The Development of CDC25A-Derived Phosphoseryl Peptides That Bind 14-3-3ε with High Affinities.

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.

A Comprehensive Overview of the Developments of Cdc25 Phosphatase Inhibitors.

Cdc25 phosphatases have been considered promising targets for anticancer development due to the correlation of their overexpression with a wide variety of cancers. In the last two decades, the interest in this subject has considerably increased and many publications have been launched concerning this issue. An overview is constructed based on data analysis of the results of the previous publications covering the years from 1992 to 2021. Thus, the main objective of the current review is to report the chemical structures of Cdc25s inhibitors and answer the question, how to design an inhibitor with better efficacy and lower toxicity?

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

Transition metal dependent regulation of the signal transduction cascade driving oocyte meiosis.

The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis.

Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations.

Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface.

Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

The design of novel inhibitors for treating cancer by targeting CDC25B through disruption of CDC25B-CDK2/Cyclin A interaction using computational approaches.

Cell division cycle 25B is a key cell cycle regulator and widely considered as potent clinical drug target for cancers. This research focused on identifying potential compounds in theory which are able to disrupt transient interactions between CDC25B and its CDK2/Cyclin A substrate.By using the method of ZDOCK and RDOCK, the most optimized 3D structure of CDK2/Cyclin A in complex with CDC25B was constructed and validated using two methods: 1) the superimposition of proteins; 2) analysis of the hydrogen bond distances of Arg 488(N1)-Asp 206(OD1), Arg 492(NE)-Asp 206(OD1), Arg 492(N1)-Asp 206(OD2) and Tyr 497(NE)-Asp 210(OD1). A series of new compounds was gained through searching the fragment database derived from ZINC based on the known inhibitor-compound 7 by the means of "replace fragment" technique. The compounds acquired via meeting the requirements of the absorption, distribution, metabolism, and excretion (ADME) predictions. Finally, 12 compounds with better binding affinity were identified. The comp#1, as a representative, was selected to be synthesized and assayed for their CDC25B inhibitory activities. The comp#1 exhibited mild inhibitory activities against human CDC25B with IC50 values at about 39.02 µM. Molecular Dynamic (MD) simulation revealed that the new inhibitor-comp#1 had favorable conformations for binding to CDC25B and disturbing the interactions between CDC25B and CDK2/Cyclin A.

Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif).

Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity.

Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2-CDC25 and CDC25-14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity.

Insights into the interaction of high potency inhibitor IRC-083864 with phosphatase CDC25.

CDC25 phosphatases play a crucial role in cell cycle regulation. They have been found to be over-expressed in various human tumours and to be valuable targets for cancer treatment. Here, we report the first model of binding of the most potent CDC25 inhibitor to date, the bis-quinone IRC-083864, into CDC25B obtained by combining molecular modeling and NMR studies. Our study provides new insights into key interactions of the catalytic site inhibitor and CDC25B in the absence of any available experimental structure of CDC25 with a bound catalytic site inhibitor. The docking model reveals that IRC-083864 occupies both the active site and the inhibitor binding pocket of the CDC25B catalytic domain. NMR saturation transfer difference and WaterLOGSY data indicate the binding zones of the inhibitor and support the docking model. Probing interactions of analogues of the two quinone units of IRC-083864 with CDC25B demonstrate that IRC-083864 competes with each monomer. Proteins 2017; 85:593-601. © 2016 Wiley Periodicals, Inc.

Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry.

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.

Conformational flexibility of the complete catalytic domain of Cdc25B phosphatases.

Cdc25B phosphatases are involved in cell cycle checkpoints and have become a possible target for developing new anticancer drugs. A more rational design of Cdc25B ligands would benefit from detailed knowledge of its tertiary structure. The conformational flexibility of the C-terminal region of the Cdc25B catalytic domain has been debated recently and suggested to play an important structural role. Here, a combination of experimental NMR measurements and molecular dynamics simulations for the complete catalytic domain of the Cdc25B phosphatase is presented. The stability of the C-terminal α-helix is confirmed, but the last 20 residues in the complete catalytic domain are very flexible, partially occlude the active site and may establish transient contacts with the protein core. This flexibility in the C-terminal tail may modulate the molecular recognition of natural substrates and competitive inhibitors by Cdc25B. Proteins 2016; 84:1567-1575. © 2016 Wiley Periodicals, Inc.

Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression.

Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1. To this end, we generated a genetically encoded FRET (Förster Resonance Energy Transfer)-based Cdc25C phosphorylation biosensor to observe Cdc25 spatiotemporal phosphorylation during cell cycle progression in live single cell assays. Because this approach proved to be powerful, we provide here guidelines for the development of biosensors for any phosphorylation site of interest.

MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma.

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3' UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one based inhibitors of the dual-specificity phosphatase Cdc25B.

The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes, particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus, Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the 3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling methodology to synthesize a new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a Ki of 1.9µM. Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint bypass. Molecular docking studies support binding near the catalytic site.

Inhibition of CDC25B phosphatase through disruption of protein-protein interaction.

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein-protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein-protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

Design novel inhibitors for treating cancer by targeting Cdc25B catalytic domain with de novo design.

The cell division cycle 25 (Cdc25) family of proteins is a group of highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent a group of attractive drug targets for anticancer therapies. To develop novel Cdc25B inhibitors, the ZINC database was screened for finding optimal fragments with de novo design approaches. As a result, top 11 compounds with higher binding affinities in flexible docking were obtained, which were derived from five novel scaffolds (scaffold C) consisting of the linker-part and two isolated scaffolds (scaffold A and B)located in the two binding domains (catalytic pocket and swimming pool), respectively. The subsequent molecular docking and molecular dynamics studies showed that these compounds not only adopt more favorable conformations but also have stronger binding interaction with receptor than the inhibitors identified previously. The additional absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions also indicted that the 11 compounds (especially Comp#1) hold a high potential to be novel lead compounds for anticarcinogen. Consequently, the findings reported here may at least provide a new strategy or useful insights for designing effective Cdc25B inhibitors.

Activation of Nrf2 pathways correlates with resistance of NSCLC cell lines to CBP501 in vitro.

CBP501 is an anticancer drug candidate that was investigated in two randomized phase II clinical trials for patients with nonsquamous non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). CBP501 has been shown to have two mechanisms of action, namely calmodulin modulation and G2 checkpoint abrogation. Here, we searched for a biomarker to predict sensitivity to CBP501. Twenty-eight NSCLC cell lines were classified into two subgroups, CBP501-sensitive and -insensitive, by quantitatively analyzing the cis-diamminedichloro-platinum (II) (CDDP)-enhancing activity of CBP501 through treatments with short-term (1 hour) coexposure to CDDP and CBP501 or to either alone. Microarray analysis was performed on these cell lines to identify gene expression patterns that correlated with CBP501 sensitivity. We found that multiple nuclear factor erythroid-2-related factor 2 (Nrf2) target genes showed high expression in CBP501-insensitive cell lines. Western blot and immunocytochemical analysis for Nrf2 in NSCLC cell lines also indicated higher protein level in CBP501-insensitive cell lines. Moreover, CBP501 sensitivity is modulated by silencing or sulforaphane-induced overexpression of Nrf2. These results indicate that Nrf2 transcription factor is a potential candidate as a biomarker for resistance to CBP501. This study might help to identify those subpopulations of patients who would respond well to the CBP501 and CDDP combination treatment of NSCLC.

Solution NMR studies reveal no global flexibility in the catalytic domain of CDC25B.

The CDC25B phosphatase is a critical regulator of the cell cycle and has been validated as an important therapeutic target in cancer. Previous studies using molecular dynamics simulations have concluded that the catalytic domain of CDC25B may experience a significant degree of dynamics or be partially disordered in solution, a finding that has a pronounced impact on the structure-based development of CDC25B inhibitors. We have probed the backbone dynamics of the CDC25B catalytic domain in solution using NMR relaxation experiments and found that the core of the protein is relatively rigid and does not experience any large-scale dynamics over a broad range of time scales. Furthermore, based on residual dipolar coupling measurements we have concluded that the conformation in solution is very similar to that observed in the crystal form. Importantly, these findings rationalize the application of the CDC25B crystal structure in structure-based drug development.