Molecular dynamics simulations on the inhibition of Cyclin-Dependent Kinases 2 and 5 in the presence of activators

Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.     

Structural determinants of CDK4 inhibition and design of selective ATP competitive inhibitors

A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.     

Selective ATP competitive leads of CDK4: Discovery by 3D-QSAR pharmacophore mapping and molecular docking approach

The discovery of ATP competitive CDK4 inhibitors is an on-going challenging task in cancer therapy. Here, an attempt has been made to develop new leads targeting ATP binding site of CDK4 by applying 3D-QSAR pharmacophore mapping and molecular docking methods The outcome of 6 leads offers a significant contribution for selective CDK4 inhibition, since they show potential binding interactions with Val⁹⁶, Arg¹⁰¹, and Glu¹⁴⁴ residues of CDK4, that are unique and from other kinases. It is worth noting that there is a striking similarity in binding interactions of the leads and known CDK4 inhibitors, namely Abemaciclib, Palbociclib and Ribociclib. Further key features, including high dock score value, good predicted activity, scaffold diversity, and the acceptable ADME profile of leads, provide a great opportunity for the development of highly potent and selective ATP competitive inhibitors of CDK4.     

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

Summary Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.     

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein–ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein–ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.     

CDK9 inhibitors push cancer cells over the edge

The trouble with CDK active-site inhibitors is their tendency to have off-target effects. This is not surprising, as the ATP binding sites of most protein kinases are very similar. Wang et?al. (2010) have used some clever screening approaches to identify selective CDK9 inhibitors that drive cancer cells into apoptosis.     

Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent

BACKGROUND: Over 2000 protein kinases regulate cellular functions. Screening for inhibitors of some of these kinases has already yielded some potent and selective compounds with promising potential for the treatment of human diseases. RESULTS: The marine sponge constituent hymenialdisine is a potent inhibitor of cyclin-dependent kinases, glycogen synthase kinase-3beta and casein kinase 1. Hymenialdisine competes with ATP for binding to these kinases. A CDK2-hymenialdisine complex crystal structure shows that three hydrogen bonds link hymenialdisine to the Glu81 and Leu83 residues of CDK2, as observed with other inhibitors. Hymenialdisine inhibits CDK5/p35 in vivo as demonstrated by the lack of phosphorylation/down-regulation of Pak1 kinase in E18 rat cortical neurons, and also inhibits GSK-3 in vivo as shown by the inhibition of MAP-1B phosphorylation. Hymenialdisine also blocks the in vivo phosphorylation of the microtubule-binding protein tau at sites that are hyperphosphorylated by GSK-3 and CDK5/p35 in Alzheimer's disease (cross-reacting with Alzheimer's-specific AT100 antibodies). CONCLUSIONS: The natural product hymenialdisine is a new kinase inhibitor with promising potential applications for treating neurodegenerative disorders.     

Development of Dual and Selective Degraders of Cyclin‐Dependent Kinases 4 and 6

Cyclin‐dependent kinases 4 and 6 (CDK4/6) are key regulators of the cell cycle, and there are FDA‐approved CDK4/6 inhibitors for treating patients with metastatic breast cancer. However, due to conservation of their ATP‐binding sites, development of selective agents has remained elusive. Here, we report imide‐based degrader molecules capable of degrading both CDK4/6, or selectively degrading either CDK4 or CDK6. We were also able to tune the activity of these molecules against Ikaros (IKZF1) and Aiolos (IKZF3), which are well‐established targets of imide‐based degraders. We found that in mantle cell lymphoma cell lines, combined IKZF1/3 degradation with dual CDK4/6 degradation produced enhanced anti‐proliferative effects compared to CDK4/6 inhibition, CDK4/6 degradation, or IKZF1/3 degradation. In summary, we report here the first compounds capable of inducing selective degradation of CDK4 and CDK6 as tools to pharmacologically dissect their distinct biological functions.     

Differential binding of inhibitors to active and inactive CDK2 provides insights for drug design

The cyclin-dependent kinases (CDKs) have been characterized in complex with a variety of inhibitors, but the majority of structures solved are in the inactive form. We have solved the structures of six inhibitors in both the monomeric CDK2 and binary CDK2/cyclinA complexes and demonstrate that significant differences in ligand binding occur depending on the activation state. The binding mode of two ligands in particular varies substantially in active and inactive CDK2. Furthermore, energetic analysis of CDK2/cyclin/inhibitors demonstrates that a good correlation exists between the in vitro potency and the calculated energies of interaction, but no such relationship exists for CDK2/inhibitor structures. These results confirm that monomeric CDK2 ligand complexes do not fully reflect active conformations, revealing significant implications for inhibitor design while also suggesting that the monomeric CDK2 conformation can be selectively inhibited.     

3D-QSAR和分子对接研究吲哚咔唑类细胞周期蛋白激酶抑制剂的选择性

细胞周期蛋白激酶(cyclin-dependent kinases, CDKs)是近年来治疗肿瘤的重要靶标. 由于大多数激酶ATP结合位点的保守性, CDK选择性激酶抑制剂的开发成为当前的研发难点和热点. 针对吲哚咔唑类CDK抑制剂, 我们采用比较分子力场分析方法(CoMFA)建立了CDK2-QSAR(quantitative structure-activity relationship)和CDK4-QSSR(quantitative structure-selectivity relationship)模型. 所建模型的交叉验证系数q2分别为0.722和0.703; 非交叉验证系数r2分别为0.977和0.946, 表明其具有较好的预测能力. 同时, 用分子对接的方法分析了这类化合物与CDK4同源模建结构的作用模式, 根据这两个模型发现, 吲哚咔唑类化合物的R5和R6位长链取代对CDK4的选择性具有一定的影响, 而且结合其作用模式比较合理地解释了这类抑制剂的选择性原因, 这对CDKs的选择性研究具有一定的指导意义.     

New scoring functions for virtual screening from molecular dynamics simulations with a quantum-refined force-field (QRFF-MD). Application to cyclin-dependent kinase 2

A recently introduced new methodology based on ultrashort (50-100 ps) molecular dynamics simulations with a quantum-refined force-field (QRFF-MD) is here evaluated in its ability both to predict protein-ligand binding affinities and to discriminate active compounds from inactive ones. Physically based scoring functions are derived from this approach, and their performance is compared to that of several standard knowledge-based scoring functions. About 40 inhibitors of cyclin-dependent kinase 2 (CDK2) representing a broad chemical diversity were considered. The QRFF-MD method achieves a correlation coefficient, R(2), of 0.55, which is significantly better than that obtained by a number of traditional approaches in virtual screening but only slightly better than that obtained by consensus scoring (R(2) = 0.50). Compounds from the Available Chemical Directory, along with the known active compounds, were docked into the ATP binding site of CDK2 using the program Glide, and the 650 ligands from the top scored poses were considered for a QRFF-MD analysis. Combined with structural information extracted from the simulations, the QRFF-MD methodology results in similar enrichment of known actives compared to consensus scoring. Moreover, a new scoring function is introduced that combines a QRFF-MD based scoring function with consensus scoring, which results in substantial improvement on the enrichment profile.     

Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors

Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins.     

Computational Analysis for Residue-Speci c CDK2-Inhibitor Bindings

Cyclin-dependent kinase 2 (CDK2) is a key macromolecule in cell cycle regulation. In cancer cells, CDK2 is often overexpressed and its inhibition is an effective therapy of many cancers including breast carcinomas, leukemia, and lymphomas. Quantitative characterization of the interactions between CDK2 and its inhibitors at atomic level may provide a deep understanding of protein-inhibitor interactions and clues for more effective drug discovery. In this study, we have used the computational alanine scanning approach in combination with an efficient interaction entropy method to study the microscopic mechanism of binding between CDK2 and its 13 inhibitors. The total binding free energy from the method shows a correlation of 0.76?0.83 with the experimental values. The free energy component reveals two binding mode in the 13 complexes, namely van der Waals dominant, and electrostatic dominant. Decomposition of the total energy to per-residue contribution allows us to identify five hydrophobic residues as hot spots during the binding. Residues that are responsible for determining the strength of the binding were also analyzed.     

Molecular Docking and Design of Novel Heterodimers of Donepezil and Huperzine Fragments as Acetylcholinesterase Inhibitors

To provide hints for the design of new acetylcholinesterase(ACh E) inhibitors with higher potency and specificity, the binding modes of novel heterodimers comprised of donepezil and huperzine A fragments with ACh E were explored by employing the docking simulations. The results show that the binding mode of S-17b(the most potent inhibitor in Ref. 2, i.e., Bioorg. Med. Chem. 2013, 21, 676-683) is clearly different from that of donepezil, while the binding modes of other heterodimers in Ref. 2 are the same as that of donepezil. In addition, based on the binding mode and structure modification of S-17 b, two novel inhibitors(S-17b1 and S-17bb1) with much higher inhibitory potency than S-17 b were obtained. Our design strategy was to replace the hupyridone moiety of S-17 b with the bulky group, and to replace the dimethoxyindanone moiety of S-17 b with more hydrophobic and bulky group with a highly positive charge, which would result in generating potent and selective AChE inhibitors.     

Cyclin-Dependent Kinase 4 and 6 Inhibitors in Cell Cycle Dysregulation for Breast Cancer Treatment

In cell development, the cell cycle is crucial, and the cycle progression’s main controllers are endogenous CDK inhibitors, cyclin-dependent kinases (CDKs), and cyclins. In response to the mitogenic signal, cyclin D is produced and retinoblastoma protein (Rb) is phosphorylated due to activated CDK4/CDK6. This causes various proteins required in the cell cycle progression to be generated. In addition, complexes of CDK1-cyclin A/B, CDK2-cyclin E/A, and CDK4/CDK6-cyclin D are required in each phase of this progression. Cell cycle dysregulation has the ability to lead to cancer. Based on its role in the cell cycle, CDK has become a natural target of anticancer therapy. Therefore, understanding the CDK structures and the complex formed with the drug, helps to foster the development of CDK inhibitors. This development starts from non-selective CDK inhibitors to selective CDK4/CDK6 inhibitors, and these have been applied in clinical cancer treatment. However, these inhibitors currently require further development for various hematologic malignancies and solid tumors, based on the results demonstrated. In drug development, the main strategy is primarily to prevent and asphyxiate drug resistance, thus a determination of specific biomarkers is required to increase the therapy’s effectiveness as well as patient selection suitability in order to avoid therapy failure. This review is expected to serve as a reference for early and advanced-stage researchers in designing new molecules or repurposing existing molecules as CDK4/CDK6 inhibitors to treat breast cancer.     

CDK5-dependent inhibitory phosphorylation of Drp1 during neuronal maturation

Mitochondrial functions are essential for the survival and function of neurons. Recently, it has been demonstrated that mitochondrial functions are highly associated with mitochondrial morphology, which is dynamically changed by the balance between fusion and fission. Mitochondrial morphology is primarily controlled by the activation of dynamin-related proteins including dynamin-related protein 1 (Drp1), which promotes mitochondrial fission. Drp1 activity is regulated by several post-translational modifications, thereby modifying mitochondrial morphology. Here, we found that phosphorylation of Drp1 at serine 616 (S616) is mediated by cyclin-dependent kinase 5 (CDK5) in post-mitotic rat neurons. Perturbation of CDK5 activity modified the level of Drp1^S616 phosphorylation and mitochondrial morphology in neurons. In addition, phosphorylated Drp1^S616 preferentially localized as a cytosolic monomer compared with total Drp1. Furthermore, roscovitine, a chemical inhibitor of CDKs, increased oligomerization and mitochondrial translocation of Drp1, suggesting that CDK5-dependent phosphorylation of Drp1 serves to reduce Drp1''s fission-promoting activity. Taken together, we propose that CDK5 has a significant role in the regulation of mitochondrial morphology via inhibitory phosphorylation of Drp1^S616 in post-mitotic neurons.     

Virtual screening studies to design potent CDK2-cyclin A inhibitors

The cell division cycle is controlled by cyclin-dependent kinases (CDK), which consist of a catalytic subunit (CDK1-CDK8) and a regulatory subunit (cyclin A-H). Pharmacophore analysis indicates that the best inhibitor model consists of (1) two hydrogen bond acceptors, (2) one hydrogen bond donor, and (3) one hydrophobic feature. The HypoRefine pharmacophore model gave an enrichment factor of 1.31 and goodness of fit score of 0.76. Docking studies were carried out to explore the structural requirements for the CDK2-cyclin A inhibitors and to construct highly predictive models for the design of new inhibitors. Docking studies demonstrate the important role of hydrogen bond and hydrophobic interactions in determining the inhibitor-receptor binding affinity. The validated pharmacophore model is further used for retrieving the most active hits/lead from a virtual library of molecules. Subsequently, docking studies were performed on the hits, and novel series of potent leads were suggested based on the interaction energy between CDK2-cyclin A and the putative inhibitors.