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

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 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

Mycobacterial carbonic anhydrase inhibition with phenolic acids and esters: kinetic and computational investigations

4A series of phenolic acids and some of their esters, derivatives of caffeic, ferulic, and p-coumaric acid, was investigated for the inhibition of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Mycobacterium tuberculosis, Rv1248, Rv3588 and Rv3273 β-CAs. Some of these compounds were low micromolar inhibitors of the pathogenic enzymes and they did not show inhibitory activity against the human widespread cytosolic isoforms CA I and II. The binding mode of these inhibitors to two of the bacterial enzymes was investigated by computational approaches. We propose that the inhibitors anchor to the zinc-coordinated water molecule from the CA active site interfering with the nucleophilic attack of the zinc hydroxide on the substrate CO2. These compounds may be considered as interesting anti-mycobacterial lead compounds. © 2016 The Royal Society of Chemistry.nonenoneCau, Ylenia; Mori, Mattia; Supuran, Claudiu T; Botta, MaurizioCau, Ylenia; Mori, Mattia; Supuran, Claudiu T; Botta, Maurizi

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

Molecular Dynamics Simulations and Structural Analysis of Giardia duodenalis 14-3-3 Protein-Protein Interactions

Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite <i>Giardia duodenalis</i>, 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 <i>G. duodenalis</i> (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

Potent and selective carboxylic acid inhibitors of tumor-associated carbonic anhydrases IX and XII

Selective inhibition of tumor-associated carbonic anhydrase (CA; EC 4.2.1.1) isoforms IX and XII is a crucial prerequisite to develop successful anticancer therapeutics. Herein, we confirmed the efficacy of the 3-nitrobenzoic acid substructure in the design of potent and selective carboxylic acid derivatives as CAs inhibitors. Compound 10 emerged as the most potent inhibitor of the tumor-associated hCA IX and XII (Ki= 16 and 82.1 nM, respectively) with a significant selectivity with respect to the wide spread hCA II. Other 3-nitrobenzoic acid derivatives showed a peculiar CA inhibition profile with a notable potency towards hCA IX

Molecular insights to the bioactive form of BV02, a reference inhibitor of 14-3-3σ protein-protein interactions

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

Hit Recycling: Discovery of a Potent Carbonic Anhydrase Inhibitor by in Silico Target Fishing

In silico target fishing is an emerging tool in drug discovery, which is mostly used for primary target or off-target prediction and drug repositioning. In this work, we developed an in silico target fishing protocol to identify the primary target of GV2-20, a false-positive hit highlighted in a cell-based screen for 14-3-3 modulators. Although GV2-20 does not bind to 14-3-3 proteins, it showed remarkable antiproliferative effects in CML cells, thus raising interest toward the identification of its primary target. Six potential targets of GV2-20 were prioritized in silico and tested in vitro. Our results show that the molecule is a potent inhibitor of carbonic anhydrase 2 (CA2), thus confirming the predictive capability of our protocol. Most notably, GV2-20 experienced a remarkable selectivity for CA2, CA7, CA9, and CA12, and its scaffold was never explored before as a chemotype for CA inhibition, thus becoming an interesting lead candidate for further development

Molecular Dynamics Simulations and Structural Analysis of <i>Giardia duodenalis</i> 14-3‑3 Protein–Protein Interactions

Giardiasis is a gastrointestinal diarrheal illness caused by the protozoan parasite <i>Giardia duodenalis</i>, 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 <i>G. duodenalis</i> (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