Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes.

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

Can Precision Electrophile Signaling Make a Meaningful and Lasting Impression in Drug Design?

For several years, drugs with reactive electrophilic appendages have been developed. These units typically confer prolonged residence time of the drugs on their protein targets, and may assist targeting shallow binding sites and/or improving the drug-protein target spectrum. Studies on natural electrophilic molecules have indicated that, in many instances, natural electrophiles use similar mechanisms to alter signaling pathways. However, natural reactive species are also endowed with other important mechanisms to hone signaling properties that are uncommon in drug design. These include ability to be active at low occupancy and elevated inhibitor kinetics. Herein, we discuss how we have begun to harness these properties in inhibitor design.

Redox-guided small molecule antimycobacterials.

The emergence of drug resistance has posed a major challenge to treatment of tuberculosis worldwide. The new drug candidates in the pipeline are few and therefore there is an urgent need to develop antimycobacterials with novel mechanisms of action. Maintenance of redox homeostasis is integral to mycobacterial survival and growth. Therefore, perturbation of this equilibrium can result in irreversible stress induction and inhibition of growth. Herein, we review a number of small molecules that have either been designed to induce redox stress or were found to do so after their discovery. A number of these small molecules are quite effective against drug-resistant mycobacterial strains and thus offer scope for exploration of potentially new mechanism of action. The progress in redox-guided antimycobacterial compounds and the challenges towards clinical applications are reviewed. © 2018 IUBMB Life, 70(9):826-835, 2018.

Facile Functionalization of Carbon Electrodes for Efficient Electroenzymatic Hydrogen Production.

Enzymatic electrocatalysis holds promise for new biotechnological approaches to produce chemical commodities such as molecular hydrogen (H2). However, typical inhibitory limitations include low stability and/or low electrocatalytic currents (low product yields). Here we report a facile single-step electrode preparation procedure using indium-tin oxide nanoparticles on carbon electrodes. The subsequent immobilization of a model [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on the functionalized carbon electrode permits comparatively large quantities of H2 to be produced in a stable manner. Specifically, we observe current densities of >8 mA/cm2 at -0.8 V vs the standard hydrogen electrode (SHE) by direct electron transfer (DET) from cyclic voltammetry, with an onset potential for H2 production close to its standard potential at pH 7 (approximately -0.4 V vs. SHE). Importantly, hydrogenase-modified electrodes show high stability retaining ∼92% of their electrocatalytic current after 120 h of continuous potentiostatic H2 production at -0.6 V vs. SHE; gas chromatography confirmed ∼100% Faradaic efficiency. As the bioelectrode preparation method balances simplicity, performance, and stability, it paves the way for DET on other electroenzymatic reactions as well as semiartificial photosynthesis.

Enzymatic and Microbial Electrochemistry: Approaches and Methods.

The coupling of enzymes and/or intact bacteria with electrodes has been vastly investigated due to the wide range of existing applications. These span from biomedical and biosensing to energy production purposes and bioelectrosynthesis, whether for theoretical research or pure applied industrial processes. Both enzymes and bacteria offer a potential biotechnological alternative to noble/rare metal-dependent catalytic processes. However, when developing these biohybrid electrochemical systems, it is of the utmost importance to investigate how the approaches utilized to couple biocatalysts and electrodes influence the resulting bioelectrocatalytic response. Accordingly, this tutorial review starts by recalling some basic principles and applications of bioelectrochemistry, presenting the electrode and/or biocatalyst modifications that facilitate the interaction between the biotic and abiotic components of bioelectrochemical systems. Focus is then directed toward the methods used to evaluate the effectiveness of enzyme/bacteria-electrode interaction and the insights that they provide. The basic concepts of electrochemical methods widely employed in enzymatic and microbial electrochemistry, such as amperometry and voltammetry, are initially presented to later focus on various complementary methods such as spectroelectrochemistry, fluorescence spectroscopy and microscopy, and surface analytical/characterization techniques such as quartz crystal microbalance and atomic force microscopy. The tutorial review is thus aimed at students and graduate students approaching the field of enzymatic and microbial electrochemistry, while also providing a critical and up-to-date reference for senior researchers working in the field.

Chemoproteomics of an Indole-Based Quinone Epoxide Identifies Druggable Vulnerabilities in Vancomycin-Resistant Staphylococcus aureus.

The alarming global rise in fatalities from multidrug-resistant Staphylococcus aureus (S. aureus) infections has underscored a need to develop new therapies to address this epidemic. Chemoproteomics is valuable in identifying targets for new drugs in different human diseases including bacterial infections. Targeting functional cysteines is particularly attractive, as they serve critical catalytic functions that enable bacterial survival. Here, we report an indole-based quinone epoxide scaffold with a unique boat-like conformation that allows steric control in modulating thiol reactivity. We extensively characterize a lead compound (4a), which potently inhibits clinically derived vancomycin-resistant S. aureus. Leveraging diverse chemoproteomic platforms, we identify and biochemically validate important transcriptional factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus, the chemical tools and biological targets that we describe here prospect new therapeutic paradigms in combatting S. aureus infections.

Visible-Light Controlled Release of a Fluoroquinolone Antibiotic for Antimicrobial Photopharmacology.

Owing to the dwindling arsenal of antibiotics, new methodologies for their effective and localized delivery are necessary. The use of optical control over delivery of drugs, also known as photopharmacology, has emerged as an important option for the spatiotemporally controlled generation of drugs and bioactive molecules. In the field of antimicrobial photopharmacology, most strategies utilize ultraviolet light for triggering release of the antibiotic. The use of such short wavelength light may have limitations such as phototoxicity. Here, a small molecule that is activated by visible light to release a fluoroquinolone, a broad-spectrum antibiotic, is reported. A boron-dipyrromethene, which is sensitive to cleavage at 470 nm, was used, and levofloxacin was used as a model fluoroquinolone. BDP-Levo was found to undergo cleavage in the presence of visible light to release the active antibiotic. Using growth inhibitory studies in Gram-positive as well as Gram-negative bacteria, the efficacy of BDP-Levo is demonstrated. Together, our study demonstrates that visible light can be used for optical control over antibiotic release and lays the foundation for visible-light-mediated antimicrobial photopharmacology.