Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

Cyclin-dependent kinase 2 (CDK2) is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP) binding site (Site I) and two non-competitive binding sites (Site II and III). In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV). All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate). In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.     

Comparative analysis of the surface interaction properties of the binding sites of CDK2, CDK4, and ERK2

Recently developed hydrogen-bonding and hydrophobic analysis algorithms were used to investigate the interaction properties of the ATP binding sites of CDK2, CDK4, and ERK2. We were able to prioritise those hydrogen-bonding groups that are observed to bind the native ATP ligand, as well as to identify other important groups found to bind inhibitors of these enzymes. However, as the hydrogen-bonding groups in the ATP binding sites of these enzymes are fairly well-conserved, we have confirmed that inhibitor selectivity may be predominantly due to differences in either the hydrophobic or steric properties of their binding sites. In particular, the hydrophobic properties of regions outside the specificity surface were observed to provide a rationale for the differences in specificity between various inhibitors to these enzymes. Our method was thus able to identify variations in hydrophobicity. The greater hydrophobicity of certain regions of CDK4 over analogous regions in CDK2 was detectable; likewise, it was possible to distinguish variations in hydrophobicity for regions of CDK2 against those in ERK2, despite the fact that these regions are largely composed of similar residue types.     

Biomarkers of Response and Resistance to CDK4/6 Inhibitors in Breast Cancer: Hints from Liquid Biopsy and microRNA Exploration

New evidence on the impact of dysregulation of the CDK4/6 pathway on breast cancer (BC) cell proliferation has led to the development of selective CDK4/6 inhibitors, which have radically changed the management of advanced BC. Despite the improved outcomes obtained by CDK4/6 inhibitors, approximately 10% of tumors show primary resistance, whereas acquired resistance appears to be an almost ubiquitous occurrence, leading to treatment failure. The identification of differentially expressed genes or genomic mutational signatures able to predict sensitivity or resistance to CDK4/6 inhibitors is critical for medical decision making and for avoiding or counteracting primary or acquired resistance against CDK4/6 inhibitors. In this review, we summarize the main mechanisms of resistance to CDK4/6 inhibitors, focusing on those associated with potentially relevant biomarkers that could predict patients’ response/resistance to treatment. Recent advances in biomarker identification are discussed, including the potential use of liquid biopsy for BC management and the role of multiple microRNAs as molecular predictors of cancer cell sensitivity and resistance to CDK4/6 inhibitors.     

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)‐2‐Benzyl‐3‐dehydroquinic Acids

The binding mode of several substrate analogues, (2R)‐2‐benzyl‐3‐dehydroquinic acids 4 , which are potent reversible competitive inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway, has been investigated by structural and computational studies. The crystal structures of Mycobacterium tuberculosis and Helicobacter pylori DHQ2 in complex with one of the most potent inhibitor, p‐methoxybenzyl derivative 4 a , have been solved at 2.40 Å and 2.75 Å, respectively. This has allowed the resolution of the M. tuberculosis DHQ2 loop containing residues 20–25 for the first time. These structures show the key interactions of the aromatic ring in the active site of both enzymes and additionally reveal an important change in the conformation and flexibility of the loop that closes over substrate binding. The loop conformation and the binding mode of compounds 4 b – d has been also studied by molecular dynamics simulations, which suggest that the benzyl group of inhibitors 4 prevent appropriate orientation of the catalytic tyrosine of the loop for proton abstraction and disrupts its basicity.     

CDK4, CDK6/cyclin-D1 Complex Inhibition and Radiotherapy for Cancer Control: A Role for Autophagy

The expanding clinical application of CDK4- and CDK6-inhibiting drugs in the managements of breast cancer has raised a great interest in testing these drugs in other neoplasms. The potential of combining these drugs with other therapeutic approaches seems to be an interesting work-ground to explore. Even though a potential integration of CDK4 and CDK6 inhibitors with radiotherapy (RT) has been hypothesized, this kind of approach has not been sufficiently pursued, neither in preclinical nor in clinical studies. Similarly, the most recent discoveries focusing on autophagy, as a possible target pathway able to enhance the antitumor efficacy of CDK4 and CDK6 inhibitors is promising but needs more investigations. The aim of this review is to discuss the recent literature on the field in order to infer a rational combination strategy including cyclin-D1/CDK4-CDK6 inhibitors, RT, and/or other anticancer agents targeting G1-S phase cell cycle transition.     

Macrocyclic aminopyrimidines as multitarget CDK and VEGF-R inhibitors with potent antiproliferative activities

X-ray structures from CDK2-aminopyrimidine inhibitor complexes led to the idea to stabilize the active conformation of aminopyrimidine inhibitors by incorporating the recognition site into a macrocyclic framework. A modular synthesis approach that relies on a new late-stage macrocyclization protocol that enables fast and efficient synthesis of macrocyclic aminopyrimidines was developed. A set of structurally diverse derivatives was prepared. Macrocyclic aminopyrimidines were shown to be multitarget inhibitors of CDK1/2 and VEGF-RTKs. In addition, potent antiproliferative activities toward various human tumor cells and a human tumor xenograft model were demonstrated.     

Inhibitors of Cyclin-Dependent Kinases: Types and Their Mechanism of Action

Recent studies on cyclin-dependent kinase (CDK) inhibitors have revealed that small molecule drugs have become very attractive for the treatment of cancer and neurodegenerative disorders. Most CDK inhibitors have been developed to target the ATP binding pocket. However, CDK kinases possess a very similar catalytic domain and three-dimensional structure. These features make it difficult to achieve required selectivity. Therefore, inhibitors which bind outside the ATP binding site present a great interest in the biomedical field, both from the fundamental point of view and for the wide range of their potential applications. This review tries to explain whether the ATP competitive inhibitors are still an option for future research, and highlights alternative approaches to discover more selective and potent small molecule inhibitors.     

A Novel Approach to the Discovery of Small-Molecule Ligands of CDK2

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 μM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.     

Molecular Modeling Studies of 4,5-Dihydro-1H-pyrazolo[4,3-h] quinazoline Derivatives as Potent CDK2/Cyclin A Inhibitors Using 3D-QSAR and Docking

CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r(2) (cv) values of 0.747 and 0.518 and r(2) values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity.     

A 3D QSAR model of 17beta-HSD1 inhibitors based on a thieno[2,3-d]pyrimidin-4(3H)-one core applying molecular dynamics simulations and ligand-protein docking

The 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) enzyme plays a crucial role in female hormonal regulation by catalysing the NADPH-dependent reduction of the less potent estrone E1 into the biologically active estradiol E2. Because 17beta-HSD1 is a key enzyme in E2 biosynthesis, it has emerged as an attractive drug target for inhibitor development. Herein we report the plausible binding modes and a 3D QSAR model of 17beta-HSD1 inhibitors based on a (di)cycloalkenothieno[2,3-d]pyrimidin-4(3H)-one core. Two generated enzyme complexes with potent inhibitors were subjected to molecular dynamics simulation to mimic the dynamic process of inhibitor binding. A set of 17beta-HSD1 inhibitors based on the thieno[2,3-d]pyrimidin-4(3H)-one core were docked into the resulting active site, and a CoMFA model employing the most extensive training set to date was generated. The model was validated with an external test set. Active site residues involved in inhibitor binding and CoMFA fields for steric and electrostatic interactions were identified. The model will be used to guide structural modifications of 17beta-HSD1 inhibitors based on a thieno[2,3-d]pyrimidin-4(3H)-one core in order to improve the biological activity as well as in the design of novel 17beta-HSD1 inhibitors.     

Designing and Synthesis of New Isatin Derivatives as Potential CDK2 Inhibitors

Tumors are still one of the main causes of death; therefore, the search for new therapeutic agents that will enable the implementation of effective treatment is a significant challenge for modern pharmacy. One of the important factors contributing to the development of neoplastic diseases is the overexpression of enzymes responsible for the regulation of cell division processes such as cyclin-dependent kinases. Numerous studies and examples of already-developed drugs confirm that isatin is a convenient basis for the development of new groups of inhibitors for this class of enzyme. Therefore, in this work, a new group of potential inhibitors of the CDK2 enzyme, utilizing isatin derivatives and substituted benzoylhydrazines, has been designed based on the application of computational chemistry methods, such as docking and molecular dynamics, and their inhibiting ability was assessed. In the cases of the selected compounds, a synthesis method was developed, and the selected physicochemical properties of the newly synthesized derivatives were estimated. As part of the completed project, new compounds are developed which are potential inhibitors of the CDK2 enzyme.     

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.     

A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer

In recent years, three PARP inhibitors and three CDK4/6 inhibitors have been approved by the FDA for the treatment of recurrent ovarian cancer and advanced ER-positive breast cancer, respectively. However, the clinical benefits of the PARPi or CDK4/6i monotherapy are not as satisfied as expected and benefit only a fraction of patients. Current studies have shown therapeutic synergy for combinations of PARPi and CDK4/6i in breast and ovarian cancers with homologous recombination (HR) proficiency, which represents a new synthetic lethal strategy for treatment of these cancers regardless HR status. Thus, any compounds or strategies that can combine PARP and CDK4/6 inhibition will likely have great potential in improving clinic outcomes and in benefiting more patients. In this study, we developed a novel compound, ZC-22, that effectively inhibited both PARP and CDK4/6. This dual-targeting compound significantly inhibited breast and ovarian cancer cells by inducing cell cycle arrest and severe DNA damage both in vitro and in vivo. Interestingly, the efficacy of ZC-22 is even higher than the combination of PARPi Olaparib and CDK4/6i Abemaciclib in most breast and ovarian cancer cells, suggesting that it may be an effective alternative for the PARPi and CDK4/6i combination therapy. Moreover, ZC-22 sensitized breast and ovarian cancer cells to cisplatin treatment, a widely used chemotherapeutic agent. Altogether, our study has demonstrated the potency of a novel CDK4/6 and PARP dual inhibitor, which can potentially be developed into a monotherapy or combinatorial therapy with cisplatin for breast and ovarian cancer patients with HR proficiency.     

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

A monomeric variant of triosephosphate isomerase (TIM) witha new engineered binding groove has been characterized further.In this variant (ml8bTIM), the phosphate binding loop had beenshortened, causing the binding site to be much more extended.Here, it is reported that in the V233A variant of ml8bTIM (A-TIM),three important properties of the wild-type TIM active sitehave been restored: (i) the structural properties of loop-7,(ii) the binding site of a conserved water molecule betweenloop-7 and loop-8 and (iii) the binding site of the phosphatemoiety. It is shown that the active site of A-TIM can bind TIMtransition state analogs and suicide inhibitors competently.It is found that the active site geometry of the A-TIM complexesis less compact and more solvent exposed, as in wild-type TIM.This correlates with the observation that the catalytic efficiencyof A-TIM for interconverting the TIM substrates is too low tobe detected. It is also shown that the A-TIM active site canbind compounds which do not bind to wild-type TIM and whichare completely different from the normal TIM substrate, likea citrate molecule. The binding of this citrate molecule isstabilized by hydrogen bonding interactions with the new bindinggroove.     

Understanding Kinase Selectivity Through Energetic Analysis of Binding Site Waters

Kinases remain an important drug target class within the pharmaceutical industry; however, the rational design of kinase inhibitors is plagued by the complexity of gaining selectivity for a small number of proteins within a family of more than 500 related enzymes. Herein we show how a computational method for identifying the location and thermodynamic properties of water molecules within a protein binding site can yield insight into previously inexplicable selectivity and structure–activity relationships. Four kinase systems (Src family, Abl/c‐Kit, Syk/ZAP‐70, and CDK2/4) were investigated, and differences in predicted water molecule locations and energetics were able to explain the experimentally observed binding selectivity profiles. The successful predictions across the range of kinases studied here suggest that this methodology could be generally applicable for predicting selectivity profiles in related targets.     

De Novo Design,Synthesis and Evaluation of Benzylpiperazine Derivatives as Highly Selective Binders of Mcl‐1

Considerable efforts have been made to the development of small‐molecule inhibitors of antiapoptotic B‐cell lymphoma 2 (Bcl‐2) family proteins (such as Bcl‐2, Bcl‐x_L, and Mcl‐1) as a new class of anticancer therapies. Unlike general inhibitors of the entire family, selective inhibitors of each member protein can hopefully reduce the adverse side effects in chemotherapy treatments of cancers overexpressing different Bcl‐2 family proteins. In this study, we designed four series of benzylpiperazine derivatives as plausible Bcl‐2 inhibitors based on the outcomes of a computational algorithm. A total of 81 compounds were synthesized, and their binding affinities to Bcl‐2, Bcl‐x_L, and Mcl‐1 measured. Encouragingly, 22 compounds exhibited binding affinities in the micromolar range (K_i<20 μM ) to at least one target protein. Moreover, some compounds were observed to be highly selective binders to Mcl‐1 with no detectable binding to Bcl‐2 or Bcl‐x_L, among which the most potent one has a K_i value of 0.18 μM for Mcl‐1. Binding modes of four selected compounds to Mcl‐1 and Bcl‐x_L were derived through molecular docking and molecular dynamics simulations. It seems that the binding affinity and selectivity of these compounds can be reasonably interpreted with these models. Our study demonstrated the possibility for obtaining selective Mcl‐1 inhibitors with relatively simple chemical scaffolds. The active compounds identified by us could be used as lead compounds for developing even more potent selective Mcl‐1 inhibitors with potential pharmaceutical applications.