Cooperative stabilisation of 14-3-3σ protein-protein interactions via covalent protein modification.

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein-protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like 'molecular glues' is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth.

Discovering protein-protein interaction stabilisers by native mass spectrometry.

Protein-protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers.

Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker.

A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer's and other age-related pathologies. Consistent with this, clearance of senescent cells can prolong healthspan and lifespan in in vivo models. This provided a rationale for developing a new class of drugs, called senolytics, designed to selectively eliminate senescent cells in human tissues. The senolytics tested so far lack specificity and have significant off-target effects, suggesting that a targeted approach could be more clinically relevant. Here, we propose to use an extracellular epitope of B2M, a recently identified membrane marker of senescence, as a target for the specific delivery of toxic drugs into senescent cells. We show that an antibody-drug conjugate (ADC) against B2M clears senescent cells by releasing duocarmycin into them, while an isotype control ADC was not toxic for these cells. This effect was dependent on p53 expression and therefore more evident in stress-induced senescence. Non-senescent cells were not affected by either antibody, confirming the specificity of the treatment. Our results provide a proof-of-principle assessment of a novel approach for the specific elimination of senescent cells using a second generation targeted senolytic against proteins of their surfaceome, which could have clinical applications in pathological ageing and associated diseases.

Electrophysiological and Clinical Improvement in Non-Invasive Treatment of Carpal Tunnel Syndrome.

BACKGROUND AND OBJECTIVE: Carpal tunnel syndrome (CTS) is the most common form of nerve entrapment. Clinically, various signs and symptoms compare due to overexposure to mechanical vibrations transmitted to the wrist bones and cartilage, resulting in compression of the sensory and motor nerve fibers of the median nerve. Early symptoms include nocturnal paresthesia and electromyography reveals reduced sensory nerve conduction velocity. Aim of this study was to evaluate the efficacy of a dietary integrator composed of acetyl-L-carnitine, α-lipoic acid, quercetin, bromelain, pantothenic acid, C and B1 and B2 and B6 and B12 vitamins in patients with early (minimal) carpal tunnel syndrome. METHODS: 36 patients (28 female and 8 male) with early CTS characterized by sensory nerve demyelination and inflammation of the transverse carpal ligament. Patients were divided into two groups, group A (18 patients received physical therapy) and group B (18 patients received physical therapy and an oral integrator). Clinical (sleep quality questionnaire to measure severity of paresthesia) and neurophysiological assessment (Sensory Nerve Conduction Velocity) performed at baseline, and then at 30 and 60 days after treatment. RESULTS: Sleep quality and Sensory Nerve Conduction Velocity data analysis show improvement in both groups at 30 and 60 days, with statistical difference between them in both time of analysis. CONCLUSION: In the early CTS, with sensory fibers damage, use of dietary integrator, such as Micronil Dol®, composed of acetyl-L-carnitine, α-lipoic acid, quercetin, bromelain, pantothenic acid, C and B1 and B2 and B6 and B12 vitamins can be effective in quick recovery of median nerve sensory.

Regulation of p53 by the 14-3-3 protein interaction network: new opportunities for drug discovery in cancer.

Most cancers evolve to disable the p53 pathway, a key tumour suppressor mechanism that prevents transformation and malignant cell growth. However, only ~50% exhibit inactivating mutations of p53, while in the rest its activity is suppressed by changes in the proteins that modulate the pathway. Therefore, restoring p53 activity in cells in which it is still wild type is a highly attractive therapeutic strategy that could be effective in many different cancer types. To this end, drugs can be used to stabilise p53 levels by modulating its regulatory pathways. However, despite the emergence of promising strategies, drug development has stalled in clinical trials. The need for alternative approaches has shifted the spotlight to the 14-3-3 family of proteins, which strongly influence p53 stability and transcriptional activity through direct and indirect interactions. Here, we present the first detailed review of how 14-3-3 proteins regulate p53, with special emphasis on the mechanisms involved in their binding to different members of the pathway. This information will be important to design new compounds that can reactivate p53 in cancer cells by influencing protein-protein interactions. The intricate relationship between the 14-3-3 isoforms and the p53 pathway suggests that many potential drug targets for p53 reactivation could be identified and exploited to design novel antineoplastic therapies with a wide range of applications.

Artemisinin and its derivatives; ancient tradition inspiring the latest therapeutic approaches against malaria.

Artemisinin (ART) is an endoperoxide sesquiterpene lactone, commonly used in the treatment of malaria. Although it was isolated from Artemisia annuaL., a plant widely applied in Chinese Traditional Medicine, its mechanism of action remains uncertain and its clinical use is still limited due to its low solubility, its poor bioavailability and short in vivo half-life. Over time, several studies have been aimed towards the discovery of potent ART derivatives that could overcome clinical drawbacks. In this review, we focus on the multifaced aspects of ART and on the efforts spent to improve its pharmacological profile that so far culminated in the discovery of more effective drugs. Lastly, we outline the new perspectives in the ART-derivatives scenario.