Protein vicinal thiols as intrinsic probes of brain redox states in health, aging, and ischemia.

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism.

Structural basis of the acyl-transfer mechanism of human GPAT1.

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states.

Understanding Data Noise and Uncertainty through Analysis of Replicate Samples in DNA-Encoded Library Selection.

By analyzing data sets of replicate DNA-Encoded Library (DEL) selections, an approach for estimating the noise level of the experiment has been developed. Using a logarithm transformation of the number of counts associated with each compound and a subset of compounds with the highest number of counts, it is possible to assess the quality of the data through normalizing the replicates and use this same data to estimate the noise in the experiment. The noise level is seen to be dependent on sequencing depth as well as specific selection conditions.

Toward the assembly and characterization of an encoded library hit confirmation platform: Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS).

The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNA-encoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures.

Fluorophosphonates on-Demand: A General and Simplified Approach toward Fluorophosphonate Synthesis.

Herein, we report a general and simplified synthesis of fluorophosphonates directly from p-nitrophenylphosphonates. This FP on-demand reaction is mediated by a commercially available polymer-supported fluoride reagent that produces a variety (25 examples) of fluorophosphonates in high yields while only requiring reagent filtration for pure fluorophosphonate isolation. This reaction protocol facilitates the rapid profiling of serine hydrolases with diverse and novel sets of activated phosphonates with differential proteome reactivity.

Selecting Approaches for Hit Identification and Increasing Options by Building the Efficient Discovery of Actionable Chemical Matter from DNA-Encoded Libraries.

Over the past 20 years, the toolbox for discovering small-molecule therapeutic starting points has expanded considerably. Pharmaceutical researchers can now choose from technologies that, in addition to traditional high-throughput knowledge-based and diversity screening, now include the screening of fragment and fragment-like libraries, affinity selection mass spectrometry, and selection against DNA-encoded libraries (DELs). Each of these techniques has its own unique combination of advantages and limitations that makes them more, or less, suitable for different target classes or discovery objectives, such as desired mechanism of action.

A simple method for determining compound affinity and chemical yield from DNA-encoded library selections.

DNA-encoded libraries (DELs) can contain billions of unique chemical species; selecting against such large inputs, it is typical to find more candidate binders than is reasonable to pursue for follow-up synthesis and testing. Given this wealth of choices, common practice is to limit synthesis to only those compounds estimated to have the greatest chance of being high-affinity binders; of the many potential factors contributing to this estimation, the strength of the selection signal of a candidate binder is always important. We define here methods and equations which relate the theoretical selection signal of a compound to its affinity and chemical yield.

A method for estimating binding affinity from primary DEL selection data.

DEL selections are binding assays conducted with mixtures of chemically diverse DNA-tagged ligands and a screening target. DEL selections use DNA sequence counts to measure target binding, where ideally higher affinity ligands will have higher counts than weaker affinity ligands. However, there is not always a clear relationship between DNA sequence count (assay signal) and binding affinity.

The Reducible Disulfide Proteome of Synaptosomes Supports a Role for Reversible Oxidations of Protein Thiols in the Maintenance of Neuronal Redox Homeostasis.

The mechanisms by which neurons maintain redox homeostasis, disruption of which is linked to disease, are not well known. Hydrogen peroxide, a major cellular oxidant and neuromodulator, can promote reversible oxidations of protein thiols but the scope, targets, and significance of such oxidations occurring in neurons, especially in vivo, are uncertain. Using redox phenylarsine oxide (PAO)-affinity chromatography, which exploits the high-affinity of trivalent arsenicals for protein dithiols, this study investigated the occurrence of reducible and, therefore, potentially regulatory, protein disulfide bonds in Triton X-100-soluble protein fractions from isolated nerve-endings (synaptosomes) prepared from rat brains.

A Solution Phase Platform to Characterize Chemical Reaction Compatibility with DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis.

Reductive Reprogramming: A Not-So-Radical Hypothesis of Neurodegeneration Linking Redox Perturbations to Neuroinflammation and Excitotoxicity.

Free radical-mediated oxidative stress, neuroinflammation, and excitotoxicity have long been considered insults relevant to the progression of Alzheimer's disease and other aging-related neurodegenerative disorders (NDD). Among these phenomena, the significance of oxidative stress and, more generally, redox perturbations, for NDD remain ill-defined and unsubstantiated. Here, I argue that (i) free radical-mediated oxidations of biomolecules can be dissociated from the progression of NDD, (ii) oxidative stress fails as a descriptor of cellular redox states under conditions relevant to disease, and (iii) aberrant upregulation of compensatory reducing activities in neural cells, resulting in reductive shifts in thiol-based redox potentials, may be an overlooked and paradoxical contributor to disease progression.

Potential widespread denitrosylation of brain proteins following prolonged restraint: proposed links between stress and central nervous system disease.

The biochemical pathways by which aberrant psychophysiological stress promotes neuronal damage and increases the risks for central nervous system diseases are not well understood. In light of previous findings that psychophysiological stress, modeled by animal restraint, can increase the activities and expression levels of nitric oxide synthase isoforms in multiple brain regions, we examined the effects of restraint, for up to 6 h, on levels of S-nitrosylated proteins and NOx (nitrite + nitrate), a marker for high-level nitric oxide generation, in the brains of rats. Results identify functionally-diverse protein targets of S-nitrosylation in the brain, in vivo, and demonstrate the potential for widespread loss of protein nitrosothiols following prolonged restraint despite a concomitant increase in NOx levels.

Small molecule inhibitors reveal PTK6 kinase is not an oncogenic driver in breast cancers.

Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability.

Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors of Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates.

In vitro and in vivo effects of 2,4 diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Context-specific modulation of SMN transcript levels.

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective.

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA).

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes.

Disulfide Stress Targets Modulators of Excitotoxicity in Otherwise Healthy Brains.

Oxidative stress is a long-hypothesized cause of diverse neurological and psychiatric disorders but the pathways by which physiological redox perturbations may detour healthy brain development and aging are unknown. We reported recently (Foley et al., Neurochem Res 39:2030-2039, 2014) that two-electron oxidations, to disulfides, of protein vicinal thiols can vary markedly in association with more modest oxidations of the glutathione redox couple in brains from healthy adolescent rats whereas levels of protein S-glutathionylation were low and unchanged.

A Platform to Enable the Pharmacological Profiling of Small Molecules in Gel-Based Electrophoretic Mobility Shift Assays.

We describe a polyacrylamide gel casting cassette that overcomes limitations of commercially available gel electrophoresis equipment. This apparatus molds a single polyacrylamide gel that can evaluate more than 200 samples in parallel, is loaded with a multichannel pipettor, and is flexible with respect to composition of the separating matrix. We demonstrate its use to characterize inhibitors of enzymes that modify protein and nucleic acid substrates.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

Background: Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard.

Results: We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis.

Covalent small molecule inhibitors of Ca(2+)-bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca(2+)-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA(S100B)) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer.

Protein vicinal thiol oxidations in the healthy brain: not so radical links between physiological oxidative stress and neural cell activities.

Reversible oxidations of protein thiols have emerged as alternatives to free radical-mediated oxidative damage with which to consider the impacts of oxidative stress on cellular activities but the scope and pathways of such oxidations in tissues, including the brain, have yet to be fully defined. We report here a characterization of reversible oxidations of glutathione and protein thiols in extracts from rat brains, from two sources, which had been (1) frozen quickly after euthanasia to preserve in vivo redox states and (2) subjected to alkylation upon tissue disruption to trap reduced thiols. Brains were defined, relatively, as Reduced and Moderately Oxidized based on measured ratios of reduced (GSH) to oxidized (GSSG) glutathione.

4-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), a potent inhibitor of bacterial phosphopantetheinyl transferase that attenuates secondary metabolism and thwarts bacterial growth.

4'-Phosphopantetheinyl transferases (PPTases) catalyze a post-translational modification essential to bacterial cell viability and virulence. We present the discovery and medicinal chemistry optimization of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides, which exhibit submicromolar inhibition of bacterial Sfp-PPTase with no activity toward the human orthologue. Moreover, compounds within this class possess antibacterial activity in the absence of a rapid cytotoxic response in human cells.

Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.

Phosphopantetheinyl transferase (PPTase; E.C. 2.