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Computational techniques are valuable tools for the discovery of protein-protein interaction inhibitors: The 14-3-3σ case
Abstract
Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.
... In addition, studies have shown that 14-3-3σ also induces overexpression of matrix metalloproteinase 1 (MMP-1), a proteolytic enzyme that degrades native fibrillar collagens, and is often associated with poor prognosis in malignant tumor [79,93,94]. Furthermore, 14-3-3σ has also been reported to bind to the c-Abl protein, preventing its nuclear translocation and subsequently interfering with its pro-apoptotic effect [95,96]. ...
... To overcome this issue, Corradi and his group used computational techniques, in combination with biophysical and biochemical techniques, to investigate a new set of promising hits with a stable scaffold at room temperature, while Iralde-Lorente and colleagues proposed a synthetic scheme of compound 14 and its chemically stable derivatives. These studies successfully identified two synthesizable and chemically stable compounds, BV01 (15) and 16 ( Figure 8a) which showed antiproliferative activity against IM-resistant cells expressing the T315I Bcr-Abl mutation, and a K-562 erythroleukemia cell line at low micromolar concentrations, respectively [95,114]. ...
... Compound 13 was reported to be able to disrupt the interaction between 14-3-3σ and c-Abl protein and subsequently promotes c-Abl translocation into the nucleus and provide antiproliferative effects in CML cells expressing the imatinib-resistant T315I Bcr-Abl construct [111,112]. Unfortunately, further studies using NMR techniques showed that 13 undergoes spontaneous chemical rearrangement at room temperature and exists in equilibrium between 2-carbamoyl benzoic form (13) and its bioactive phthalimidic form, 9 (14) [95,113]. To overcome this issue, Corradi and his group used computational techniques, in combination with biophysical and biochemical techniques, to investigate a new set of promising hits with a stable scaffold at room temperature, while Iralde-Lorente and colleagues proposed a synthetic scheme of compound 14 and its chemically stable derivatives. ...
14-3-3σ is an acidic homodimer protein with more than one hundred different protein
partners associated with oncogenic signaling and cell cycle regulation. This review aims to highlight the crucial role of 14-3-3σ in controlling tumor growth and apoptosis and provide a detailed discussion on the structure-activity relationship and binding interactions of the most recent 14-3-3σ protein-protein interaction (PPI) modulators reported to date, which has not been reviewed previously. This includes the new fusicoccanes stabilizers (FC-NAc, DP-005), fragment stabilizers (TCF521-123,
TCF521-129, AZ-003, AZ-008), phosphate-based inhibitors (IMP, PLP), peptide inhibitors (2a–d), as well as inhibitors from natural sources (85531185, 95911592). Additionally, this review will also include the discussions of the recent efforts by a different group of researchers for understanding the binding mechanisms of existing 14-3-3σ PPI modulators. The strategies and state-of-the-art techniques applied by various group of researchers in the discovery of a different chemical class of 14-3-3σ modulators for cancer are also briefly discussed in this review, which can be used as a guide in the development of new 14-3-3σ modulators in the near future
... Following the discovery of 12, two additional 14-3-3 PPI inhibitors have been reported. These molecules, namely BV01 (10) and BV101 (11) (Figure 9C), were also initially discovered by an in silico approach, 126 and promoted c-Abl nuclear translocation in Ba/F3 cells expressing the WT and the Imatinib-resistant T315I-mutated Bcr-Abl constructs. Furthermore, the interaction of 10 with 14-3-3σ was supported by transfer NOE experiments. ...
... Furthermore, the interaction of 10 with 14-3-3σ was supported by transfer NOE experiments. 126 In 2014, the same group published the discovery of compound 9 (13, Figure 9C), a phthalimide derivative of 12 that is able to promote c-Abl nuclear translocation as well as to sensitize multidrug-resistant (MDR) cancer stem cells. 127 This discovery was facilitated by in silico docking of a virtual library of 12 and 10 analogues to a 14-3-3σ crystal structure using a well-established computational protocol. ...
... (C) Chemical structure of 14-3-3 PPI inhibitors 10−13 identified by the group of Botta. The reversible hydration pathway converts 12 to 13 and vice versa.124,126,127 ...
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 a 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.
... Despite these challenges, pharmacophore models have still successfully been applied to protein-protein interactions, in certain cases. Most notably, several groups have recently developed related computational approaches that select some of the side chains from the natural protein partner (the "hotspot" or "anchor" residues), and then use this collection of functional groups to build a pharmacophore [38][39][40][41][42][43][44][45] . However, this approach comes with several important limitations. ...
... Given the overabundance of certain sidechains as "anchor residues" (phenylalanine, tyrosine, tryptophan, valine, and leucine), this approach lends itself naturally to construction of custom screening libraries (accessible through multi-component reaction chemistry) containing compounds mimicking these sidechains 39 . Others have also used similar methods to extract specific sidechains -or smaller functional groups -from protein complexes, and defined pharmacophores based on these as templates for virtual screening [40][41][42][43][44][45] . ...
... Prior studies have sought to incorporate multiple conformations when using sidechain mimicry [42][43][44] or pharmacophores built from docking probe compounds [84][85][86] . Typically, these approaches then consolidate pharmacophores into a small number of consensus templates for virtual screening. ...
Protein-protein interactions play important roles in virtually all cellular processes, making them enticing targets for modulation by small-molecule therapeutics: specific examples have been well validated in diseases ranging from cancer and autoimmune disorders, to bacterial and viral infections. Despite several notable successes, however, overall these remain a very challenging target class. Protein interaction sites are especially challenging for computational approaches, because the target protein surface often undergoes a conformational change to enable ligand binding: this confounds traditional approaches for virtual screening. Through previous studies, we demonstrated that biased "pocket optimization" simulations could be used to build collections of low-energy pocket-containing conformations, starting from an unbound protein structure. Here, we demonstrate that these pockets can further be used to identify ligands that complement the protein surface. To do so, we first build from a given pocket its "exemplar": a perfect, but non-physical, pseudo-ligand that would optimally match the shape and chemical features of the pocket. In our previous studies, we used these exemplars to quantitatively compare protein surface pockets to one another. Here, we now introduce this exemplar as a template for pharmacophore-based screening of chemical libraries. Through a series of benchmark experiments, we demonstrate that this approach exhibits comparable performance as traditional docking methods for identifying known inhibitors acting at protein interaction sites. However, because this approach is predicated on ligand/exemplar overlays, and thus does not require explicit calculation of protein-ligand interactions, exemplar screening provides a tremendous speed advantage over docking: 6 million compounds can be screened in about 15 minutes on a single 16-core, dual-GPU computer. The extreme speed at which large compound libraries can be traversed easily enables screening against a "pocket-optimized" ensemble of protein conformations, which in turn facilitates identification of more diverse classes of active compounds for a given protein target.
... To overcome this issue, we developed an additional 14-3-3 PPI inhibitor, BV01, which has anti-proliferative activity in vitro at low micromolar concentration and was confirmed by NMR to bind directly to 14-3-3s. [17] Moreover, we have optimized a high-throughput cellular imaging assay that monitors nuclear/cytoplasmic compartmentalization and translocation of a fluorescent GPF-FOXO3A fusion protein in tumor cells, which is a valuable tool to effectively screen 14-3-3 PPI inhibitors in vitro. [18] Here, with the aim of providing 14-3-3 PPI inhibitors with improved potency as lead candidates for anticancer therapy and affording structure-activity relationships (SAR) for this class of molecules, we generated and screened in silico a focused library of compounds based on BV01 and BV02. ...
... [19] Accordingly, these molecules are thought to inhibit 14-3-3 PPIs by binding directly to 14-3-3, as confirmed by previous NMR studies. [17] With the aims of providing lead candidates characterized by enhanced activity and affording SAR that may be useful for improving ligand binding affinity to 14-3-3, we generated a focused virtual library of compounds in silico by combining 400 different heads with eight different tails by means of a custom script in Python language, based on the OpenEye OEChem Python Toolkit for library generation (OELibrary-Gen). [20] When generating the virtual library, the synthetic feasibility of the compounds was taken into account to allow straightforward and accessible synthesis of the most promising virtual hits. ...
... BV02 has been characterized as the first non-peptidic or peptidomimetic 14-3-3 PPI inhibitor. [16] However, we noticed that it underwent spontaneous cyclization at room temperature, producing the opened 2-carbamoyl benzoic derivative in equilibrium with its closed phthalimide form, as reported in Scheme 1, [17] thus limiting the identification of the chemical structure responsible for the observed biological activity. HPLC and LC-MS analyses clearly confirmed the presence of both species in solution (see Supporting Information for details). ...
14-3-3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small-molecule inhibitors of 14-3-3 protein–protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14-3-3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14-3-3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image-based high-content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c-Abl and FOXO pro-apoptotic factors into the nucleus and sensitized multidrug-resistant cancer cells to apoptotic inducers such as doxorubicin and the pan-Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14-3-3 PPI inhibitors in anticancer combination therapies.
... BV02 was identified out of 200 000 compounds screened in silico for inhibition of the 14-3-3σ/c-Abl interaction [80]. BV01 and BV101 were similarly discovered [82]. Further BV02 modifications have led to more stable compounds that disrupt the 14-3-3σ/c-Abl interaction [79]. ...
... PPI modulation continues to prove difficult compared with more traditional drug discovery methods, yet ,as technology examining 14-3-3 PPI interactions advances, the potential for targeting PPIs for therapeutics increases. Rational drug design using structural information from X-ray crystallography along with computational molecular modeling techniques and even proteomics, as illustrated in several examples earlier, will lead to more potent and specific compounds [82,86,92,[102][103][104][105]. ...
14-3-3 proteins are a family of proteins expressed throughout the body and implicated in many diseases, from cancer to neurodegenerative disorders. While these proteins do not have direct enzymatic activity, they form a hub for many signaling pathways via protein–protein interactions (PPIs). 14-3-3 interactions have proven difficult to target with traditional pharmacological methods due to the unique nature of their binding. However, recent advances in compound development utilizing a range of tools, from thermodynamic binding site analysis to computational molecular modeling techniques, have opened the door to targeting these interactions. Compounds are already being developed targeting 14-3-3 interactions with potential therapeutic implication for neurodegenerative disorders, but challenges still remain in optimizing specificity and target engagement to avoid unintended negative consequences arising from targeting 14-3-3 signaling networks.
... Our research group has long been involved in targeting 14-3-3 proteins as a strategy to treat chronic myeloid leukaemia (CML) [8][9][10] . The first non-peptidic inhibitor of 14-3-3/c-Abl interaction (namely, BV02, Figure 1) has been discovered by virtual screening of a commercial library of compounds. ...
... The crystallographic structure of 14-3-3r in complex with a phosphopeptide coded by PDB ID: 1YWT was used as rigid receptor in molecular docking simulations, upon removal of the coordinates of the phosphopeptide and crystallographic water molecules 18 . Docking was carried out by the GOLD docking program version 5.0.1 (The Cambridge Crystallographic Data Centre, Cambridge, UK) 19,20 using settings described previously 10,11 . ...
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.
... Over the years, the implementation of computational tools has led to the development of non-peptidic 14-3-3 inhibitors. The group of Botta described the first non-phosphonate small-molecule inhibitors of 14-3-3 PPIs (BV02, BV101) ( Figure 7D) by applying structure-based pharmacophore modeling, virtual screening, and molecular docking simulations [59]. Other examples are the phosphonate inhibitors identified from a virtual screening, with follow-up analysis, synthesis, and crystallization [60]. ...
... Over the years, the implementation of computational tools has led to the development of non-peptidic 14-3-3 inhibitors. The group of Botta described the first nonphosphonate small-molecule inhibitors of 14-3-3 PPIs (BV02, BV101) ( Figure 7D) by applying structure-based pharmacophore modeling, virtual screening, and molecular docking simulations [59]. Other examples are the phosphonate inhibitors identified from a virtual screening, with followup analysis, synthesis, and crystallization [60]. ...
In recent years, targeting the complex network of protein⁻protein interactions (PPIs) has been identified as a promising drug-discovery approach to develop new therapeutic strategies. 14-3-3 is a family of eukaryotic conserved regulatory proteins which are of high interest as potential targets for pharmacological intervention in human diseases, such as cancer and neurodegenerative and metabolic disorders. This viewpoint is built on the “hub” nature of the 14-3-3 proteins, binding to several hundred identified partners, consequently implicating them in a multitude of different cellular mechanisms. In this review, we provide an overview of the structural and biological features of 14-3-3 and the modulation of 14-3-3 PPIs for discovering small molecular inhibitors and stabilizers of 14-3-3 PPIs.
... Cette inhibition pourrait être directe, grâce à l'utilisation d'inhibiteurs d'interaction protéine-protéine (2P2I). Il a en effet été démontré très récemment que l'inhibition spécifique d'une interaction protéine-protéine était possible grâce à ces molécules, réalisées à l'aide d'analyses computationnelles et structurale (Corradi et al., 2011). Ainsi, plusieurs 2P2I ont déjà été mis au point avec succès (Corradi et al., 2011;Morelli et al., 2011). ...
... Il a en effet été démontré très récemment que l'inhibition spécifique d'une interaction protéine-protéine était possible grâce à ces molécules, réalisées à l'aide d'analyses computationnelles et structurale (Corradi et al., 2011). Ainsi, plusieurs 2P2I ont déjà été mis au point avec succès (Corradi et al., 2011;Morelli et al., 2011). Cette technique pourrait permettre de déterminer avec précision le rôle de chaque interaction, et donc de savoir à quel niveau du système ERQC intervient Gα 12 . ...
F508del, the most frequent mutation found in cystic fibrosis (CF) population, impacts CFTR (Cystic Fibrosis Transmembrane conductance Regulator) trafficking and causes its rapid degradation at the endoplasmic compartment, resulting in a significant decrease in Cl- secretion at the apical membrane of epithelial cells. F508del has two main features, significant thickening of the bronchial mucus and a reduction in the integrity of the luminal barrier of the bronchial epithelium. These two phenomena are involved in the invasion and infection of lung tissue by pathogenic bacteria such as Pseudomonas aeruginosa, exacerbating the inflammation and lung destruction. The objective of this study was to determine the role of two proteins member of the heterotrimeric G proteins family, G12 and G13, in the degradation of the F508del CFTR, and in the control of junctional complexes in the normal and CF bronchial epithelium. Our results show for the first time that G12 and G13 are down expressed in CF. G12, but not G13, is involved in the control of F508del-CFTR degradation through its interaction with Calnexin and HSP90 chaperones. Unlike kidney epithelia cells, G12 promotes the formation and maintenance of cell junctions in the bronchial epithelium by affecting E-cadherine and ZO-1 stability. Altogether, our results set therefore G12 as a significant actor of the CF disease.
... The 14-3-3 proteins can be pharmacological targets, and various compounds have been developed to target their interactions in signaling pathways involved in cancers and other diseases. Using computational methods, Corradi and colleagues [28] discovered BV01 and BV101 as inhibitors of 14-3-3σ -c-Abl interactions that have anti-proliferative activity in chronic myeloid leukemia. BV02 [29] and its derivatives [30] are among other inhibitors of 14-3-3 -c-Abl interaction that induce apoptosis in leukemia. ...
Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cuta-neous 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 Gln 176 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.
... The structural domain on 14-3-3η that used for screening hit compound P6 from ChemDiv database contained amino acid residues LYS-50, ARG-57 and ARG-132, which were determined by molecular dynamics simulation [18]. Similar binding sites were also reported to participate in the interactions between 14-3-3σ or 14-3-3ζ with their small-molecule inhibitors [36,37], which might be attributed to the identity and conservation of amino acid sequence of 14-3-3 proteins [9]. ...
In this work, a series of novel 1H-indole-2-carboxylic acid derivatives targeting 14-3-3η protein were designed and synthesized for treatment of liver cancer. After structural optimization for several rounds, C11 displayed a relatively better affinity with 14-3-3η, as well as the best inhibitory activities against several typical human liver cancer cell lines, including Bel-7402, SMMC-7721, SNU-387, Hep G2 and Hep 3B cells. Compound C11 also displayed best inhibitory activity against chemotherapy-resistant Bel-7402/5-Fu cells. Besides, C11 was rather safe against hERG and possessed moderate T1/2 and CL values in liver microsomes. In anti-proliferation, trans-well and cell apoptosis assays, C11 also showed its huge potential as a potent antitumor agent. Then, Western blot assay was conducted, following analyzed by molecular docking, the anti-proliferative mechanisms of this small-molecule inhibitor were revealed. Moreover, C11 was demonstrated to induce G1-S phase cell cycle arrest in liver cancer cells.
... A number of 14− 3-3 PPI modulators have been reported; molecules that act as inhibitors or stabilisers of the interaction have both been described [122]. Peptidic and non-peptidic inhibitors have been developed including the R18 peptide [123], phosphonate-containing small molecules [124] and non-phosphate inhibitors such as BV02 and BV101 [125]. 14− 3-3 PPI stabilisers have received particular attention. ...
Regulation of inflammation is a central part of the maintenance of homeostasis by the immune system. One important class of regulatory protein that has been shown to have effects on the inflammatory process are the 14−3-3 proteins. Herein we describe the roles that have been identified for 14−3-3 in regulation of the inflammatory response. These roles encompass regulation of the response that affect inflammation at the genetic, molecular and cellular levels. At a genetic level 14−3-3 is involved in the regulation of multiple transcription factors and affects the transcription of key effectors of the immune response. At a molecular level many of the constituent parts of the inflammatory process, such as pattern recognition receptors, protease activated receptors and cytokines are regulated through phosphorylation and recognition by 14−3-3 whilst disruption of the recognition processes has been observed to result in clinical syndromes. 14−3-3 is also involved in the regulation of cell proliferation and differentiation, this has been shown to affect the immune system, particularly T- and B-cells. Finally, we discuss how abnormal levels of 14−3-3 contribute to undesirable immune responses and chronic inflammatory conditions.
... Several small-molecule 14-3-3 inhibitors have been developed, but, to our knowledge, few have moved past in vitro proof-of-concept. Botta and colleagues developed a non-peptidic 14-3-3 inhibitor that effectively induces apoptosis of imatinib-resistant BCR-Abl-expressing leukemia cells [48,49]. The same group further optimized their compounds and settled on two 14-3-3 inhibitors that sensitize multi-drug resistant non-small-cell lung carcinoma and colon carcinoma cell lines to doxorubicin-induced apoptosis [50]. ...
14-3-3 proteins are a family of structurally similar phospho-binding proteins that regulate essentially every major cellular function. Decades of research on 14-3-3s have revealed a remarkable network of interacting proteins that demonstrate how 14-3-3s integrate and control multiple signaling pathways. In particular, these interactions place 14-3-3 at the center of the signaling hub that governs critical processes in cancer, including apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. Historically, the majority of 14-3-3 interactions have been identified and studied under nutrient-replete cell culture conditions, which has revealed important nutrient driven interactions. However, this underestimates the reach of 14-3-3s. Indeed, the loss of nutrients, growth factors, or changes in other environmental conditions (e.g., genotoxic stress) will not only lead to the loss of homeostatic 14-3-3 interactions, but also trigger new interactions, many of which are likely stress adaptive. This dynamic nature of the 14-3-3 interactome is beginning to come into focus as advancements in mass spectrometry are helping to probe deeper and identify context-dependent 14-3-3 interactions—providing a window into adaptive phosphorylation-driven cellular mechanisms that orchestrate the tumor cell’s response to a variety of environmental conditions including hypoxia and chemotherapy. In this review, we discuss emerging 14-3-3 regulatory mechanisms with a focus on post-translational regulation of 14-3-3 and dynamic protein–protein interactions that illustrate 14-3-3’s role as a stress-adaptive signaling hub in cancer.
... Each compound is scored against how well it matches these elements and ranked. The highest ranked compounds can then be assessed in an appropriate bioassay 47,[55][56][57][58][59] . An example of the protein-based pharmacophore approach is the identification of substituted 1,2,4-triazoles as inhibitors of the S100A10-annexin A2 protein interaction (see Supplementary information S4 (table)) 55 . ...
Historically, targeting protein-protein interactions with small molecules was not thought possible because the corresponding interfaces were considered mostly flat and featureless and therefore 'undruggable'. Instead, such interactions were targeted with larger molecules, such as peptides and antibodies. However, the past decade has seen encouraging breakthroughs through the refinement of existing techniques and the development of new ones, together with the identification and exploitation of unexpected aspects of protein-protein interaction surfaces. In this Review, we describe some of the latest techniques to discover modulators of protein-protein interactions and how current drug discovery approaches have been adapted to successfully target these interfaces.
... A second virtual screening run based on highthroughput docking with GOLD allowed the discovery of two other inhibitors with improved potency and chemical stability [80]. ...
As the pivotal role of protein-protein interactions in cell growth, transcriptional activity, intracellular trafficking, signal transduction and pathological conditions has been assessed, experimental and in silico strategies have been developed to design protein-protein interaction modulators. State-of-the-art structure-based design methods, mainly pharmacophore modeling and docking, which have succeeded in the identification of enzyme inhibitors, receptor agonists and antagonists, and new tools specifically conceived to target protein-protein interfaces (e.g., hot-spot and druggable pocket prediction methods) have been applied in the search for small-molecule protein-protein interaction modulators. Many successful applications of structure-based design approaches that, despite the challenge of targeting protein-protein interfaces with small molecules, have led to the identification of micromolar and submicromolar hits are reviewed here.
... This procedure is aimed at identifying compounds with a certain conformation and chemical composition that matches the requirements of a pharmacophore model. The pharmacophore-based approach has been successfully utilized to identify novel inhibitors of the MDM2/p53 interaction [50,51], PPIs of BCL2 family proteins [52], and 14-3-3 inhibitors [53]. Conversely, the structure-based approach relies on the structural information of binding site on the target protein. ...
The emergence and convergence of cancer genomics, targeted therapies, and network oncology have significantly expanded the landscape of protein-protein interaction (PPI) networks in cancer for therapeutic discovery. Extensive biological and clinical investigations have led to the identification of protein interaction hubs and nodes that are critical for the acquisition and maintenance of characteristics of cancer essential for cell transformation. Such cancer-enabling PPIs have become promising therapeutic targets. With technological advances in PPI modulator discovery and validation of PPI-targeting agents in clinical settings, targeting of PPI interfaces as an anticancer strategy has become a reality. Future research directed at genomics-based PPI target discovery, PPI interface characterization, PPI-focused chemical library design, and patient-genomic subpopulation-driven clinical studies is expected to accelerate the development of the next generation of PPI-based anticancer agents for personalized precision medicine. Here we briefly review prominent PPIs that mediate cancer-acquired properties, highlight recognized challenges and promising clinical results in targeting PPIs, and outline emerging opportunities.
We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host–virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.
The anticancer therapeutic effects of usnic acid (UA), a lichen secondary metabolite, have been demonstrated in vitro and in vivo. However, the mechanism underlying the anticancer effect of UA remains to be clarified. In this study, the target protein of UA was identified using a UA-linker-Affi-Gel molecule, which showed that UA binds to the 14-3-3 protein. UA binds to 14-3-3, causing the degradation of proteasomal and autophagosomal proteins. The interaction of UA with 14-3-3 isoforms modulated cell invasion, cell cycle progression, aerobic glycolysis, mitochondrial biogenesis, and the Akt/mTOR, JNK, STAT3, NF-κB, and AP-1 signaling pathways in colorectal cancer. A peptide inhibitor of 14-3-3 blocked or regressed the activity of UA and inhibited its effects. The results suggest that UA binds to 14-3-3 isoforms and suppresses cancer progression by affecting 14-3-3 targets and phosphorylated proteins.
Protein–protein interactions (PPIs) have been sought as putative therapeutic targets for the advancement of various new treatments. This chapter deals with the various studies that have successfully discovered small-molecule inhibitors (SMIs) associated with particular disease-causing PPI. The employed methodologies in these studies as well as the conclusive results have been thoroughly discussed. Further, other aspects of the discovery like optimization of the process, strategizing drug binding, selection of targets have also been delineated. This chapter thus provides the reader with a comprehensive account of the current state-of-art in the discovery of small molecules inhibiting PPIs. It also throws light on the future potential of these small molecules as commercial drug candidates.
Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein–protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.
The chemistry of chalcone has been recognized as a significant field of study. Chalcone serve as to prepare starting materials for the synthesis of various heterocyclic compounds. From the backbone of reported literature, we have developed an alternative heterogeneous and simple catalytic system for the synthesis of chalcones via the oxidative condensation of benzyl alcohol with substituted acetophenone using metal nitrate supported HY-Zeolite as a catalyst. 30 mol% CAN supported HY-zeolite has been efficiently used as a catalyst for the oxidative condensation reaction of benzyl alcohol with substituted acetophenones in the presence of hydrogen peroxide as an oxidant in toluene to afford the corresponding chalcones in good to moderate yields. Docking studies were carried out for the synthesized compounds towards the protein Lysine aminotransferase using the software.
The 14-3-3 family comprises multifunctional proteins that play a role in neurogenesis, neuronal migration, neuronal differentiation, synaptogenesis and dopamine synthesis. 14-3-3 members function as adaptor proteins and impact a wide variety of cellular and physiological processes involved in the pathophysiology of neurological disorders. Schizophrenia is a psychiatric disorder and knowledge about its pathophysiology is still limited. 14-3-3 have been proven to be linked with the dopaminergic, glutamatergic and neurodevelopmental hypotheses of schizophrenia. Further, research using genetic models has demonstrated the role played by 14-3-3 proteins in neurodevelopment and neuronal circuits, however a more integrative and comprehensive approach is needed for a better understanding of their role in schizophrenia. For instance, we still lack an integrated assessment of the processes affected by 14-3-3 proteins in the dopaminergic and glutamatergic systems. In this context, it is also paramount to understand their involvement in the biology of brain cells other than neurons. Here, we present previous and recent research that has led to our current understanding of the roles 14-3-3 proteins play in brain development and schizophrenia, perform an assessment of their functional protein association network and discuss the use of protein-protein interaction modulators to target 14-3-3 as a potential therapeutic strategy.
We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease.
One sentence summary:
A host-targeted drug to treat all respiratory viruses without viral resistance development.
Candida albicans, a polymorphic fungal species of human microflora, is pathogenic and known to cause immense damage to the host organism which includes biofilm formation, oral and skin infections in immune deficient individuals. Secreted aspartic proteinase (SAP) enzyme plays a major role in promoting virulence to C. albicans, and thus could be established as a drug target for Candida infections. As a result, inhibiting the enzyme's active center using phytochemicals would reduce the severity of the enzyme's virulence. The present work focuses on the in silico analysis of about 15 plant phytochemicals against the SAP enzyme using the AutoDock 4.2.6 software. The docking results were found to be promising with emodin having the highest binding score of −6.44 kCal/mol followed by the isoflavonoid equol with the binding score of −6.29 kCal/mol. Thus, these bioactive compounds could be used as leads for drugs targeting SAP enzymes in treating resistant Candida infections.
The 14-3-3 protein family is implicated in several diseases and biological processes. Several recent reviews have summarized knowledge on certain aspects of 14-3-3 proteins, ranging from a historic overview to the structure, function and regulation. This review focuses on the structures and molecular recognition of the modulators by the 14-3-3 proteins, and small modifications of certain modulators are proposed where cocrystal structures have been reported. Our analysis opens up possibilities for the optimisation of the reported compounds. It is very timely to analyse the current status of recently developed modulators given that the field has seen a lot of activity in recent years. This review provides an overview combined with a critical analysis of each class of modulators, keeping their suitability for future development in mind.
Protein-protein interaction is one of the key events in the signal transduction pathway. The interaction changes the conformations, activities, localization and stabilities of the proteins, and transduces the signal to the next step. Frequently, this interaction occurs upon the protein phosphorylation. When upstream signals are stimulated, protein kinase(s) is/are activated and phosphorylate(s) their substrates, and induce the phosphorylation dependent protein-protein interaction. For this interaction, several domains in proteins are known to specifically recognize the phosphorylated residues of target proteins. These specific domains for interaction are important in the progression of the diseases caused by disordered signal transduction such as cancer. Thus small molecules that modulate this interaction are attractive lead compounds for the treatment of such diseases. In this review, we focused on three examples of phosphorylation dependent protein-protein interaction modules (14-3-3, polo box domain of Plk1 and F-box proteins in SCF ubiquitin ligases) and summarize small molecules that modulate their interaction. We also introduce our original screening system to identify such small molecules.
BV02 is a reference inhibitor of 14-3-3 protein–protein interactions, which is currently used as chemical biology tool to understand the role of 14-3-3 proteins in pathological contexts. Due to chemical instability in certain conditions, its bioactive form has remained unclear. Here, we use NMR spectroscopy to prove for the first time the direct interaction between the molecule and 14-3-3σ, and to depict its bioactive form, namely the phthalimide derivative 9. Our work provides molecular insights to the bioactive form of the 14-3-3 PPI inhibitor and facilitates further development as candidate therapeutic agent.
Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite Giardia duodenalis, which affects annually over 200 million people worldwide. The limited antigiardial drug arsenal and the emergence of clinical cases refractory to standard treatments dictate the need for new chemotherapeutics. The 14-3-3 family of regulatory proteins, extensively involved in protein-protein interactions (PPIs) with pSer/pThr clients, represents a highly promising target. Despite homology with human counterparts, the single 14-3-3 of G. duodenalis (g14-3-3) is characterized by a constitutive phosphorylation in a region critical for target binding, thus affecting the function and the conformation of g14-3-3/clients interaction. However, to approach the design of specific small molecule modulators of g14-3-3 PPIs, structural elucidations are required. Here, we present a detailed computational and crystallographic study exploring the implications of g14-3-3 phosphorylation on protein structure and target binding. Self-Guided Langevin Dynamics and classical molecular dynamics simulations show that phosphorylation affects locally and globally g14-3-3 conformation, inducing a structural rearrangement more suitable for target binding. Profitable features for g14-3-3/clients interaction were highlighted using a hydrophobicity-based descriptor to characterize g14-3-3 client peptides. Finally, the X-ray structure of g14-3-3 in complex with a mode-1 prototype phosphopeptide was solved and combined with structure-based simulations to identify molecular features relevant for clients binding to g14-3-3. The data presented herein provide a further and structural understanding of g14-3-3 features and set the basis for drug design studies.
A new class of target molecules containing p-aminobenzoic acid and benzenesulfonamide moiety is reported, based on the structural features of a series of analogues with strong biological activities previously synthesized by the group. By taking 4 discrete synthetic routes, the practical procedures and facile preparative routes for both the intermediates of IM1-IM3 and the target molecules of TM1 and TM2 were established. A total of 26 designed compounds were synthesized smoothly using the established synthetic approaches under mild reaction conditions, with low cost and high yields (64%∼ 95%). The chemical structures of 21 new compounds were confirmed by 1H NMR, 13C NMR and HRMS techniques. The bioassay test demonstrates weak antidiabetic activity for all the target molecules. This study has further expanded the application of alcohol/SOCI2 system in the deacylation of N-arylacetamides and chloro-N-arylacetamides as well as esterification of carboxy group concomitantly, which is supportive to the structure optimization of novel molecules containing p-aminobenzoic acid and benzenesulfonamide moiety.
One of the proteins that is found in a diverse range of eukaryotic protein-protein interactions is the adaptor protein 14-3-3. As 14-3-3 is a hub protein with very diverse interactions, it is a good model to study various protein-protein interactions. A wide range of classes of molecules, peptides, small molecules or natural products, has been used to modify the protein interactions, providing both stabilization or inhibition of the interactions of 14-3-3 with its binding partners. The first protein crystal structures were solved in 1995 and gave molecular insights for further research. The plant analog of 14-3-3 binds to a plant plasma membrane H(+)-ATPase and this protein complex is stabilized by the fungal phytotoxin fusicoccin A. The knowledge gained from the process in plants was transferred to and applied in human models to find stabilizers or inhibitors of 14-3-3 interaction in human cellular pathways.
14-3-3 is a family of highly conserved regulatory proteins which is attracting a significant interest due to its potential role as target for pharmacological intervention against cancer and neurodegenerative disorders. Although modulating protein–protein interactions (PPI) is still conceived as a challenging task in drug discovery, in past few years peptide inhibitors and small molecular modulators of 14-3-3 PPI have been described. Here we examine structural and biological features of 14-3-3 and propose an overview on techniques used for discovering small molecular inhibitors and stabilizers of 14-3-3 PPI.
14-3-3 Proteins are eukaryotic adapter proteins that regulate a plethora of physiological processes by binding to several hundred partner proteins. They play a role in biological activities as diverse as signal transduction, cell cycle regulation, apoptosis, host-pathogen interactions and metabolic control. As such, 14-3-3s are implicated in disease areas like cancer, neurodegeneration, diabetes, pulmonary disease, and obesity. Targeted modulation of 14-3-3 protein-protein interactions (PPIs) by small molecules is therefore an attractive concept for disease intervention. In recent years a number of examples of inhibitors and stabilizers of 14-3-3 PPIs have been reported promising a vivid future in chemical biology and drug development for this remarkable class of proteins.
Small-molecule modulation of protein-protein interactions (PPIs) is one of the most exciting but also difficult fields in chemical biology and drug development. As one of the most important "hub" proteins with at least 200 - 300 interaction partners the 14-3-3 proteins are an especially fruitful case for PPI intervention. Here, we summarize recent success stories in small-molecule modulation - both inhibition and stabilization - of 14-3-3 PPIs. The chemical breath of modulators includes natural products such as fusicoccin A and derivatives. But also compounds identified via high throughput- and in silico-screening, which has yielded a toolbox of useful inhibitors and stabilizers for this interesting class of adapter proteins.
Pharmacophore searches have become a popular tool for virtual screening of libraries to identify novel active substances that can be potentially developed into drugs. While they have been applied for years on common drug targets, their application in the discovery of protein-protein interaction inhibitors remains limited. This review describes current pharmacophore modelling methods applied in the discovery of novel inhibitors targeting protein-protein interactions. We first address the mimicry of protein-protein interactions with their respective inhibitors as observed in crystal structure complexes. This mimicry can be exploited to derive a pharmacophore query from protein-protein complex structures. We then discuss several cases where pharmacophore queries were utilized for the discovery of first-in-class inhibitors of their respective protein-protein interaction targets. These examples have demonstrated the usefulness of pharmacophore modelling in the quest for protein-protein interaction inhibitors.
During the last decades, a large amount of evidence has been gathered on the importance of protein-protein interactions in tuning and regulating most important biological processes. Since many of these pathways are deeply involved in diseases, big research efforts have been undertaken towards the modulation of protein-protein interactions. At the early stage of this challenge most of the attention was drawn to the drawbacks of such a therapeutic approach. Encouragingly, however, several recent studies provided a proof of concept that protein-protein interactions are actually valuable targets and that they do have a promising therapeutic potential. This review is divided into three sections. In the first section we summarize the general features of protein-protein interfaces, focusing on the characteristics that make them different from classical protein-ligand binding sites, as well as on problematic aspects that hamper the application of classical drug discovery approaches. In the second section, we present how some of the characteristics of protein-protein interactions can be exploited fruitfully in drug design, hence focusing on the druggability of protein-protein interfaces. Methods successfully applied to protein-protein interactions will be introduced, giving special attention to the computational ones. In the third section, three case studies are presented. First, we describe protein-protein interaction modulators targeting HDM2 and the computational methods applied to identify them. Next, we present the retrospective application of the discussed approaches on the well-examined target IL 2. We conclude with a prospective application to the NHR2 protein, a target just recently validated experimentally with the aid of computational methods.
14-3-3 proteins regulate many cellular processes that are important in cancer biology, such as apoptosis and cell-cycle checkpoints. There are seven human 14-3-3 genes and one of these, 14-3-3σ, has been directly implicated in the aetiology of human cancer. Loss of 14-3-3σ expression sensitizes cancer cells to conventional anticancer agents, so its inhibition could be exploited for therapeutic purposes. Interference with 14-3-3 function as a therapeutic approach is being evaluated at present and, in the case of UCN-01, is under clinical investigation.
Protein–protein interactions have a key role in most biological processes, and offer attractive opportunities for therapeutic intervention. Developing small molecules that modulate protein–protein interactions is difficult, owing to issues such as the lack of well-defined binding pockets. Nevertheless, there has been important progress in this endeavour in recent years. Here, we use illustrative examples to discuss general strategies for addressing the challenges inherent in the discovery and characterization of small-molecule inhibitors of protein–protein interactions.
The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.
Fusicoccin, a naturally occurring compound, stabilizes a 14-3-3 protein–protein interaction and induces wilting of plants by opening the gas-exchanging stomatal pores (gray). C. Ottmann and co-workers describe in their Communication on page 4129 ff. how easily available small molecules have been identified that mimic the action of fusicoccin. Crystal-structure and functional biophysical analyses reveal the binding modes of these molecules.
This article describes the implementation of a new docking approach. The method uses a Tabu search methodology to dock flexibly ligand molecules into rigid receptor structures. It uses an empirical objective function with a small number of physically based terms derived from fitting experimental binding affinities for crystallographic complexes. This means that docking energies produced by the searching algorithm provide direct estimates of the binding affinities of the ligands. The method has been tested on 50 ligand-receptor complexes for which the experimental binding affinity and binding geometry are known. All water molecules are removed from the structures and ligand molecules are minimized in vacuo before docking. The lowest energy geometry produced by the docking protocol is within 1.5 Å root-mean square of the experimental binding mode for 86% of the complexes. The lowest energies produced by the docking are in fair agreement with the known free energies of binding for the ligands. Proteins 33:367–382, 1998. © 1998 Wiley-Liss, Inc.
This paper reviews the use of similarity searching in chemical databases. It begins by introducing the concept of similarity searching, differentiating it from the more common substructure searching, and then discusses the current generation of fragment-based measures that are used for searching chemical structure databases. The next sections focus upon two of the principal characteristics of a similarity measure: the coefficient that is used to quantify the degree of structural resemblance between pairs of molecules and the structural representations that are used to characterize molecules that are being compared in a similarity calculation. New types of similarity measure are then compared with current approaches, and examples are given of several applications that are related to similarity searching.
A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.
Resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitor imatinib mesylate (IM) is most often due to point mutations in the Bcr-Abl fusion gene. T315I mutation (resulting in substitution of Ile for a Thr residue at the "gatekeeper" position 315) raises particular concern, because it also provides resistance to second-generation kinase inhibitors already approved for clinical use (nilotinib and dasatinib). Much effort is therefore focused on alternative molecular-based strategies. Previous studies proved that binding to 14-3-3 scaffolding proteins leads to cytoplasmic compartmentalization and suppression of proapoptotic and antiproliferative signals associated with Bcr-Abl protein kinase, hence contributing to leukemic clone expansion. Here we investigated the effect of 14-3-3 inhibition disruption on hematopoietic cells expressing the IM-sensitive wild type Bcr-Abl and the IM-resistant T315I mutation. Using a virtual screening protocol and docking simulations, we identified a nonpeptidic inhibitor of 14-3-3, named BV02, that exhibits a remarkable cytotoxicity against both cell types. c-Abl release from 14-3-3σ, promoting its relocation to nuclear compartment (where it triggers transcription of p73-dependent proapoptotic genes) and to mitochondrial membranes (where it induces the loss of mitochondrial transmembrane potential) combined with c-Abl enhanced association with caspase 9 (a critical step of sequential caspase activation further contributing to c-Abl pro-apoptotic function) has a prominent role in the effect of BV02 on Bcr-Abl-expressing cells. In conclusion, BV02 may be considered as a treatment option for CML and, in particular, for more advanced phases of the disease that developed IM resistance as a consequence of Bcr-Abl point mutations.
An in silico structure-based ligand design approach resulted in the identification of the first non-peptidic small molecule able to inhibit protein-protein interactions between 14-3-3 and c-Abl. This compound shows an anti-proliferative effect on human leukemia cells either sensitive or resistant to Imatinib, in consequence of the T315I mutation. It also mediates c-Abl release from 14-3-3 in a way similar to that found in response to Imatinib treatment.
The way to a 14-3-3 binder: A fragment-based combinatorial small-molecule microarray generates affinity-based fingerprints of the 14-3-3σ protein. One small molecule (see picture; in red box) that disrupts the 14-3-3/protein interaction (green/blue) has been identified. The compound is cell-permeable and possesses both in vitro and in-cell activities.
Here we demonstrated that the 'loss of function' of not-rearranged c-ABL in chronic myeloid leukemia (CML) is promoted by its cytoplasmic compartmentalization bound to 14-3-3 sigma scaffolding protein. In particular, constitutive tyrosine kinase (TK) activity of p210 BCR-ABL blocks c-Jun N-terminal kinase (JNK) phosphorylation leading to 14-3-3 sigma phosphorylation at a critical residue (Ser(186)) for c-ABL binding in response to DNA damage. Moreover, it is associated with 14-3-3 sigma over-expression arising from epigenetic mechanisms (promoter hyper-acetylation). Accordingly, p210 BCR-ABL TK inhibition by the TK inhibitor Imatinib mesylate (IM) evokes multiple events, including JNK phosphorylation at Thr(183), p38 mitogen-activated protein kinase (MAPK) phosphorylation at Thr(180), c-ABL de-phosphorylation at Ser residues involved in 14-3-3 binding and reduction of 14-3-3 sigma expression, that let c-ABL release from 14-3-3 sigma and nuclear import, and address BCR-ABL-expressing cells towards apoptotic death. Informational spectrum method (ISM), a virtual spectroscopy method for analysis of protein interactions based on their structure, and mathematical filtering in cross spectrum (CS) analysis identified 14-3-3 sigma/c-ABL binding sites. Further investigation on CS profiles of c-ABL- and p210 BCR-ABL-containing complexes revealed the mechanism likely involved 14-3-3 precluded phosphorylation in CML cells.
Docking simulations were used to predict the most favorable interaction between the T315I mutated form of Abl (invariably associated with resistance to the tyrosine kinase inhibitor imatinib mesylate, IM) and C6-unsubstituted and substituted pyrazolo[3,4-d]pyrimidines previously found to be dual Src/Abl inhibitors. Two C6-unsubstituted (1 and 2) and eight C6-substituted compounds (3-10) were selected and assayed for their effects on the Ba/F3 cell line transducing the wild-type p210Bcr-Abl construct, which is IM-sensitive, or three of the most common mutations associated with IM resistance in vivo (T315I, Y253F, and E255K), and driven to drug resistance by saturating doses of IL-3 or by the expression of the Bcr-Abl construct coding for the p185 protein of acute lymphoblastic leukemia. Compounds 1 and 2 were active against all cell lines assayed (LD(50) range: 0.7-4.3 microM), whereas C6-substituted compounds exhibited lower activity (LD(50) approximately 8 microM for compound 3 toward the T315I mutant). Notably, 1 and 2 were also effective toward the T315I mutation, which is insensitive to dual Src/Abl inhibitors. The cytotoxic effects of 1 and 2 on IM-sensitive and IM-resistant Ba/F3 cells were attributable, at least in part, to their pro-apoptotic activity. Taken together, such findings suggest that C6-unsubstituted pyrazolo[3,4-d]pyrimidines may represent useful inhibitors to target IM-resistant chronic myeloid leukemia.
Understanding the principles whereby macromolecular biological receptors can recognise small molecule substrates or inhibitors is the subject of a major effort. This is of paramount importance in rational drug design where the receptor structure is known (the "docking" problem). Current theoretical approaches utilise models of the steric and electrostatic interaction of bound ligands and recently conformational flexibility has been incorporated. We report results based on software using a genetic algorithm that uses an evolutionary strategy in exploring the full conformational flexibility of the ligand with partial flexibility of the protein, and which satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Results are reported on five test systems showing excellent agreement with experimental data. The design of the algorithm offers insight into the molecular recognition mechanism.
This paper describes the development of a simple empirical scoring function designed to estimate the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to estimate lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalises flexibility. The coefficients of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coefficients obtained are stable to changes in the composition of the training set. The function is also tested on two test sets containing a further 20 and 10 complexes, respectively. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.
The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K(D) values of 7-9 x 10(-)(8) M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3-R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3zeta revealed the occupancy of the general binding groove of 14-3-3zeta by R18, explaining the potent inhibitory effect of R18 on 14-3-3-ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications.
Many human diseases are the result of abnormal protein-protein interactions involving endogenous proteins, proteins from pathogens or both. The inhibition of these aberrant associations is of obvious clinical significance. Because of the diverse nature of protein-protein interactions, however, the successful design of therapeutics requires detailed knowledge of each system at a molecular and atomic level. Several recent studies have identified and/or characterised specific interactions from various disease systems, including cervical cancer, bacterial infection, leukaemia and neurodegenerative disease. A range of approaches are being developed to generate inhibitors of protein-protein interactions that may form useful therapeutics for human disease.
Co-ordinated progression through the cell cycle is essential for the maintenance of genomic integrity. Several checkpoint mechanisms guarantee that the next step in cell cycle progression is only entered after error-free completion of the previous phase. Cell cycle deregulation caused by changes in 14-3-3 expression has been implicated in cancer formation. 14-3-3 proteins function at several key points in G(1)/S- and G(2)/M-transition by binding to regulatory proteins and modulating their function. In most cases, the association with 14-3-3 proteins requires a specific phosphorylation of the protein ligand and mediates cell cycle arrest. 14-3-3 binding may lead to cytoplasmic sequestration of the protein ligand but may also have other functional consequences. The 14-3-3sigma gene is induced by p53 and its product inhibits G(2)/M progression by cytoplasmatic sequestration of CDC2-cyclin B complexes. In addition, 14-3-3 proteins have been implicated in the transcriptional regulation of CDK-inhibitors as they modulate the transcription factors p53, FOXO and MIZ1. Effects of 14-3-3 proteins on cell cycle progression and the regulation of 14-3-3 activity during the cell cycle are reviewed in this chapter.
14-3-3 proteins are a family of highly conserved cellular proteins that play key roles in the regulation of central physiological pathways. More than 200 14-3-3 target proteins have been identified, including proteins involved in mitogenic and cell survival signaling, cell cycle control and apoptotic cell death. Importantly, the involvement of 14-3-3 proteins in the regulation of various oncogenes and tumor suppressor genes points to a potential role in human cancer. The present review summarizes current findings implicating a 14-3-3 role in cancer while discussing potential mechanisms and points of action of 14-3-3 during cancer development and progression.
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