Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

Unveiling Conformational States of CDK6 Caused by Binding of Vcyclin Protein and Inhibitor by Combining Gaussian Accelerated Molecular Dynamics and Deep Learning.

CDK6 plays a key role in the regulation of the cell cycle and is considered a crucial target for cancer therapy. In this work, conformational transitions of CDK6 were identified by using Gaussian accelerated molecular dynamics (GaMD), deep learning (DL), and free energy landscapes (FELs). DL finds that the binding pocket as well as the T-loop binding to the Vcyclin protein are involved in obvious differences of conformation contacts. This result suggests that the binding pocket of inhibitors (LQQ and AP9) and the binding interface of CDK6 to the Vcyclin protein play a key role in the function of CDK6. The analyses of FELs reveal that the binding pocket and the T-loop of CDK6 have disordered states. The results from principal component analysis (PCA) indicate that the binding of the Vcyclin protein affects the fluctuation behavior of the T-loop in CDK6. Our QM/MM-GBSA calculations suggest that the binding ability of LQQ to CDK6 is stronger than AP9 with or without the binding of the Vcyclin protein. Interaction networks of inhibitors with CDK6 were analyzed and the results reveal that LQQ contributes more hydrogen binding interactions (HBIs) and hot interaction spots with CDK6. In addition, the binding pocket endures flexibility changes from opening to closing states and the Vcyclin protein plays an important role in the stabilizing conformation of the T-loop. We anticipate that this work could provide useful information for further understanding the function of CDK6 and developing new promising inhibitors targeting CDK6.

Palbociclib and Michael-acceptor hybrid compounds as CDK4/6 covalent inhibitors: Improved potency, broad anticancer spectrum and overcoming drug resistance.

To search for potent CDK4/6 covalent inhibitors, total 14 compounds have been designed and synthesized by connecting different Michael-acceptor to the piperazine moiety of palbociclib. All the compounds displayed good antiproliferative activity against human hepatoma cell (HepG2), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231 and MCF-7) cell lines. In particular, compound A4 showed the highest inhibitory activity to MDA-MB-231 and MCF-7 cells with IC50 values of 0.51 µM and 0.48 µM, respectively. More importantly, A4 also showed strong inhibition against MDA-MB-231/palbociclib cells, indicating that A4 could effectively avoid the resistance of palbociclib. In the enzyme test, A4 showed selective inhibitory activity against CDK4/6, with the IC50 value of 18 nM and 13 nM, respectively. It was also found that A4 could efficiently induce apoptosis and arrest the cell cycle at G0/G1 phase. Moreover, A4 could significantly decrease the phosphorylation level of CDK4 and CDK6. HPLC and molecular modeling studies suggested that A4 could form a covalent bond with the target protein.

Targeting cyclin-dependent kinase 6 by vanillin inhibits proliferation of breast and lung cancer cells: Combined computational and biochemical studies.

Cyclin-dependent kinase 6 (CDK6) is a member of serine/threonine kinase family, and its overexpression is associated with cancer development. Thus, it is considered as a potential drug target for anticancer therapies. This study showed the CDK6 inhibitory potential of vanillin using combined experimental and computational methods. Structure-based docking and 200 ns molecular dynamics simulation studies revealed that the binding of vanillin stabilizes the CDK6 structure and provides mechanistic insights into the binding mechanism. Enzyme inhibition and fluorescence-binding studies showed that vanillin inhibits CDK6 with an half maximal inhibitory concentration = 4.99 µM and a binding constant (K) 4.1 × 107 M-1 . Isothermal titration calorimetry measurements further complemented our observations. Studies on human cancer cell lines (MCF-7 and A549) showed that vanillin decreases cell viability and colonization properties. The protein expression studies have further revealed that vanillin reduces the CDK6 expression and induces apoptosis in the cancer cells. In conclusion, our study presents the CDK6-mediated therapeutic implications of vanillin for anticancer therapies.

Discovery of new small-molecule cyclin-dependent kinase 6 inhibitors through computational approaches.

Excessive cell proliferation due to cell cycle disorders is one of the hallmarks of breast cancer. Cyclin-dependent kinases (CDKs), which are involved in the transition of the cell cycle from G1 phase to S phase by combining CDKs with cyclin, are considered promising targets with broad therapeutic potential based on their critical role in cell cycle regulation. Pharmacological evidence has shown that abnormal cell cycle due to the overexpression of CDK6 is responsible for the hyperproliferation of cancer cells. Blocking CDK6 expression inhibits tumour survival and growth. Therefore, CDK6 can be regarded as a potential target for anticancer therapeutics. Thus, small molecules that can be considered CDK inhibitors have been developed into promising anticancer drugs. In this study, combined structure-based and ligand-based in silicon models were created to identify new chemical entities against CDK6 with the appropriate pharmacokinetic properties. The database used to screen drug-like compounds in this thesis was based on the best E-pharmacophore hypothesis and the best ligand-based drug hypothesis. As a result, 147 common compounds were identified by further molecular docking. Surprisingly, the in vitro evaluation results of 20 of those compounds showed that the two had good CDK6 inhibitory effects. The best compound was subjected to kinase panel screening, followed by molecular dynamic simulations. The 50-ns MD studies revealed the pivotal role of VAL101 in the binding of inhibitors to CDK6. Overall, the identification of two new chemical entities with CDK6 inhibitory activity demonstrated the feasibility and potential of the new method.

A Search for Cyclin-Dependent Kinase 4/6 Inhibitors by Pharmacophore-Based Virtual Screening, Molecular Docking, and Molecular Dynamic Simulations.

The G1 phase of cell cycle progression is regulated by Cyclin-Dependent Kinase 4 (CDK4) as well as Cyclin-Dependent Kinase 6 (CDK6), and the acivities of these enzymes are regulated by the catalytic subunit, cyclin D. Cell cycle control through selective pharmacological inhibition of CDK4/6 has proven to be beneficial in the treatment of estrogen receptor-positive (ER-positive) breast cancer, particularly improving the progression-free survival of patients. Thus, targeting specific inhibition on CDK4/6 is bound to increase therapeutic efficiency. This study aimed to obtain CDK4/6 inhibitors through a pharmacophore-based virtual screening of the ZINC15 purchasable compound database using the in silico method. The pharmacophore model was designed based on the FDA-approved cdk4/6 inhibitor structures, and molecular docking was performed to further screen the hit compounds obtained. A total of eight compounds were selected based on docking results and interactions with CDK4 and CDK6, using palbociclib as the reference drug. According to the results, the compounds of ZINC585292724 and ZINC585291674 were the best compounds based on free binding energy, as well as hydrogen bond stability, and, therefore, exhibit potential as starting points in the development of CDK4/6 inhibitors.

Ellagic Acid Controls Cell Proliferation and Induces Apoptosis in Breast Cancer Cells via Inhibition of Cyclin-Dependent Kinase 6.

Cyclin-Dependent Kinase 6 (CDK6) plays an important role in cancer progression, and thus, it is considered as an attractive drug target in anticancer therapeutics. This study presents an evaluation of dietary phytochemicals, capsaicin, tocopherol, rosmarinic acid, ursolic acid, ellagic acid (EA), limonene, caffeic acid, and ferulic acid for their potential to inhibit the activity of CDK6. Molecular docking and fluorescence binding studies revealed appreciable binding affinities of these compounds to the CDK6. Among them, EA shows the highest binding affinity for CDK6, and thus a molecular dynamics simulation study of 200 ns was performed to get deeper insights into the binding mechanism and stability of the CDK6-EA complex. Fluorescence binding studies revealed that EA binds to the CDK6 with a binding constant of K = 107 M-1 and subsequently inhibits its enzyme activity with an IC50 value of 3.053 µM. Analysis of thermodynamic parameters of CDK6-EA complex formation suggested a hydrophobic interaction driven process. The treatment of EA decreases the colonization of cancer cells and induces apoptosis. Moreover, the expression of CDK6 has been downregulated in EA-treated human breast cancer cell lines. In conclusion, this study establishes EA as a potent CDK6 inhibitor that can be further evaluated in CDK6 directed anticancer therapies.

2-Methylpyridine-1-ium-1-sulfonate as an Inducer of Apoptosis and Cell Cycle Arrest: A comparative in vitro and Computational Study.

"Let food be thy medicine and thy medicine be thy food" was expressed by Hippocrates and the health benefits of medicinal plants and natural products have been considered by humans since historic times. The current study aims to investigate the anti-cancer activity of 2-Methylpyridine-1-ium-1-sulfonate (MPS) isolated from bulbs of Allium hirtifolium. The MPS compound (in a dose-dependent manner) induced arrest the AGS cells in G1 and G2/M phases, and Caco-2 cells in G1 and S phases. These findings were associated with the down-regulation of cyclin D1, CDK4, and up-regulation of p21, p27 and p53. According to the morphological observations and DNA fragmentation assay, the MPS compound induced apoptosis in both cell lines, and also cause a significant increase in the expression of Bax/Bcl-2. In this context, our molecular docking results unveiled that the MPS compound has considerable affinity to interact with the minor groove of ctDNA and also with cell cycle kinases. To approve and find the accurate MPS mode of action against cancer cell lines (especially in gastrointestinal cancer) further studies is highly recommended.

Reduced state transition barrier of CDK6 from open to closed state induced by Thr177 phosphorylation and its implication in binding modes of inhibitors.

BACKGROUND: CDK6 is considered as a highly validated anticancer drug target due to its essential role in regulating cell cycle progression at G1 restriction point. Activation of CDK6 requires the phosphorylation of Thr177 on A-loop, but the structural insights of the activation mechanism remain unclear. METHODS: Herein, all-atoms molecular dynamics (MD) simulations were used to study the effects of Thr177 phosphorylation on the dynamic structure of CDK6-Vcyclin complex. RESULTS: MD results indicated that the free energy barrier of the transition from open to closed state decreased ~47.2% after Thr177 phosphorylation. Key steps along the state transition process were obtained from a cluster analysis. Binding preference of ten different inhibitors to open or closed state were also investigated through molecular docking along with MD simulations methods. CONCLUSIONS: Our results indicated that Thr177 phosphorylation increased the flexibility around the ATP-binding pocket. The transition of the ATP-binding pocket between open and closed states should be considered for understanding the binding of CDK6 inhibitors. GENERAL SIGNIFICANCE: This work could deepen the understanding of CDKs activation mechanism, and provide useful information for the discovery of new CDKs inhibitors with high affinity and specificity.

Target fishing of glycopentalone using integrated inverse docking and reverse pharmacophore mapping approach.

Glycopentalone isolated from Glycosmis pentaphylla (family Rutaceae) has cytotoxic and apoptosis inducing effects in various human cancer cell lines; however, its mode of action is not known. Therefore, target fishing of glycopentalone using a combined approach of inverse docking and reverse pharmacophore mapping approach was used to identify potential targets of glycopentalone, and gain insight into its binding modes against the selected molecular targets, viz., CDK-2, CDK-6, Topoisomerase I, Bcl-2, VEGFR-2, Telomere:G-quadruplex and Topoisomerase II. These targets were chosen based on their key roles in the progression of cancer via regulation of cell cycle and DNA replication. Molecular docking analysis revealed that glycopentalone displayed binding energies ranging from -6.38 to -8.35 kcal/mol and inhibition constants ranging from 0.758 to 20.90 µM. Further, the binding affinities of glycopentalone to the targets were in the order: Telomere:G-quadruplex > VEGFR-2 > CDK-6 > CDK-2 > Topoisomerase II > Topoisomerase I > Bcl-2. Binding mode analysis revealed critical hydrogen bonds as well as hydrophobic interactions with the targets. The targets were validated by reverse pharmacophore mapping of glycopentalone against a set of 2241 known human target proteins which revealed CDK-2 and VEGFR-2 as the most favorable targets. The glycopentalone was well mapped to CDK-2 and VEGFR-2 which involve six pharmacophore features (two hydrophobic centers and four hydrogen bond acceptors) and nine pharmacophore features (five hydrophobic, two hydrogen bond acceptors and two hydrogen bond donors), respectively. The present computational approach may aid in rational identification of targets for small molecules against large set of candidate macromolecules before bioassays validation.

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Molecular docking and MD simulation studies of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as novel inhibitors targeted to CDK2/4/6.

PURPOSE: Nowadays, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have been approved for treating metastatic breast cancer and have achieved inspiring curative effects. But some discoveries have indicated that CDK 4/6 are not the requisite factors in some cell types because CDK2 partly compensates for the inhibition of CDK4/6. Thus, it is urgent to design CDK2/4/6 inhibitors for significantly enhancing their potency. This study aims to explore the mechanism of the binding of CDK2/4/6 kinases and their inhibitors to design novel CDK2/4/6 inhibitors for significantly enhancing their potency in different kinds of cancers. MATERIALS AND METHODS: A series of 72 disparately functionalized 4-substituted N-phenylpyrimidin-2-amine derivatives exhibiting potent inhibitor activities against CDK2, CDK4 and CDK6 were collected to apply to this research. The total set of these derivatives was divided into a training set (54 compounds) and a test set (18 compounds). The derivatives were constructed through the sketch molecule module in SYBYL 6.9 software. A Powell gradient algorithm and Tripos force field were used to calculate the minimal structural energy and the minimized structure was used as the initial conformation for molecular docking. By the means of 3D-QSAR models, partial least squares (PLS) analysis, molecular dynamics (MD) simulations and binding free energy calculations, we can find the relationship between structure and biological activity. RESULTS: In this study, we used molecular docking, 3D-QSAR and molecular dynamics simulation methods to comprehensively analyze the interaction and structure-activity relationships of 72 new CDK2/4/6 inhibitors. We used detailed statistical data to reasonably verify the constructed 3D-QSAR models for three receptors (q2 of CDK2 = 0.714, R2pred = 0.764, q2 = 0.815; R2pred of CDK4 = 0.681, q2 = 0.757; R2pred of CDK6 = 0.674). MD simulations and decomposition energy analysis validated the reasonability of the docking results and identified polar interactions as crucial factors that influence the different bioactivities of the studied inhibitors of CDK2/4/6 receptors, especially the electrostatic interactions of Lys33/35/43 and Asp145/158/163. The nonpolar interaction with Ile10/12/19 was also critical for the differing potencies of the CDK2/4/6 inhibitors. We concluded that the following probably enhanced the bioactivity against CDK2/4/6 kinases: (1) electronegative groups at the N1-position and electropositive and moderate-sized groups at ring E; (2) electrogroups featured at R2; (3) carbon atoms at the X-position or ring C replaced by a benzene ring; and (4) an electrogroup as R4. CONCLUSION: Previous studies, to our knowledge, only utilized a single approach of 3D-QSAR and did not integrate this method with other sophisticated techniques such as molecular dynamics simulations to discover new potential inhibitors of CDK2, CDK4, or CDK6. So we applied the intergenerational technology, such as 3D-QSAR technology, molecular docking simulation techniques, molecular dynamics simulations and MMPBSA19/MMGBSA20-binding free energy calculations to statistically explore the correlations between the structure with biological activities. The constructed 3D-QSAR models of the three receptors were reasonable and confirmed by the excellent statistical data. We hope the results obtained from this work will provide some useful references for the development of novel CDK2/4/6 inhibitors.

A novel p16(INK4A) mutation associated with esophageal squamous cell carcinoma in a high risk population.

This study describes identification of p16(INK4A) sequence variants and their potential association with esophageal squamous cell carcinoma (ESCC) in a high risk population from Kashmir, India. We report a novel 7 base pair exon 2 deletion in 22 out of 106 (~20%) surgically resected tumor samples. The deletion beginning at the second base of codon 103, results in a frame shift causing premature termination of the protein at codon 142, with structural and functional consequences predicted by insilico analysis. The described mutation is a previously unreported variant of p16(INK4A), perhaps representing a founder mutation unique to the population.

Molecular dynamic behavior and binding affinity of flavonoid analogues to the cyclin dependent kinase 6/cyclin D complex.

The cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FST > AGN > CHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme.

Effect of pH on the structure and function of cyclin-dependent kinase 6.

Cyclin-dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0-4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0-11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0-8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.

Naringenin as a potential inhibitor of human cyclin-dependent kinase 6: Molecular and structural insights into anti-cancer therapeutics.

Cancer is one of the major causes of global deaths and needs immediate therapeutic development. So far, several strategies have been undertaken to prevent cancer, including kinase targeting by small-molecule inhibitors. Cyclin dependent kinase 6 (CDK6) plays an essential role in cancer progression and development as its overexpression is associated with tumor development and progression. The present study demonstrated that Naringenin (NAG) binds strongly to CDK6 with a binding affinity of -7.51 kcal/mol. ATPase assay of CDK6 in the presence of NAG shows that it inhibits CDK6 with an IC50 = 3.13 µM. Fluorescence and isothermal titration calorimetry studies demonstrated that NAG binds to CDK6 with the binding constant (K) values of 3.55 × 106 M-1 and 7.06 ± 2.70 × 106 M-1, respectively. The cell-based functional studies showed that NAG decreases the cell viability of human cancer cell lines, induces apoptosis, and reduces their colonization ability. Outcomes of the present in silico and in vitro studies highlighted the significance of NAG for the development of anti-cancer leads in terms of CDK6 inhibitors and provided future implications for combinatorial anti-cancer therapies.

TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression.

TDP-43 (for TAR DNA binding protein) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in specific pre-mRNA splicing and transcription events. TDP-43 recently has been identified as the main component of cytoplasmic inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders. The cellular role of this protein remains to be identified. Here, we show that loss of TDP-43 results in dysmorphic nuclear shape, misregulation of the cell cycle, and apoptosis. Removal of TDP-43 in human cells significantly increases cyclin-dependent kinase 6 (Cdk6) protein and transcript levels. The control of Cdk6 expression mediated by TDP-43 involves GT repeats in the target gene sequence. Cdk6 up-regulation in TDP-43-depleted cells is accompanied by an increase in phosphorylation of two of its major targets, the retinoblastoma protein pRb and pRb-related protein pRb2/p130. TDP-43 silencing also is followed by changes in the expression levels of several factors that control cell proliferation. Morphological nuclear defects and increased apoptosis upon TDP-43 loss are mediated via the pRb pathway because pRb-negative cells (Saos-2) do not undergo programmed cell death or nuclear shape deformation upon TDP-43 removal. Our results identify a regulatory target of TDP-43 and show that TDP-43 depletion has important consequences in essential metabolic processes in human cells.

A bioinformatics investigation into molecular mechanism of Yinzhihuang granules for treating hepatitis B by network pharmacology and molecular docking verification.

Yinzhihuang granules (YZHG) is a patented Chinese medicine for the treatment of hepatitis B. This study aimed to investigate the intrinsic mechanisms of YZHG in the treatment of hepatitis B and to provide new evidence and insights for its clinical application. The chemical compounds of YZHG were searched in the CNKI and PUBMED databases, and their putative targets were then predicted through a search of the SuperPred and Swiss Target Prediction databases. In addition, the targets of hepatitis B were obtained from TTD, PharmGKB and DisGeNET. The abovementioned data were visualized using Cytoscape 3.7.1, and network construction identified a total of 13 potential targets of YZHG in the treatment of hepatitis B. Molecular docking verification showed that CDK6, CDK2, TP53 and BRCA1 might be strongly correlated with hepatitis B treatment. Furthermore, GO and KEGG analyses indicated that the treatment of hepatitis B by YZHG might be related to positive regulation of transcription, positive regulation of gene expression, the hepatitis B pathway and the viral carcinogenesis pathway. Network pharmacology intuitively shows the multicomponent, multitarget and multichannel pharmacological effects of YZHG in the treatment of hepatitis B and provides a scientific basis for its mechanism of action.

Discovery and SARs of 5-Chloro-N4-phenyl-N2-(pyridin-2-yl)pyrimidine-2,4-diamine Derivatives as Oral Available and Dual CDK 6 and 9 Inhibitors with Potent Antitumor Activity.

Cyclin-dependent kinases (CDKs) are promising therapeutic targets for cancer therapy. Herein, we describe our efforts toward the discovery of a series of 5-chloro-N4-phenyl-N2-(pyridin-2-yl)pyrimidine-2,4-diamine derivatives as dual CDK6 and 9 inhibitors. Intensive structural modifications lead to the identification of compound 66 as the most active dual CDK6/9 inhibitor with balancing potency against these two targets and good selectivity over CDK2. Further biological studies revealed that compound 66 was directly bound to CDK6/9, resulting in suppression of their downstream signaling pathway and inhibition of cell proliferation by blocking cell cycle progression and inducing cellular apoptosis. More importantly, compound 66 significantly inhibited tumor growth in a xenograft mouse model with no obvious toxicity, indicating the promising therapeutic potential of CDK6/9 dual inhibitors for cancer treatment. Therefore, the above results are of great importance in the development of dual CDK6/9 inhibitors for cancer therapy.

Artificial Intelligence Approach to Find Lead Compounds for Treating Tumors.

It has been demonstrated that MMP13 enzyme is related to most cancer cell tumors. The world's largest traditional Chinese medicine database was applied to screen for structure-based drug design and ligand-based drug design. To predict drug activity, machine learning models (Random Forest (RF), AdaBoost Regressor (ABR), Gradient Boosting Regressor (GBR)), and Deep Learning models were utilized to validate the Docking results, and we obtained an R2 of 0.922 on the training set and 0.804 on the test set in the RF algorithm. For the Deep Learning algorithm, R2 of the training set is 0.90, and R2 of the test set is 0.810. However, these TCM compounds fly away during the molecular dynamics (MD) simulation. We seek another method: peptide design. All peptide database were screened by the Docking process. Modification peptides were optimized the interaction modes, and the affinities were assessed with ZDOCK protocol and Refine Docked protein protocol. The 300 ns MD simulation evaluated the stability of receptor-peptide complexes. The double-site effect appeared on S2, a designed peptide based on a known inhibitor, when complexed with BCL2. S3, a designed peptide referred from endogenous inhibitor P16, competed against cyclin when binding with CDK6. The MDM2 inhibitors S5 and S6 were derived from the P53 structure and stable binding with MDM2. A flexible region of peptides S5 and S6 may enhance the binding ability by changing its own conformation, which was unforeseen. These peptides (S2, S3, S5, and S6) are potentially interesting to treat cancer; however, these findings need to be affirmed by biological testing, which will be conducted in the near future.