ACE Phenotyping in Human Blood and Tissues: Revelation of ACE Outliers and Sex Differences in ACE Sialylation.

Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by blood ACE levels) is associated with an increased risk of cardiovascular diseases. Elevated blood ACE is also a marker for granulomatous diseases. Decreased blood ACE activity is becoming a new risk factor for Alzheimer's disease. We applied our novel approach-ACE phenotyping-to characterize pairs of tissues (lung, heart, lymph nodes) and serum ACE in 50 patients. ACE phenotyping includes (1) measurement of ACE activity with two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of these substrates (ZPHL/HHL ratio); (3) determination of ACE immunoreactive protein levels using mAbs to ACE; and (4) ACE conformation with a set of mAbs to ACE. The ACE phenotyping approach in screening format with special attention to outliers, combined with analysis of sequencing data, allowed us to identify patient with a unique ACE phenotype related to decreased ability of inhibition of ACE activity by albumin, likely due to competition with high CCL18 in this patient for binding to ACE. We also confirmed recently discovered gender differences in sialylation of some glycosylation sites of ACE. ACE phenotyping is a promising new approach for the identification of ACE phenotype outliers with potential clinical significance, making it useful for screening in a personalized medicine approach.

Carriers of Heterozygous Loss-of-Function ACE Mutations Are at Risk for Alzheimer's Disease.

We hypothesized that subjects with heterozygous loss-of-function (LoF) ACE mutations are at risk for Alzheimer's disease because amyloid Aß42, a primary component of the protein aggregates that accumulate in the brains of AD patients, is cleaved by ACE (angiotensin I-converting enzyme). Thus, decreased ACE activity in the brain, either due to genetic mutation or the effects of ACE inhibitors, could be a risk factor for AD. To explore this hypothesis in the current study, existing SNP databases were analyzed for LoF ACE mutations using four predicting tools, including PolyPhen-2, and compared with the topology of known ACE mutations already associated with AD. The combined frequency of >400 of these LoF-damaging ACE mutations in the general population is quite significant-up to 5%-comparable to the frequency of AD in the population > 70 y.o., which indicates that the contribution of low ACE in the development of AD could be under appreciated. Our analysis suggests several mechanisms by which ACE mutations may be associated with Alzheimer's disease. Systematic analysis of blood ACE levels in patients with all ACE mutations is likely to have clinical significance because available sequencing data will help detect persons with increased risk of late-onset Alzheimer's disease. Patients with transport-deficient ACE mutations (about 20% of damaging ACE mutations) may benefit from preventive or therapeutic treatment with a combination of chemical and pharmacological (e.g., centrally acting ACE inhibitors) chaperones and proteosome inhibitors to restore impaired surface ACE expression, as was shown previously by our group for another transport-deficient ACE mutation-Q1069R.

Targeting EB3-IP3R3 Interface with Cognate Peptide Protects from Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a lung disease characterized by acute onset of noncardiogenic pulmonary edema, hypoxemia, and respiratory insufficiency. The current treatment for ARDS is mainly supportive in nature, providing a critical need for targeted pharmacological management. We addressed this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor EB3 (end binding protein 3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells in response to inflammatory stimuli. EB3 interacts with IP3R3 (inositol 1,4,5-trisphosphate receptor 3) and orchestrates calcium release from endoplasmic reticulum stores. Here, we designed and tested the therapeutic benefits of a 14-aa peptide named CIPRI (cognate IP3 receptor inhibitor), which disrupted EB3-IP3R3 interaction in vitro and in lungs of mice challenged with endotoxin. Treatment with CIPRI or depletion of IP3R3 in lung microvascular endothelial monolayers mitigated calcium release from endoplasmic reticulum stores and prevented a disassembly of vascular endothelial cadherin junctions in response to the proinflammatory mediator α-thrombin. Furthermore, intravenous administration of CIPRI in mice mitigated inflammation-induced lung injury, blocked pulmonary microvascular leakage, prevented activation of NFAT (nuclear factor of activated T cells) signaling, and reduced production of proinflammatory cytokines in the lung tissue. CIPRI also improved survival of mice from endotoxemia and polymicrobial sepsis. Together, these data demonstrate that targeting EB3-IP3R3 interaction with a cognate peptide is a promising strategy to address hyperpermeability of microvessels in inflammatory lung diseases.

Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

Urinary ACE Phenotyping as a Research and Diagnostic Tool: Identification of Sex-Dependent ACE Immunoreactivity.

BACKGROUND: Angiotensin-converting enzyme (ACE) is highly expressed in renal proximal tubules, but ACE activity/levels in the urine are at least 100-fold lower than in the blood. Decreased proximal tubular ACE has been associated with renal tubular damage in both animal models and clinical studies. Because ACE is shed into urine primarily from proximal tubule epithelial cells, its urinary ACE measurement may be useful as an index of tubular damage. OBJECTIVE AND METHODOLOGY: We applied our novel approach-ACE phenotyping-to characterize urinary ACE in volunteer subjects. ACE phenotyping includes (1) determination of ACE activity using two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of the two substrates (ZPHL/HHL ratio); (3) quantification of ACE immunoreactive protein levels; and (4) fine mapping of local ACE conformation with mAbs to ACE. PRINCIPAL FINDINGS: In normal volunteers, urinary ACE activity was 140-fold less than in corresponding plasma/serum samples and did not differ between males and females. However, urinary ACE immunoreactivity (normalized binding of 25 mAbs to different epitopes) was strongly sex-dependent for the several mAbs tested, an observation likely explained by differences in tissue ACE glycosylation/sialylation between males and females. Urinary ACE phenotyping also allowed the identification of ACE outliers. In addition, daily variability of urinary ACE has potential utility as a feedback marker for dieting individuals pursuing weight loss. CONCLUSIONS/SIGNIFICANCE: Urinary ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques.

Blood ACE Phenotyping for Personalized Medicine: Revelation of Patients with Conformationally Altered ACE.

Background: The angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated blood ACE is a marker for granulomatous diseases and elevated ACE expression in tissues is associated with increased risk of cardiovascular diseases. Objective and Methodology: We applied a novel approach -ACE phenotyping-to find a reason for conformationally impaired ACE in the blood of one particular donor. Similar conformationally altered ACEs were detected previously in 2-4% of the healthy population and in up to 20% of patients with uremia, and were characterized by significant increase in the rate of angiotensin I hydrolysis. Principal findings: This donor has (1) significantly increased level of endogenous ACE inhibitor in plasma with MW less than 1000; (2) increased activity toward angiotensin I; (3) M71V mutation in ABCG2 (membrane transporter for more than 200 compounds, including bilirubin). We hypothesize that this patient may also have the decreased level of free bilirubin in plasma, which normally binds to the N domain of ACE. Analysis of the local conformation of ACE in plasma of patients with Gilbert and Crigler-Najjar syndromes allowed us to speculate that binding of mAbs 1G12 and 6A12 to plasma ACE could be a natural sensor for estimation of free bilirubin level in plasma. Totally, 235 human plasma/sera samples were screened for conformational changes in soluble ACE. Conclusions/Significance: ACE phenotyping of plasma samples allows us to identify individuals with conformationally altered ACE. This type of screening has clinical significance because this conformationally altered ACE could not only result in the enhancement of the level of angiotensin II but could also serve as an indicator of free bilirubin levels.

Probing the Surface of a Parasite Drug Target Thioredoxin Glutathione Reductase Using Small Molecule Fragments.

Fragment screening is a powerful drug discovery approach particularly useful for enzymes difficult to inhibit selectively, such as the thiol/selenol-dependent thioredoxin reductases (TrxRs), which are essential and druggable in several infectious diseases. Several known inhibitors are reactive electrophiles targeting the selenocysteine-containing C-terminus and thus often suffering from off-target reactivity in vivo. The lack of structural information on the interaction modalities of the C-terminus-targeting inhibitors, due to the high mobility of this domain and the lack of alternative druggable sites, prevents the development of selective inhibitors for TrxRs. In this work, fragments selected from actives identified in a large screen carried out against Thioredoxin Glutathione Reductase from Schistosoma mansoni (SmTGR) were probed by X-ray crystallography. SmTGR is one of the most promising drug targets for schistosomiasis, a devastating, neglected disease. Utilizing a multicrystal method to analyze electron density maps, structural analysis, and functional studies, three binding sites were characterized in SmTGR: two sites are close to or partially superposable with the NADPH binding site, while the third one is found between two symmetry related SmTGR subunits of the crystal lattice. Surprisingly, one compound bound to this latter site stabilizes, through allosteric effects mediated by the so-called guiding bar residues, the crucial redox active C-terminus of SmTGR, making it finally visible at high resolution. These results further promote fragments as small molecule probes for investigating functional aspects of the target protein, exemplified by the allosteric effect on the C-terminus, and providing fundamental chemical information exploitable in drug discovery.

Epitope mapping of novel monoclonal antibodies to human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.

Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition.

Migraine is the sixth most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, α-tubulin. We use validated mouse models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity.

Migraines are a common brain disorder that affects 14% of the world's population. For many people the main symptom of a migraine is a painful headache, often on one side of the head. Other symptoms include increased sensitivity to light or sound, disturbed vision, and feeling sick. These sensory disturbances are called aura and they often occur before the headache begins. One particularly debilitating subset of migraines are chronic migraines, in which patients experience more than 15 headache days per month. Migraine therapies are often only partially effective or poorly tolerated, making it important to develop new drugs for this condition, but unfortunately, little is known about the molecular causes of migraines. To bridge this gap, Bertels et al. used two different approaches to cause migraine-like symptoms in mice. One approach consisted on giving mice nitroglycerin, which dilates blood vessels, produces hypersensitivity to touch, and causes photophobia in both humans and mice. In the second approach, mice underwent surgery and potassium chloride was applied onto the dura, a thick membrane that surrounds the brain. This produces cortical spreading depression, an event that is linked to migraine auras and involves a wave of electric changes in brain cells that slowly propagates across the brain, silencing brain electrical activity for several minutes. Using these approaches, Bertels et al. studied whether causing chronic migraine-like symptoms in mice is associated with changes in the structures of neurons, focusing on the effects of migraines on microtubules. Microtubules are cylindrical protein structures formed by the assembly of smaller protein units. In most cells, microtubules assemble and disassemble depending on what the cell needs. Neurons need stable microtubules to establish connections with other neurons. The experiments showed that provoking chronic migraines in mice led to a reduction in the numbers of connections between different neurons. Additionally, Bertels et al. found that inhibiting HDAC6 (a protein that destabilizes microtubules) reverses the structural changes in neurons caused by migraines and decreases migraine symptoms. The same effects are seen when a known migraine treatment strategy, known as CGRP receptor blockade, is applied. These results suggest that chronic migraines may involve decreased neural complexity, and that the restoration of this complexity by HDAC6 inhibitors could be a potential therapeutic strategy for migraine.

Novel ACE mutations mimicking sarcoidosis by increasing blood ACE levels.

An elevated blood angiotensin I-converting enzyme (ACE) supports diagnosis of sarcoidosis and Gaucher disease. However, some ACE mutations increase ACE shedding, and patients with these mutations are therefore at risk of being incorrectly diagnosed with sarcoidosis because of elevated serum ACE levels. We applied a novel approach called "ACE phenotyping" to identify possible ACE mutations in 3 pulmonary clinic patients that had suspected sarcoidosis based on elevated blood ACE levels. Conformational fingerprinting of ACE indicated that these mutations may be localized in the stalk region of the protein and these were confirmed by whole exome sequencing. Index patient 1 (IP1) had a mutation (P1199L) that had been previously identified, while the other 2 patients had novel ACE mutations. IP2 had 2 mutations, T887M and N1196K (eliminating a putative glycosylation site), while IP3 had a stop codon mutation Q1124X (eliminating the transmembrane anchor). We also performed a comprehensive analysis of the existing database of all ACE mutations to estimate the proportion of mutations increasing ACE shedding. The frequency of ACE mutations resulting in increased blood ACE levels may be much higher than previously estimated. ACE phenotyping, together with whole exome sequencing, is a diagnostic approach that could prevent unnecessary invasive and/or costly diagnostic procedures, or potentially harmful treatment for patients misdiagnosed on the basis of elevated blood ACE levels.

Modulation of hepatitis B virus pregenomic RNA stability and splicing by histone deacetylase 5 enhances viral biosynthesis.

Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development.

Characterization of Lead Compounds Targeting the Selenoprotein Thioredoxin Glutathione Reductase for Treatment of Schistosomiasis.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1-5 are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1-5, 7, and 8 displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds 1-4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds 4-6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

Ectopic suicide inhibition of thioredoxin glutathione reductase.

Selective suicide inhibitors represent a seductively attractive approach for inactivation of therapeutically relevant enzymes since they are generally devoid of off-target toxicity in vivo. While most suicide inhibitors are converted to reactive species at enzyme active sites, theoretically bioactivation can also occur in ectopic (secondary) sites that have no known function. Here, we report an example of such an "ectopic suicide inhibition", an unprecedented bioactivation mechanism of a suicide inhibitor carried out by a non-catalytic site of thioredoxin glutathione reductase (TGR). TGR is a promising drug target to treat schistosomiasis, a devastating human parasitic disease. Utilizing hits selected from a high throughput screening campaign, time-resolved X-ray crystallography, molecular dynamics, mass spectrometry, molecular modeling, protein mutagenesis and functional studies, we find that 2-naphtholmethylamino derivatives bound to this novel ectopic site of Schistosoma mansoni (Sm)TGR are transformed to covalent modifiers and react with its mobile selenocysteine-containing C-terminal arm. In particular, one 2-naphtholmethylamino compound is able to specifically induce the pro-oxidant activity in the inhibited enzyme. Since some 2-naphtholmethylamino analogues show worm killing activity and the ectopic site is not conserved in human orthologues, a general approach to development of novel and selective anti-parasitic therapeutics against schistosoma is proposed.

Correction to Design, Synthesis, and Biological Evaluation of Tetrahydroisoquinoline-Based Histone Deacetylase 8 Selective Inhibitors.

[This corrects the article DOI: 10.1021/acsmedchemlett.7b00126.].

Analysis of Toxigenic Fusarium Species Associated with Wheat Grain from Three Regions of Russia: Volga, Ural, and West Siberia.

Wheat grains collected in three regions of Russia-Volga, Ural, and West Siberia-were analyzed for triangulation of methods in analysis of toxigenic Fusarium species. The presence of fungi and quantitative content of their biomass were detected by using various analytical methods, including a mycological and immunochemical methods, and quantitative PCR. Additionally, an enzyme-linked immunosorbent assay and high-performance liquid chromatography with tandem mass spectrometry were applied for determination of mycotoxins. Regional differences were found regarding the contamination of wheat grain by Fusarium fungi and their toxins. The most important observation was the detection of F. graminearum in the Ural and West Siberian regions, where this pathogen had not been found previously. A maximum damaged grains by F. graminearum and F. sporotrichioides was found in the grain samples from West Siberia. The DNA of F. graminearum was detected in 19.2% and DNA of F. sporotrichioides was found in 84.1% of the analyzed grain samples. The amount of Fusarium antigens in the grain samples from the West Siberian region was 7-8 times higher than in the grain samples from the other two regions. Significant contamination of the grain with deoxynivalenol and T-2/HT-2 toxins (maximum contents were 2239 ppb and 199 ppb, respectively) was detected in the West Siberian region.

Structurally Diverse Histone Deacetylase Photoreactive Probes: Design, Synthesis, and Photolabeling Studies in Live Cells and Tissue.

Histone deacetylase (HDAC) activity is modulated in vivo by post-translational modifications and formation of multiprotein complexes. Novel chemical tools to study how these factors affect engagement of HDAC isoforms by HDAC inhibitors (HDACi) in cells and tissues are needed. In this study, a synthetic strategy to access chemically diverse photoreactive probes (PRPs) was developed and used to prepare seven novel HDAC PRPs 9-15. The class I HDAC isoform engagement by PRPs was determined in biochemical assays and photolabeling experiments in live SET-2, HepG2, HuH7, and HEK293T cell lines and in mouse liver tissue. Unlike the HDAC protein abundance and biochemical activity against recombinant HDACs, the chemotype of the PRPs and the type of cells were key in defining the engagement of HDAC isoforms in live cells. Our findings suggest that engagement of HDAC isoforms by HDACi in vivo may be substantially modulated in a cell- and tissue-type-dependent manner.

Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

BACKGROUND: Platelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions. METHODS: Bioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions. RESULTS: Deletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage. CONCLUSIONS: Our results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

Histone deacetylase inhibitor-based chromatin precipitation for identification of targeted genomic loci.

Histone deacetylase (HDAC) catalyzes the removal of acetyl marks from histones, effectively regulating gene expression. Genome wide chromatin immunoprecipitation (ChIP) studies have shown HDACs are present on numerous active and repressed genes. However, HDAC inhibitors (HDACi) only regulate a small subset of this population in a cell type dependent fashion. To determine genomic locations directly targeted by HDACi, we developed a chromatin precipitation method using a photoreactive HDAC inhibitor probe (photomate). We validate this method by analyzing several canonical HDACi regulated genes, CDKN1A and FOSL1, and compare it to traditional ChIP using HDAC1 antibodies. We show that HDACi target HDACs bound at the promoter regions but not gene bodies, differing from HDAC1 antibody-based ChIP in the case of CDKN1A. This approach is anticipated to be useful for genome wide studies to identify the subset of genes directly regulated by an HDACi in a given cell type.

2-Arylidene Hydrazinecarbodithioates as Potent, Selective Inhibitors of Cystathionine γ-Lyase (CSE).

Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS.