Phosphorylated full-length Tau interacts with 14-3-3 proteins via two short phosphorylated sequences, each occupying a binding groove of 14-3-3 dimer.

Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer's disease. The interaction of 14-3-3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer's disease. When Tau is phosphorylated by PKA (Tau-PKA), several phosphorylation sites are generated, including two known 14-3-3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3σ interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.

Selectivity via Cooperativity: Preferential Stabilization of the p65/14-3-3 Interaction with Semisynthetic Natural Products.

Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein-protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.

Fragment-based Differential Targeting of PPI Stabilizer Interfaces.

Stabilization of protein-protein interactions (PPIs) holds great potential for therapeutic agents, as illustrated by the successful drugs rapamycin and lenalidomide. However, how such interface-binding molecules can be created in a rational, bottom-up manner is a largely unanswered question. We report here how a fragment-based approach can be used to identify chemical starting points for the development of small-molecule stabilizers that differentiate between two different PPI interfaces of the adapter protein 14-3-3. The fragments discriminately bind to the interface of 14-3-3 with the recognition motif of either the tumor suppressor protein p53 or the oncogenic transcription factor TAZ. This X-ray crystallography driven study shows that the rim of the interface of individual 14-3-3 complexes can be targeted in a differential manner with fragments that represent promising starting points for the development of specific 14-3-3 PPI stabilizers.

Design of Drug-Like Protein-Protein Interaction Stabilizers Guided By Chelation-Controlled Bioactive Conformation Stabilization.

Protein-protein interactions (PPIs) of 14-3-3 proteins are a model system for studying PPI stabilization. The complex natural product Fusicoccin A stabilizes many 14-3-3 PPIs but is not amenable for use in SAR studies, motivating the search for more drug-like chemical matter. However, drug-like 14-3-3 PPI stabilizers enabling such studies have remained elusive. An X-ray crystal structure of a PPI in complex with an extremely low potency stabilizer uncovered an unexpected non-protein interacting, ligand-chelated Mg2+ leading to the discovery of metal-ion-dependent 14-3-3 PPI stabilization potency. This originates from a novel chelation-controlled bioactive conformation stabilization effect. Metal chelation has been associated with pan-assay interference compounds (PAINS) and frequent hitter behavior, but chelation can evidently also lead to true potency gains and find use as a medicinal chemistry strategy to guide compound optimization. To demonstrate this, we exploited the effect to design the first potent, selective, and drug-like 14-3-3 PPI stabilizers.

Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ.

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.

Set-up and screening of a fragment library targeting the 14-3-3 protein interface.

Protein-protein interactions (PPIs) are at the core of regulation mechanisms in biological systems and consequently became an attractive target for therapeutic intervention. PPIs involving the adapter protein 14-3-3 are representative examples given the broad range of partner proteins forming a complex with one of its seven human isoforms. Given the challenges represented by the nature of these interactions, fragment-based approaches offer a valid alternative for the development of PPI modulators. After having assembled a fragment set tailored on PPIs' modulation, we started a screening campaign on the sigma isoform of 14-3-3 adapter proteins. Through the use of both mono- and bi-dimensional nuclear magnetic resonance spectroscopy measurements, coupled with differential scanning fluorimetry, three fragment hits were identified. These molecules bind the protein at two different regions distant from the usual binding groove highlighting new possibilities for selective modulation of 14-3-3 complexes.

Backbone chemical shift assignments of human 14-3-3σ.

14-3-3 proteins are a group of seven dimeric adapter proteins that exert their biological function by interacting with hundreds of phosphorylated proteins, thus influencing their sub-cellular localization, activity or stability in the cell. Due to this remarkable interaction network, 14-3-3 proteins have been associated with several pathologies and the protein-protein interactions (PPIs) established with a number of partners are now considered promising drug targets. The activity of 14-3-3 proteins is often isoform specific and to our knowledge only one out of seven isoforms, 14-3-3[Formula: see text], has been assigned. Despite the availability of the crystal structures of all seven isoforms of 14-3-3, the additional NMR assignments of 14-3-3 proteins are important for both biological mechanism studies and chemical biology approaches. Herein, we present a robust backbone assignment of 14-3-3σ, which will allow advances in the discovery of potential therapeutic compounds. This assignment is now being applied to the discovery of both inhibitors and stabilizers of 14-3-3 PPIs.

Nuclear Magnetic Resonance Spectroscopy Insights into Tau Structure in Solution: Impact of Post-translational Modifications.

Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure. The cis or trans conformation of X-Proline peptide bonds is an additional characteristic parameter of Tau structure that is targeted and modified by prolyl cis/trans isomerases. The challenge in molecular characterization of Tau lies in being able to link structural parameters to functional consequences in normal functions and dysfunctions of Tau, including potential misfolding on the path to aggregation and/or perturbation of the interactions of Tau with its many molecular partners. Phosphorylation of Ser and Thr residues has the potential to impact the local and global structure of Tau.

Inhibition of 14-3-3/Tau by Hybrid Small-Molecule Peptides Operating via Two Different Binding Modes.

Current molecular hypotheses have not yet delivered marketable treatments for Alzheimer's disease (AD), arguably due to a lack of understanding of AD biology and an overreliance on conventional drug modalities. Protein-protein interactions (PPIs) are emerging drug targets, which show promise for the treatment of, e.g., cancer, but are still underexploited for treating neurodegenerative diseases. 14-3-3 binding to phosphorylated Tau is a promising PPI drug target based on its reported destabilizing effect on microtubules, leading to enhanced neurofibrillary tangle formation as a potential cause of AD-related neurodegeneration. Inhibition of 14-3-3/Tau may therefore be neuroprotective. Previously, we reported the structure-guided development of modified peptide inhibitors of 14-3-3/Tau. Here, we report further efforts to optimize the binding mode and activity of our modified Tau peptides through a combination of chemical synthesis, biochemical assays, and X-ray crystallography. Most notably, we were able to characterize two different high-affinity binding modes, both of which inhibited 14-3-3-binding to full-length PKA-phosphorylated Tau protein in vitro as measured by NMR spectroscopy. Our findings, besides producing useful tool inhibitor compounds for studying 14-3-3/Tau, have enhanced our understanding of the molecular parameters for inhibiting 14-3-3/Tau, which are important milestones toward the establishment of our 14-3-3 PPI hypothesis.

Repeated subcutaneous administrations of krokodil causes skin necrosis and internal organs toxicity in Wistar rats: putative human implications.

OBJECTIVE: "Krokodil" is the street name for an impure homemade drug mixture used as a cheap substitute for heroin, containing desomorphine as the main opioid. Abscesses, gangrene, thrombophlebitis, limb ulceration and amputations, jaw osteonecrosis, skin discoloration, ulcers, skin infections, and bleeding are some of the typical reported signs in humans. This study aimed to understand the toxicity of krokodil using Wistar male rats as experimental model. METHODS: Animals were divided into seven groups and exposed subcutaneously to NaCl 0.9% (control), krokodil mixture free of psychotropic substances (blank krokodil), pharmaceutical grade desomorphine 1 mg/kg, and four different concentrations of krokodil (containing 0.125, 0.25, 0.5, and 1 mg/kg of desomorphine) synthesized accordingly to a "domestic" protocol followed by people who inject krokodil (PWIK). Daily injections for five consecutive days were performed, and animals were sacrificed 24 hr after the last administration. Biochemical and histological analysis were carried out. RESULTS: It was shown that the continuous use of krokodil may cause injury at the injection area, with formation of necrotic zones. The biochemical results evidenced alterations on cardiac and renal biomarkers of toxicity, namely, creatine kinase, creatine kinase-MB, and uric acid. Significant alteration in levels of reduced and oxidized glutathione on kidney and heart suggested that oxidative stress may be involved in krokodil-mediated toxicity. Cardiac congestion was the most relevant finding of continuous krokodil administration. CONCLUSIONS: These findings contribute notably to comprehension of the local and systemic toxicological impact of this complex drug mixture on major organs and will hopefully be useful for the development of appropriate treatment strategies towards the human toxicological effects of krokodil.

Data analysis of "krokodil" samples obtained by street-like synthesis.

The data described in this work is related to be the subject of an article in the Forensic Science International, titled: "The harmful chemistry behind "krokodil": street-like synthesis and product analysis" (http://dx.doi.org/10.1016/j.forsciint.2015.07.042) [1]. The data presented here provides additional description of the chemical profile of "krokodil". Physicochemical and organoleptic characteristics, TLC profile, UV/Vis, (1)H NMR and FTIR spectrum are presented. These data validate the proposed synthetic procedure and pathway and give further information about the contaminants present in "krokodil".