG_Nacetyl-OH = ΔG_Nacetyl−ΔG_OH.</p

Electrostatic potential maps the substrate binding pocket of CDKs.

<p>Potential maps are generated for cis-N-acetyl bound (A) CDK2 (B) CDK5 (C) CDK2:L83C mutant, and (D) CDK2:H84D mutant. Red and blue represent electronegative and electropositive potentials, respectively. The inhibitor is also shown.</p

B-factors of CDKs bound with cis-OH (black) and trans-OH (red) inhibitors.

<p>Results are shown for (A) CDK2 and (B) CDK5 complexes. Highly fluctuating regions are labelled: (a) G-loop, (b) 40s loop (c) PSTAIRE helix, (d) 70s loop, (e) α-D helix, (f) substrate binding pocket, (g) T-loop, and (h) CMGC domain.</p