Cdc25 Phosphatase Inhibitors Research Articles

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?

Discovery of potent and selective Cdc25 phosphatase inhibitors via rapid assembly and in situ screening of Quinonoid-focused libraries

Cell division cycle 25 (Cdc25) phosphatase is an attractive target for drug discovery. The rapid assembly and in situ screening of focused combinatorial fragment libraries using efficient modular reactions is a highly robust strategy for discovering bioactive molecules. In this study, we have utilized miniaturized synthesis to generate several quinonoid-focused libraries, by standard CuAAC reaction and HBTU-based amide coupling chemistry. Then the enzyme inhibition screening afforded some potent and selective Cdc25s inhibitors. Compound M5N36 (Cdc25A: IC50 = 0.15 ± 0.05 μM; Cdc25B: IC50 = 0.19 ± 0.06 μM; Cdc25C: IC50 = 0.06 ± 0.04 μM) exhibited higher inhibitory activity than the initial lead NSC663284 (Cdc25A: IC50 = 0.27 ± 0.02 μM; Cdc25B: IC50 = 0.42 ± 0.01 μM; Cdc25C: IC50 = 0.23 ± 0.01 μM). Moreover, M5N36 displayed about three-fold more potent against Cdc25C than Cdc25A and B, indicating that M5N36 could act as a relatively selective Cdc25C inhibitor. Cell viability evaluation, western blotting and molecular simulations provided a mechanistic understanding of the activity of M5N36. It showed promising anti-growth activity against the MDA-MB-231 cell line and desirable predicted physicochemical properties. Overall, M5N36 was proven to be a promising novel Cdc25C inhibitor.

Anti-inflammatory functions of the CDC25 phosphatase inhibitor BN82002 via targeting AKT2

This study presents BN82002 as an anti-inflammatory drug candidate. It was found that BN82002 inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 cells and peritoneal macrophages that were activated by toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). BN82002 dose-dependently down-regulated mRNA levels of nitric oxide synthase, tumor necrosis factor-α, and cyclooxygenase-2. The nuclear translocation of nuclear factor (NF)-κB (p65 and p50) was also blocked by BN82002 in RAW265.7 cells stimulated by LPS. According to reporter gene assay performed with NF-κB construct, BN82002 clearly reduced increased level of luciferase activity mediated by transcription factor NF-κB in LPS-treated RAW264.7 cells and in MyD88- and AKT2-overexpressing HEK293 cells. However, BN82002 did not inhibit NF-κB activity in AKT1- or IKKβ-overexpressing HEK293 cells. NF-κB upstream signaling events specifically targeted AKT2 but had no effect on AKT1. The specific target of BN82002 was Tyr-178 in AKT2. BN82002 bound to Tyr-178 and interrupted the kinase activity of AKT2, according to a cellular thermal shift assay analysis of the interaction of BN82002 with AKT2 and an AKT2 mutant (Tyr-178 mutated to Ala; AKT2 Y178A). These results suggest that BN82002 could reduce inflammatory pathway by controlling NF-κB pathway and specifically targeting AKT2.

Dual-Specificity Phosphatase CDC25B Was Inhibited by Natural Product HB-21 Through Covalently Binding to the Active Site.

Cysteine 473, within the active site of the enzyme, Cdc25B, is catalytically essential for substrate activation. The most well-reported inhibitors of Cdc25 phosphatases, especially quinone-type inhibitors, function by inducing irreversible oxidation at this active site of cysteine. Here, we identified a natural product, HB-21, having a sesquiterpene lactone skeleton that could irreversibly bind to cys473 through the formation of a covalent bond. This compound inhibited recombinant human Cdc25B phosphatase with an IC50 value of 24.25 μM. Molecular modeling predicted that HB-21 not only covalently binds to cys473 of Cdc25B but also forms six hydrogen bonds with residues at the active site. Moreover, HB-21 can dephosphorylate cyclin-dependent kinase (CDK1), the natural substrate of Cdc25b, and inhibit cell cycle progression. In summary, HB-21 is a new type of Cdc25B inhibitor with a novel molecular mechanism.

Synthesis, biological evaluation and molecular modeling studies on novel quinonoid inhibitors of CDC25 phosphatases

The cell division cycle 25 phosphatases (CDC25A, B, and C; E.C. 3.1.3.48) are key regulator of the cell cycle in human cells. Their aberrant expression has been associated with the insurgence and development of various types of cancer, and with a poor clinical prognosis. Therefore, CDC25 phosphatases are a valuable target for the development of small molecule inhibitors of therapeutic relevance. Here, we used an integrated strategy mixing organic chemistry with biological investigation and molecular modeling to study novel quinonoid derivatives as CDC25 inhibitors. The most promising molecules proved to inhibit CDC25 isoforms at single digit micromolar concentration, becoming valuable tools in chemical biology investigations and profitable leads for further optimization.

Screening of indeno[1,2-b]indoloquinones by MALDI-MS: a new set of potential CDC25 phosphatase inhibitors brought to light

Quinones and quinones-like compounds are potential candidates for the inhibition of CDC25 phosphatases. The combination of MALDI-MS analyses and biological studies was used to develop a rapid screening of a targeted library of indeno[1,2-b]indoloquinone derivatives. The screening protocol using MALDI-TOFMS and MALDI-FTICRMS highlighted four new promising candidates. Biological investigations showed that only compounds 5c–f inhibited CDC25A and -C phosphatases, with IC50 values around the micromolar range. The direct use of a screening method based on MALDI-MS technology allowed achieving fast scaffold identification of a new class of potent inhibitors of CDC25 phosphatases. These four molecules appeared as novel molecules of a new class of CDC25 inhibitors. Assessment of 5c–e in an MRC5 proliferation assay provided an early indicator of toxicity to mammalian cells. Compound 5d seems the most promising hit for developing new CDC25 inhibitors.

B06 Exploring The Role Of Phosphorylation In Huntingtin Aggregation Dynamics

Background Huntington’s disease (HD) is characterised by misfolding and aggregation of mutant huntingtin (HTT), a dynamic process that starts with the association of misfolded HTT monomers into soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic HTT species while large aggregates may in fact be neuroprotective. To better understand HTT toxicity it is essential to understand the molecular mechanisms of aggregation. Aims Our goal was to elucidate the role of HTT N-terminal phosphorylation in the aggregation and toxicity of mutant HTT. Methods/techniques Bimolecular fluorescence complementation (BiFC) was employed to visualise soluble and insoluble HTT species. We used this model to study the behaviour of HTT phosphomimic and phosphoresistant mutants in living cells. Moreover, we tested several compounds that modulate phosphorylation in Drosophila expressing HTT exon 1 (HTT93Q) under the control of the GAL4/UAS system. Pan-neuronal expression of HTT93Q by elavGAL4 gives a variety of disease-relevant phenotypes, including degeneration of photoreceptor neurons. We compared the neurodegeneration of treated or non-treated HD flies using the light microscopy-based pseudopupil assay. Results/outcome When phosphomimic mutations were present in HTT103Q BiFC constructs, the generation of inclusion bodies was completely abolished. Phosphomimic and Phosphoresistant mutants had varied effects on HTT toxicity, which were not associated with the levels of oligomers or inclusion bodies. A CDC25 phosphatase inhibitor decreased aggregation in cells expressing HTT103Q and increased neurodegeneration in HTT93Q flies. Notably, a specific protein phosphatase 2A (PP2A) inhibitor completely reversed HTT93Q neurotoxicity in Drosophila. Conclusions The phosphorylation state of HTT N-terminal region plays a role in aggregation and toxicity of the protein, and this can be pharmacologically modulated by targeting cellular phosphatases.

Anticancer Activities of Cdc25 Phosphatase Inhibitors in the Proliferation of Human Lung Cancer Cells

Anticancer agent, Cdc25 phosphatase, Cell permeability, Inhibitor, Lung cancerCell division cycle 25 (Cdc25) dual-specificity phospha-tases catalyze the hydrolysis of phosphorylated threonineand tyrosine residues in proteins, and serve as key regulatorsof the cell division and the cell response to DNA damage.

Design and synthesis of novel bis-thiazolone derivatives as micromolar CDC25 phosphatase inhibitors: Effect of dimerisation on phosphatase inhibition

CDC25 phosphatases are involved in deregulated cell cycle progression and tumor development with poor prognosis. Among the most potent CDC25 inhibitors, quinonoid-based derivatives have been extensively studied. Dimerisation of heterocyclic quinones has led to IRC-083864, a bis-quinone compound with increased CDC25B inhibitory activity. Thirty-one bis-thiazolone derivatives were synthesized and assayed for CDC25 inhibitory activity. Most of the dimers displayed enhanced inhibitory activities with micromolar IC50 values lower than that observed for each thiazolone scaffold separately. Moreover, most of these compounds were selective CDC25 inhibitors. Dimer 40 showed an IC50 value of 2.9μM and could inhibit CDC25 activity without generating reactive oxygen species which is likely to occur with quinone-based inhibitors. Molecular docking studies suggested that the dimers could bind simultaneously to the active site and the inhibitor binding pocket.

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.

Environmental-benign oxidation of 2-oxazolines to oxazoles by dioxygen as the sole oxidant

A facile and environment-benign oxidation by dioxygen as the sole oxidant was applied for the conversion of 2-oxazolines to oxazoles. The substituent effect on 2-oxazoline ring was investigated. The use of this methodology for the synthesis of a key intermediate of a CDC25 phosphatase inhibitor ( SC- ααδ9) as an anticancer agent was also described.

Synthesis and biological evaluation of novel coumarin-based inhibitors of Cdc25 phosphatases

The cell division cycle 25 (Cdc25) family of proteins are dual specificity phosphatases that activate cyclin-dependent kinase (CDK) complexes, which in turn regulate progression through the cell division cycle. Overexpression of Cdc25 proteins has been reported in a wide variety of cancers; their inhibition may thus represent a novel approach for the development of anticancer therapeutics. Herein we report new coumarin-based scaffolds endowed with a selective inhibition against Cdc25A and Cdc25C, being 6a and 6d the most efficient inhibitors and worthy of further investigation as anticancer agents.

Inhibitors of Cdc25 phosphatases as anticancer agents: a patent review

Importance of the field: The cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases, the main gatekeepers of the eukaryotic cell division cycle. The three isoforms of Cdc25, including Cdc25A, Cdc25B and Cdc25C, appear to act on different cyclin-dependent kinase/cyclin complexes at different stages of the cell cycle. Overexpression of Cdc25A and/or Cdc25B, but not Cdc25C, has been detected in numerous cancers and is often correlated with a poor clinical prognosis. Thus, inhibition of these phosphatases may represent a promising therapeutic approach in oncology.Areas covered in this review: The main focus of the present review is to describe the development of Cdc25 inhibitors over the years. We describe different compounds according to the decade of discovery and focus attention on molecules that were published in patents.What the reader will gain: Insight into the most clinically relevant therapeutic Cdc25 analogues that have been published in over 40 patents over the past 19 years.Take home message: Some Cdc25 inhibitors have suppressed in vivo the growth of human tumor xenografts in animals; this confirmed the validity of using Cdc25 phosphatase inhibition as an anticancer strategy, but side effects and toxicity remain to be investigated.

Cytotoxicity of new alkylamino- and phenylamino-containing polyfluorinated derivatives of 1,4-naphthoquinone

Fluorinated derivatives of 1,4-naphthoquinone are highly potent inhibitors of Cdc25A and Cdc25 phosphatases and growth of tumor cells. Five new N-substituted polyfluorinated derivatives of 2-amino-1,4-naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as well as cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2- tert-Butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone ( 1) inhibited the growth of normal control and tumor cells at the same concentration. Three compounds: 2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone ( 2), 2-ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone ( 3), 2-phenylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone ( 4) exhibited a 50% decrease in the growth of cancer cells at low and comparable concentrations (2.4–8.6 μM) while being remarkably less cytotoxic toward normal LMTK and PMF cells. Quinones ( 1)–( 4), but not 2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone ( 5), efficiently suppressed spontaneous mutagenesis in Salmonella cells, while all compounds 1– 5 decreased the mutagenic effect of H 2O 2 on bacterial cells. Their possible perspectives as anticancer drugs are shortly discussed.

3D imaging of the response to CDC25 inhibition in multicellular spheroids

Multicellular tumor spheroids closely mimic the 3D organization of avascular microregions within tumors and thereby represent a valuable model for the evaluation of anticancer drugs. In this study, we performed a 3D analysis of the response to the CDC25 phosphatase inhibitor IRC-083864 in HCT116 spheroids. Continuous exposure to IRC-083864 strongly inhibits the growth of spheroids and is shown to correlate with a decrease in Ki-67 positive cells. The cytotoxicity induced by IRC-083864 was examined by two-photon laser microscopy imaging and 3D reconstruction. Visualization in 3D allowed us to demonstrate that IRC-083864 treatment results in the inhibition of mitosis and induces cell death specifically localized in the outer proliferative cell layers of the spheroid structure. These results emphasize the importance of 3D models and of in toto analysis for the evaluation of anticancer drugs cytotoxicity.

Rapid Discovery of Triazolobenzylidene-Thiazolopyrimidines (TBTP) as CDC25 Phosphatase Inhibitors by Parallel Click Chemistry and in Situ Screening

UniVersite Paul Sabatier Toulouse III, Faculte des Sciences Pharmaceutiques, Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox, UMR152 IRD-UT3, 31062 Toulouse cedex 09, France, UniVersite Paris Descartes, UFR Biomedicale, Laboratoire de Pharmacochimie Moleculaire et Cellulaire, 45 rue des Saints-Peres, 75270 Paris, cedex 06, France, and INSERM U648, 45 rue des Saints-Peres, 75270 Paris, cedex 06, France

Synthesis of tanshinone IIA analogues and their inhibitory activities against Cdc25 phosphatases

Two series of tanshinone IIA derivatives were synthesized and evaluated for their antitumor activities as Cdc25 phosphatase inhibitors. Most of them demonstrated potent Cdc25 inhibitory activity and powerful cytotoxicity against A549 tumor cell line, producing IC 50 values in very low micromolar range. At last, the preliminary SAR was discussed.

Discovery of Novel and Potent Cdc25 Phosphatase Inhibitors Based on the Structure-Based De Novo Design

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy due to the correlation of their overexpression with a wide variety of cancers. We have been able to identify five novel Cdc25 phosphatase inhibitors with micromolar activity by means of a structure-based de novo design method with a known inhibitor scaffold. Because the newly discovered inhibitors are structurally diverse and have desirable physicochemical properties as a drug candidate, they deserve further investigation as anticancer drugs. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are addressed in detail.

Structure-based de novo design and biochemical evaluation of novel Cdc25 phosphatase inhibitors

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy due to the correlation of their overexpression with a wide variety of cancers. We have been able to identify 32 novel Cdc25 phosphatase inhibitors with micromolar activity by means of a structure-based de novo design method with the two known inhibitor scaffolds. Because the newly discovered inhibitors are structurally diverse and have desirable physicochemical properties as a drug candidate, they deserve further investigation as anticancer drugs. The differences in binding modes of the identified inhibitors in the active sites of Cdc25A and B are addressed in detail.

Development of Novel Thiazolopyrimidines as CDC25B Phosphatase Inhibitors

The development of CDC25 phosphatase inhibitors is an interesting approach toward new antitumor agents, as CDC25 play key roles in cell-cycle regulation and are overexpressed in numerous cancers. We previously reported a novel compound belonging to the thiazolopyrimidine family that inhibits CDC25 activity with an IC(50) value of 13 microM and displays cytotoxic properties against HeLa cells. Structural modifications were subsequently conducted on this new pharmacophore which led to a library of 45 thiazolopyrimidines. Regarding the in vitro effects, 14 compounds inhibit CDC25B with IC(50)<20 microM, with the most efficient inhibitor 44 improving the potency to 4.5 microM. Steady-state kinetics were performed and showed a mixed inhibition pattern for all tested compounds. Furthermore, 44 was able to revert the bypass of genotoxicity-induced G(2) arrest upon CDC25B overexpression, indicating that this compound targets the dual-specificity phosphatase in cultured cells. Finally, the cytotoxic activities of the compounds were determined against two human cancer cell lines. The results indicate that the prostatic LNCaP cell line is more sensitive to these derivatives than the pancreatic adenocarcinoma MiaPaCa-2 line. With its interesting enzymatic and cellular properties, compound 44 appears to be a promising CDC25B inhibitor for further development.