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.     

Loop flexibility and solvent dynamics as determinants for the selective inhibition of cyclin-dependent kinase 4: comparative molecular dynamics simulation studies of CDK2 and CDK4

The design and discovery of selective cyclin-dependent kinase 4 (CDK4) inhibitors have been actively pursued in order to develop therapeutic cancer treatments. By means of a consecutive computational protocol involving homology modeling, docking experiments, and molecular dynamics simulations, we examine the characteristic structural and dynamic properties that distinguish CDK4 from CDK2 in its complexation with selective inhibitors. The results for all three CDK4-selective inhibitors under investigation show that the large-amplitude motion of a disordered loop of CDK4 is damped out in the presence of the inhibitors whereas their binding in the CDK2 active site has little effect on the loop flexibility. It is also found that the binding preference of CDK4- selective inhibitors for CDK4 over CDK2 stems from the reduced solvent accessibility in the active site of the former due to the formation of a stable hydrogen-bond triad by the Asp99, Arg101, and Thr102 side chains at the top of the active-site gorge. Besides the differences in loop flexibility and solvent accessibility, the dynamic stabilities of the hydrogen bonds between the inhibitors and the side chain of the lysine residue at the bottom of the active site also correlate well with the relative binding affinities of the inhibitors for the two CDKs. These results highlight the usefulness of this computational approach in evaluating the selectivity of a CDK inhibitor, and demonstrate the necessity of considering protein flexibility and solvent effects in designing new selective CDK4-selective 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.     

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.     

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.     

Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials

This article reviews the steps that have led us from very fundamental research on the cell division cycle, investigated with the starfish oocyte model, to the identification of drugs now being evaluated against cancer in the clinic. Among protein kinases activated during entry in M phase, the cyclin-dependent kinase CDK1/cyclin B was initially identified as a universal M-phase promoting factor. It was then used as a screening target to identify pharmacological inhibitors. The first inhibitors to be discovered were 6-dimethylaminopurine and isopentenyladenine, from which more potent and selective inhibitors were optimized (olomoucine, roscovitine, and purvalanols). All were cocrystallized with CDK2 and found to localize in the ATP-binding pocket of the kinase. Their selectivity and cellular effects have been thoroughly investigated. Following encouraging results obtained in preclinical tests and favorable pharmacological properties, one of these purines, roscovitine (CYC202), is now entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. CDK inhibitors are also being evaluated, at the preclinical level, for therapeutic use against neurodegenerative diseases, cardiovascular disorders, viral infections, and parasitic protozoa. This initially unexpected scope of potential applications and the large number and chemical diversity of pharmacological inhibitors of CDKs now available constitute a very encouraging stimulus to pursue the search for optimization and characterization of protein kinase inhibitors, from which we expect numerous therapeutic applications.     

Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations

Human cytomegalovirus (HCMV) is a pathogenic human herpesvirus associated with serious, potentially life-threatening symptoms in the immunocompromised or immunonaïve host. The limitations encountered by antiviral therapy options currently available include a narrow panel of accessible targets, the induction of viral drug resistance as well as severe drug dosage-mediated side-effects. Improved drug-targeting strategies to resolve these issues are the focus of our investigations. In particular, pharmaceutical kinase inhibitors (PKIs), either directed to host kinases or directed to the viral protein kinase pUL97, have been considered to overcome these restrictions. Recently, we reported the identification of a synergistic combination of two PKIs directed to host cyclin-dependent kinase 7 (CDK7) and viral CDK ortholog pUL97. Here, we substantiate these findings with the following results: (i) true drug synergy was exhibited by various chemical classes of PKI pairs directed to pUL97 and CDK7; (ii) no putative amplification of cytotoxicity by these drug combinations was observed; (iii) a reduction in drug dosage levels for synergistic combinations was defined on a quantitative basis and compared to monotreatments; (iv) the quantities of target proteins CDK7 and pUL97 expressed in HCMV-infected cells were assessed by confocal imaging, indicating a strong down-modulation of CDK7 levels as a result of synergistic drug treatment; (v) the functional importance of these target kinases, both binding to cyclin H, was illustrated by assessing HCMV replication under the viral genomic deletion of ORF-UL97 or cellular cyclin knock-out; (vi) new combinations of HCMV-specific drug synergy were demonstrated for solely host-directed treatments using PKIs against CDK2, CDK7, CDK8 and/or CDK9 and (vii) a triple PKI combination provided further support for the synergy approach. With these combined findings, this study highlights the potential of therapeutic drug combinations of approved, developmental and preclinical PKIs for expanding future options for anti-HCMV therapy.     

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.     

6-Substituted pyrrolo[3,4-c]pyrazoles: an improved class of CDK2 inhibitors

We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.     

Immune Checkpoint Inhibitors: A Promising Treatment Option for Metastatic Castration-Resistant Prostate Cancer?

Since 2010, several treatment options have been available for men with metastatic castration-resistant prostate cancer (mCRPC), including immunotherapeutic agents, although the clinical benefit of these agents remains inconclusive in unselected mCRPC patients. In recent years, however, immunotherapy has re-emerged as a promising therapeutic option to stimulate antitumor immunity, particularly with the use of immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 and CTLA-4 inhibitors. There is increasing evidence that ICIs may be especially beneficial in specific subgroups of patients with high PD-L1 tumor expression, high tumor mutational burden, or tumors with high microsatellite instability/mismatch repair deficiency. If we are to improve the efficacy of ICIs, it is crucial to have a better understanding of the mechanisms of resistance to ICIs and to identify predictive biomarkers to determine which patients are most likely to benefit. This review focuses on the current status of ICIs for the treatment of mCRPC (either as monotherapy or in combination with other drugs), mechanisms of resistance, potential predictive biomarkers, and future challenges in the management of mCRPC.     

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.     

Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.     

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC₅₀ value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.     

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.     

Oral Selective Estrogen Receptor Degraders (SERDs) as a Novel Breast Cancer Therapy: Present and Future from a Clinical Perspective

Estrogen receptor-positive (ER+) is the most common subtype of breast cancer. Endocrine therapy is the fundamental treatment against this entity, by directly or indirectly modifying estrogen production. Recent advances in novel compounds, such as cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), or phosphoinositide 3-kinase (PI3K) inhibitors have improved progression-free survival and overall survival in these patients. However, some patients still develop endocrine resistance after or during endocrine treatment. Different underlying mechanisms have been identified as responsible for endocrine treatment resistance, where ESR1 gene mutations are one of the most studied, outstanding from others such as somatic alterations, microenvironment involvement and epigenetic changes. In this scenario, selective estrogen receptor degraders/downregulators (SERD) are one of the weapons currently in research and development against aromatase inhibitor- or tamoxifen-resistance. The first SERD to be developed and approved for ER+ breast cancer was fulvestrant, demonstrating also interesting activity in ESR1 mutated patients in the second line treatment setting. Recent investigational advances have allowed the development of new oral bioavailable SERDs. This review describes the evolution and ongoing studies in SERDs and new molecules against ER, with the hope that these novel drugs may improve our patients’ future landscape.     

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

Cyclin‐dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small‐molecule inhibitors of CDK–CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S‐phase cell populations, and cause a decrease in CDK2, cyclin A, and p27^Kip1 levels. These molecules should help decipher the complex contributions of CDK–CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.     

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.