Discovery of a Plant 14-3-3 Inhibitor Possessing Isoform Selectivity and In Planta Activity.

Synthetic modulators of plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Herein, we describe a novel small-molecule inhibitor for 14-3-3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assays revealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide new insight into the design of potent and isoform-selective 14-3-3 modulators.

Targeting 14-3-3ε activates apoptotic signaling to prevent cutaneous squamous cell carcinoma.

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.

Ketohexokinase-A acts as a nuclear protein kinase that mediates fructose-induced metastasis in breast cancer.

Harmful effects of high fructose intake on health have been widely reported. Although fructose is known to promote cancer, little is known about the underlying mechanisms. Here, we found that fructose triggers breast cancer metastasis through the ketohexokinase-A signaling pathway. Molecular experiments showed that ketohexokinase-A, rather than ketohexokinase-C, is necessary and sufficient for fructose-induced cell invasion. Ketohexokinase-A-overexpressing breast cancer was found to be highly metastatic in fructose-fed mice. Mechanistically, cytoplasmic ketohexokinase-A enters into the nucleus during fructose stimulation, which is mediated by LRRC59 and KPNB1. In the nucleus, ketohexokinase-A phosphorylates YWHAH at Ser25 and the YWHAH recruits SLUG to the CDH1 promoter, which triggers cell migration. This study provides the effect of nutrition on breast cancer metastasis. High intake of fructose should be restricted in cancer patients to reduce the risk of metastasis. From a therapeutic perspective, the ketohexokinase-A signaling pathway could be a potential target to prevent cancer metastasis.

Structure-based evolution of a promiscuous inhibitor to a selective stabilizer of protein-protein interactions.

The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.

Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth.

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 14-3-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases.

Allosteric regulation of protein 14-3-3ζ scaffold by small-molecule editing modulates histone H3 post-translational modifications.

Background: Histone post-translational modifications (PTMs) are involved in various biological processes such as transcriptional activation, chromosome packaging, and DNA repair. Previous studies mainly focused on PTMs by directly targeting histone-modifying enzymes such as HDACs and HATs. Methods and Results: In this study, we discovered a previously unexplored regulation mechanism for histone PTMs by targeting transcription regulation factor 14-3-3ζ. Mechanistic studies revealed 14-3-3ζ dimerization as a key prerequisite, which could be dynamically induced via an allosteric effect. The selective inhibition of 14-3-3ζ dimer interaction with histone H3 modulated histone H3 PTMs by exposing specific modification sites including acetylation, trimethylation, and phosphorylation, and reprogrammed gene transcription profiles for autophagy-lysosome function and endoplasmic reticulum stress. Conclusion: Our findings demonstrate the feasibility of editing histone PTM patterns by targeting transcription regulation factor 14-3-3ζ, and provide a distinctive PTM editing strategy which differs from current histone modification approaches.

Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.

RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.

A solid-phase method for synthesis of dimeric and trimeric ligands: Identification of potent bivalent ligands of 14-3-3σ.

Multivalent protein-protein interactions including bivalent and trivalent interactions play a critical role in mediating a wide range of biological processes. Hence, there is a significant interest in developing molecules that can modulate those signaling pathways mediated by multivalent interactions. For example, multimeric molecules capable of binding to a receptor protein through a multivalent interaction could serve as modulators of such interactions. However, it is challenging to efficiently generate such multimeric ligands. Here, we have developed a facile solid-phase method that allows for the rapid generation of (homo- and hetero-) dimeric and trimeric protein ligands. The feasibility of this strategy was demonstrated by efficiently synthesizing fluorescently-labeled dimeric peptide ligands, which led to dramatically increased binding affinities (~400-fold improvement) relative to a monomeric 14-3-3σ protein ligand.

14-3-3ζ targeting induced senescence in Hep-2 laryngeal cancer cell through deneddylation of Cullin1 in the Skp1-Cullin-F-box protein complex.

OBJECTIVES: Despite of the aberrant expression of 14-3-3ζ in head and neck squamous cell carcinoma (HNSCC), little is known about the role of 14-3-3ζ in the regulation of senescence in HNSCC. This study was performed to investigate whether 14-3-3ζ is implicated in senescence evasion of Hep-2 laryngeal cancer cells. METHODS: The expression of 14-3-3ζ was suppressed using RNA interference strategy. Senescence induction was determined by senescence-associated ß-galactosidase staining and the numbers of promyelocytic leukaemia nuclear body. Real-time PCR, western blotting and immunohistochemistry were applied for the expression of corresponding proteins. Xenograft experiment was performed to show in vivo effect of 14-3-3ζ silencing on tumour growth. RESULTS: 14-3-3ζ silencing significantly induced senescence phenotypes via 27 accumulations. Subsequently, we demonstrated that p27 accumulation is linked to inactivation of SCFSkp2 complex activity, probably due to the deneddylation of cullin-1 (Cul-1) as follows. (a) Neddylated Cul-1 is decreased by 14-3-3ζ silencing. (b) Blocking neddylation using MLN4924 reproduces senescence phenotypes. (c) Knockdown of CSN5, which functions as a deneddylase, was shown to restore the senescence phenotypes induced by 14-3-3ζ depletion. Finally, we demonstrated that 14-3-3ζ depletion effectively hindered the proliferation of Hep-2 cells implanted into nude mice. CONCLUSION: 14-3-3ζ negatively regulates senescence in Hep-2 cells, suggesting that 14-3-3ζ targeting may serve to suppress the expansion of laryngeal cancer via induction of senescence through the Cul-1/SCFSkp2 /p27 axis.

YWHAE promotes proliferation, metastasis, and chemoresistance in breast cancer cells.

Breast cancer is the most common female-specific malignancy in Taiwan and developed countries worldwide, and its incidence continues to grow. 14-3-3ε (YWHAE), which belong to 14-3-3 family, it has been reported up-regulated in breast cancer tissues. However, the clinical implication and function of YWHAE in breast cancer remains unclear. In this study, we investigated the prognostic value of the YWHAE in human breast cancer. Immunohistochemistry was used to analyze YWHAE expression in breast cancer tissues. Cell model was applied to examine the functions of YWHAE. The chemotherapeutic agents were used to evaluate the effect of YWHAE in breast cancer cell lines. YWHAE expression was associated with tumor size, lymph node metastasis, and poor patient survival in patients with breast cancer. YWHAE overexpression significantly increased the proliferation, migration, and invasion abilities of breast cancer cells. Knockdown of YWHAE expression reduced the expression of Snail and Twist in breast cancer cells. We also found that YWHAE was responsible for the resistance of breast cancer cells to chemotherapeutic agents, and knockdown of YWHAE enhanced sensitivity to multiple chemotherapeutic agents in breast cancer cells. Taken together, our findings indicated that YWHAE promoted cancer progression and chemoresistance in breast cancer cells and can be a potential therapeutic target for breast cancer.

Chemically stable inhibitors of 14-3-3 protein-protein interactions derived from BV02.

14-3-3 are regulatory proteins that through protein-protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound 1 was able to promote the nuclear translocation of c-Abl at low micromolar concentrations. Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.

MiR-597 Targeting 14-3-3σ Enhances Cellular Invasion and EMT in Nasopharyngeal Carcinoma Cells.

BACKGROUND: Alterations in microRNAs (miRNAs) are related to the occurrence of nasopharyngeal carcinoma (NPC) and play an important role in the molecular mechanism of NPC. Our previous studies show low expression of 14-3-3σ (SFN) is related to the metastasis and differentiation of NPC, but the underlying molecular mechanisms remain unclear. METHODS: Through bioinformatics analysis, we find miR-597 is the preferred target miRNA of 14-3-3σ. The expression level of 14-3-3σ in NPC cell lines was detected by Western blotting. The expression of miR-597 in NPC cell lines was detected by qRT-PCR. We transfected miR-597 mimic, miR-597 inhibitor and 14-3-3σ siRNA into 6-10B cells and then verified the expression of 14-3-3σ and EMT related proteins, including E-cadherin, N-cadherin and Vimentin by western blotting. The changes of migration and invasion ability of NPC cell lines before and after transfected were determined by wound healing assay and Transwell assay. RESULTS: miR-597 expression was upregulated in NPC cell lines and repaired in related NPC cell lines, which exhibit a potent tumor-forming effect. After inhibiting the miR-597 expression, its effect on NPC cell line was obviously decreased. Moreover, 14-3-3σ acts as a tumor suppressor gene and its expression in NPC cell lines is negatively correlated with miR-597. Here 14-3-3σ was identified as a downstream target gene of miR-597, and its downregulation by miR-597 drives epithelial-mesenchymal transition (EMT) and promotes the migration and invasion of NPC. CONCLUSION: Based on these findings, our study will provide theoretical and experimental evidences for molecular targeted therapy of NPC.

Structure-based assessment and network analysis of targeting 14-3-3 proteins in prostate cancer.

Developing combination therapy for castrate-resistant prostate cancer (CRPC) may require exploiting new drug targets outside androgen receptor and PI3K / AKT / mTOR signal transduction pathways implicated in prostate cancer (PCa) progression. One such possible new target is YWHAZ of the 14-3-3 protein family as this gene has prognostic significance for metastatic CRPC patients. However, there are no small molecules targeting YWHAZ commercially available. Hence, we explored whether the small molecule BV02 targeting another 14-3-3 protein family member SFN also binds to YWHAZ. Using advanced docking algorithms we find that BV02 docks many other 14-3-3 family members. In addition, the amphipathic groove where drug binding occurs also has a high binding affinity for other drugs used to treat PCa such as docetaxel. The proteome of metastatic PCa models (LNCaP clone FGC and PC-3) was perturbed as a result of BV02 treatment. Through data integration of three proteomics data sets we found that BV02 modulates numerous protein-protein interactions involving 14-3-3 proteins in our PCa models.

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction.

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II-IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.

Inhibition of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) Overcomes Drug Resistance and Tumorigenicity in Ovarian Cancer.

BACKGROUND/AIMS: Cancer stem-like cells are the main cause of tumor occurrence, progression, and therapeutic resistance. However, the precise signals required for the maintenance of the stem-like traits of these cells in ovarian cancer remain elusive. We have thus worked to elucidate the functional role of Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, in the regulation of multidrug resistance and stem cell-like traits in ovarian cancer. METHODS: We detected the YWHAZ levels in human ovarian cancer specimens and cell lines using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blots. MTS assays, soft agar colony formation assays, migration assays, cell cycle analysis, sphere formation assays, and flow cytometry were applied to investigate the functional role of YWHAZ in ovarian cancer. RESULTS: Our data reveals substantially increased YWHAZ expression in both cisplatin- and paclitaxel-resistant ovarian cancer cells. Silencing YWHAZ restored the sensitivity of resistant ovarian cancer cells to cisplatin and paclitaxel. Furthermore, in vitro studies showed that down-regulation of YWHAZ inhibited cell cycle progression, migration, and the expression of stem cell markers. Moreover, tumorigenicity was suppressed in tumor-bearing BALB/c nude mice following YWHAZ knockdown. Additionally, we demonstrated that the expression of YWHAZ was directly down-regulated by miR-30e in resistant ovarian cancer cells. CONCLUSION: Our results have led to new insights into the essential role of YWHAZ in the regulation of tumourigenesis, stem-like traits, and drug resistance in ovarian cancer, thereby helping to identify a potential target for ovarian cancer therapy.

Mono- and Bivalent 14-3-3 Inhibitors for Characterizing Supramolecular "Lysine Wrapping" of Oligoethylene Glycol (OEG) Moieties in Proteins.

Previous studies have indicated the presence of defined interactions between oligo or poly(ethylene glycol) (OEG or PEG) and lysine residues. In these interactions, the OEG or PEG residues "wrap around" the lysine amino group, thereby enabling complexation of the amino group by the ether oxygen residues. The resulting biochemical binding affinity and thus biological relevance of this supramolecular interaction however remains unclear so far. Here, we report that OEG-containing phosphophenol ether inhibitors of 14-3-3 proteins also display such a "lysine-wrapping" binding mode. For better investigating the biochemical relevance of this binding mode, we made use of the dimeric nature of 14-3-3 proteins and designed as well as synthesized a set of bivalent 14-3-3 inhibitors for biochemical and X-ray crystallography-based structural studies. We found that all synthesized derivatives adapted the "lysine-wrapping" binding mode in the crystal structures; in solution, a different binding mode is however observed, most probably as the "lysine-wrapping" binding mode turned out to be a rather weak interaction. Accordingly, our studies demonstrate that structural studies of OEG-lysine interactions are difficult to interpret and their presence in structural studies may not automatically be correlated with a relevant interaction also in solution but requires further biochemical studies.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update.

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

Rational Design, Binding Studies, and Crystal-Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14-3-3ζ Adapter Protein.

14-3-3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14-3-3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14-3-3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X-ray crystallography.

Modulators of 14-3-3 Protein-Protein Interactions.

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as "undruggable" targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products.