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?

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.

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.

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.

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.

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.

Inhibiting transient protein-protein interactions: lessons from the Cdc25 protein tyrosine phosphatases.

Transient protein-protein interactions have key regulatory functions in many of the cellular processes that are implicated in cancerous growth, particularly the cell cycle. Targeting these transient interactions as therapeutic targets for anticancer drug development seems like a good idea, but it is not a trivial task. This Review discusses the issues and difficulties that are encountered when considering these transient interactions as drug targets, using the example of the cell division cycle 25 (Cdc25) phosphatases and their cyclin-dependent kinase (CDK)-cyclin protein substrates.

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.

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.

Cryo-EM structure of the CDK2-cyclin A-CDC25A complex.

The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.

A robust protocol to map binding sites of the 14-3-3 interactome: Cdc25C requires phosphorylation of both S216 and S263 to bind 14-3-3.

Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.

Direct roles of the signaling kinase RSK2 in Cdc25C activation during Xenopus oocyte maturation.

The induction of M phase in eukaryotic cell cycles requires robust activation of Cdc2/cyclin B by Cdc25, which itself is robustly activated by serine/threonine phosphorylations. Although multiple protein kinases that directly activate Cdc25C have been identified, whether the combination of different primary phosphorylations of Cdc25C is sufficient to fully activate Cdc25C has not been determined. By analyzing the GST-Cdc25C phosphorylating activity in Xenopus egg extracts, we previously defined roles of MAPK and Cdc2/cyclin B in partially activating Cdc25C and predicted the presence of another major Cdc25C-activating kinase. In this study, we demonstrate that this missing kinase is RSK2, which phosphorylates three sites in Cdc25C and also partially activates Cdc25C. However, the phosphorylations catalyzed by MAPK, Cdc2, and RSK2 fail to fully activate Cdc25C, suggesting that additional biochemical events are required to fully activate this key cell cycle regulator.

Carboxy-terminal phosphorylation sites in Cdc25 contribute to enforcement of the DNA damage and replication checkpoints in fission yeast.

In fission yeast and vertebrate cells, Cdc25 phosphatase is the target of checkpoint-mediated response to DNA replication blocks, DNA damage, and extracellular stress. As such, it is a key regulator of cell cycle progress and genomic stability. In fission yeast, phosphorylation of Cdc25 by the checkpoint kinases Cds1 and Chk1 and also Srk1 during stress creates a binding site for the 14-3-3 homolog Rad24; the complex is then exported from the nucleus. Cdc25 contains 12 potential serine/threonine phosphorylation sites that are phosphorylated in vitro by Cds1; 9 reside in the amino terminal half of the protein with the remaining sites are located in the extreme C-terminus. We have previously shown that deletion of the nine amino terminal sites results in degradation of the mutant protein while the checkpoint is enforced by the Mik1 kinase acting on Cdc2 tyrosine-15. Here, we examine the influence of the three C-terminal sites on the negative regulation of Cdc25. These sites are conserved in vertebrates and have been shown to be phosphorylated following DNA damage and replication blocks. We show that these three sites have a role in the negative regulation of Cdc25 following replication arrest, but perhaps more importantly they appear to particularly contribute to regulating the duration, and thus the effectiveness of the arrested state.

Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases.

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity.

BACKGROUND INFORMATION: CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)-cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. RESULTS: In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N- and C-terminal regions of CDC25B and requires CDC25B binding to its CDK-cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. CONCLUSIONS: Our results therefore suggest that CDC25B associates with a Ctn2-containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.

Discovering the distinct inhibitory effects between C4-epimeric glycosyl amino acids: new insight into the development of protein tyrosine phosphatase inhibitors.

There has been increasing interest in the development of drug candidates based on sugar templates that possess rich structural and, especially, configurational diversities. We disclose herein that the epimeric identity between methyl 3,4-bis-phenylalanyl/tyrosinyl triazolyl-alpha-D-galactopyranoside and glucopyranoside may lead to their distinct inhibitory effects on specific protein tyrosine phosphatases (PTPs). Subsequently performed molecular docking study elucidated the plausible binding behaviors of the more potent galactosyl inhibitors with their primary PTP target, i.e. Cell Division Cycle 25B (CDC25B) phosphatase.

Computational analysis of phosphopeptide binding to the polo-box domain of the mitotic kinase PLK1 using molecular dynamics simulation.

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

Absence of polo-like kinase 3 in mice stabilizes Cdc25A after DNA damage but is not sufficient to produce tumors.

The polo-like kinases (Plks1-5) are emerging as an important class of proteins involved in many facets of cell cycle regulation and response to DNA damage and stress. Here we show that Plk3 phosphorylates the key cell cycle protein phosphatase Cdc25A on two serine residues in its cyclinB/cdk1 docking domain and regulates its stability in response to DNA damage. We generated a Plk3 knock-out mouse and show that Cdc25A protein from Plk3-deficient cells is less susceptible to DNA damage-mediated degradation than cells with functional Plk3. We also show that absence of Plk3 correlates with loss of the G1/S cell cycle checkpoint. However, neither this compromised DNA damage checkpoint nor reduced susceptibility to proteasome-mediated degradation after DNA damage translated into a significant increase in tumor incidence in the Plk3-deficient mice.

Adventitious arsenate reductase activity of the catalytic domain of the human Cdc25B and Cdc25C phosphatases.

A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X(5)-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X(5)-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X(5)-Arg loop that might moonlight as arsenate reductases.