Synthesis and Biological Evaluation of an Indazole-Based Selective Protein Arginine Deiminase 4 (PAD4) Inhibitor.

Protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme that catalyzes the conversion of arginine to citrulline within target proteins. Dysregulation of PAD4 has been implicated in a number of human diseases, including rheumatoid arthritis and other inflammatory diseases as well as cancer. In this study, we report on the design, synthesis, and evaluation of a new class of haloacetamidine-based compounds as potential PAD4 inhibitors. Specifically, we describe the identification of 4,5,6-trichloroindazole 24 as a highly potent PAD4 inhibitor that displays >10-fold selectivity for PAD4 over PAD3 and >50-fold over PAD1 and PAD2. The efficacy of this compound in cells was determined by measuring the inhibition of PAD4-mediated H4 citrullination in HL-60 granulocytes.

Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases.

Dysregulation of protein tyrosine phosphorylation has been implicated in a number of human diseases, including cancer, diabetes, and neurodegenerative diseases. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here we report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, striatal-enriched protein tyrosine phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their respective targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concentrations. Additionally, active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry, and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.

Analysis of carbonaceous biomarkers with the Mars Organic Analyzer microchip capillary electrophoresis system: carboxylic acids.

The oxidizing surface chemistry on Mars argues that any comprehensive search for organic compounds indicative of life requires methods to analyze higher oxidation states of carbon with very low limits of detection. To address this goal, microchip capillary electrophoresis (µCE) methods were developed for analysis of carboxylic acids with the Mars Organic Analyzer (MOA). Fluorescent derivatization was achieved by activation with the water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) followed by reaction with Cascade Blue hydrazide in 30 mM borate, pH 3. A standard containing 12 carboxylic acids found in terrestrial life was successfully labeled and separated in 30 mM borate at pH 9.5, 20 °C by using the MOA CE system. Limits of detection were 5-10 nM for aliphatic monoacids, 20 nM for malic acid (diacid), and 230 nM for citric acid (triacid). Polyacid benzene derivatives containing 2, 3, 4, and 6 carboxyl groups were also analyzed. In particular, mellitic acid was successfully labeled and analyzed with a limit of detection of 300 nM (5 ppb). Analyses of carboxylic acids sampled from a lava tube cave and a hydrothermal area demonstrated the versatility and robustness of our method. This work establishes that the MOA can be used for sensitive analyses of a wide range of carboxylic acids in the search for extraterrestrial organic molecules.

Analysis of carbonaceous biomarkers with the Mars Organic Analyzer microchip capillary electrophoresis system: aldehydes and ketones.

A microchip CE method is developed for the analysis of two oxidized forms of carbon, aldehydes and ketones, with the Mars Organic Analyzer (MOA). Fluorescent derivitization is achieved in ∼ 15 min by hydrazone formation with Cascade Blue hydrazide in 30 mM borate pH 5-6. The microchip CE separation and analysis method is optimized via separation in 30 mM borate buffer, pH 9.5, at 20°C. A carbonyl standard consisting of ten aldehydes and ketones found in extraterrestrial matter is successfully separated; the resulting LOD depends on the reactivity of the compound and range from 70 pM for formaldehyde to 2 µM for benzophenone. To explore the utility of this method for analyzing complex samples, analyses of several fermented beverages are conducted, identifying ten aldehydes and ketones ranging from 30 nM to 5 mM. A Martian regolith simulant sample, consisting of a basalt matrix spiked with soluble ions and acetone, is designed and analyzed, but acetone is found to have a limited detectable lifetime under simulant Martian conditions. This work establishes the capability of the MOA for studying aldehydes and ketones, a critical class of oxidized organic molecules of interest in planetary and in terrestrial environmental and health studies.