A novel three base-pair deletion in domain two of the cardiac sodium channel causes Brugada syndrome.

INTRODUCTION: Mutations within SCN5A are found in a significant proportion (15-30%) of Brugada syndrome (BrS) cases and impair sodium transport across excitable cardiac cells that mediate ventricular contractions. Genetic testing offers a means to clinically assess and manage affected individuals and their family members. METHODS AND RESULTS: The proband at age 44 years old exhibited a syncopal event during exercise, and presented later with a spontaneous type-I BrS pattern on 12­lead resting electrocardiogram (ECG). Mutational analysis performed across all SCN5A exons revealed a unique three base-pair deletion p.M741_T742delinsI (c.2223_2225delGAC), in a heterozygous state in the proband and 2 siblings. This mutation was not seen in a cohort of 105 ethnicity-matched controls or in public genome databases. Patch clamp electrophysiology study conducted in TSA201 cells showed an abolishment of sodium current (INa). The proband, and several relatives, also harboured a known SCN5A variant, p.R1193Q (c.3578G>A). CONCLUSION: Our study has demonstrated the deleterious effect of a novel SCN5A mutation p.M741_T742delinsI (c.2223_2225delGAC). The findings highlight the complex effects of gender and age in phenotype manifestation. It also offers insights into improving the long-term management of BrS, and the utility of cascade genetic screening for risk stratification.

Accelerated cellular on- and off-target screening of bioactive compounds using microarrays.

In situ proteome labeling was carried out with 9 drug-like probes in live mammalian cells, with the corresponding cellular targets captured on microarrays and simultaneously screened using a diverse set of antibodies, revealing potential on- and off-targets.

A Brugada syndrome proband with compound heterozygote SCN5A mutations identified from a Chinese family in Singapore.

AIMS: Brugada syndrome (BrS) is a rare heritable ventricular arrhythmia. Genetic defects in SCN5A, a gene that encodes the α-subunit of the sodium ion channel Nav1.5, are present in 15-30% of BrS cases. SCN5A remains by far, the highest yielding gene for BrS. We studied a young male who presented with syncope at age 11. This proband was screened for possible disease causing SCN5A mutations. The inheritance pattern was also examined amongst his first-degree family members. METHODS AND RESULTS: The proband had a baseline electrocardiogram that showed Type 2 BrS changes, which escalated to a characteristic Type I BrS pattern during a treadmill test before polymorphic ventricular tachycardia onset at a cycle length of 250 ms. Mutational analysis across all 29 exons in SCN5A of the proband and first-degree relatives of the family revealed that the proband inherited a compound heterozygote mutation in SCN5A, specifically p.A226V and p.R1629X from each parent. To further elucidate the functional changes arising through these mutations, patch-clamp electrophysiology was performed in TSA201 cells expressing the mutated SCN5A channels. The p.A226V mutation significantly reduced peak sodium current (INa) to 24% of wild type (WT) whereas the p.R1629X mutation abolished the current. To mimic the functional state in our proband, functional expression of the compound variants A226V + R1629X resulted in overall peak INa of only 13% of WT (P < 0.01). CONCLUSION: Our study is the first to report a SCN5A compound heterozygote in a Singaporean Chinese family. Only the proband carrying both mutations displayed the BrS phenotype, thus providing insights into the expression and penetrance of BrS in an Asian setting.

Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism.

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Single-Vehicular Delivery of Antagomir and Small Molecules to Inhibit miR-122 Function in Hepatocellular Carcinoma Cells by using "Smart" Mesoporous Silica Nanoparticles.

MicroRNAs (miRNAs) regulate a variety of biological processes. The liver-specific, highly abundant miR-122 is implicated in many human diseases including cancer. Its inhibition has been found to result in a dramatic loss in the ability of Hepatitis C virus (HCV) to infect host cells. Both antisense technology and small molecules have been used to independently inhibit endogenous miR-122 function, but not in combination. Intracellular stability, efficient delivery, hydrophobicity, and controlled release are some of the current challenges associated with these novel therapeutic methods. Reported herein is the first single-vehicular system, based on mesoporous silica nanoparticles (MSNs), for simultaneous cellular delivery of miR-122 antagomir and small molecule inhibitors. The controlled release of both types of inhibitors depends on the expression levels of endogenous miR-122, thus enabling these drug-loaded MSNs to achieve combination inhibition of its targeted mRNAs in Huh7 cells.

Discovery of cell-permeable inhibitors that target the BRCT domain of BRCA1 protein by using a small-molecule microarray.

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50% of hereditary breast cancers. Cell-permeable, small-molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray-based platforms, we have discovered the first cell-permeable protein-protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)2 domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation-induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small-molecule inhibitor of poly-ADP-ribose polymerase) and Etoposide (a small-molecule inhibitor of topoisomerase II). Unlike previously reported peptide-based PPI inhibitors of BRCA1, our compounds are small-molecule-like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP-related pathways in DNA damage and repair response, and in cancer therapy.

A peptide aldehyde microarray for high-throughput profiling of cellular events.

Microarrays provide exciting opportunities in the field of large-scale proteomics. With the aim to elucidate enzymatic activity and profiles within native biological samples, we developed a microarray comprising a focused positional-scanning library of enzyme inhibitors. The library was diversified across P(1)-P(4) positions, creating 270 different inhibitor sublibraries which were immobilized onto avidin slides. The peptide aldehyde-based small-molecule microarray (SMM) specifically targeted cysteine proteases, thereby enabling large-scale functional assessment of this subgroup of proteases, within fluorescently labeled samples, including pure proteins, cellular lysates, and infected samples. The arrays were shown to elicit binding fingerprints consistent with those of model proteins, specifically caspases and purified cysteine proteases from parasites (rhodesein and cruzain). When tested against lysates from apoptotic Hela and red blood cells infected with Plasmodium falciparum, clear signatures were obtained that were readily attributable to the activity of constituent proteases within these samples. Characteristic binding profiles were further able to distinguish various stages of the parasite infection in erythrocyte lysates. By converting one of our brightest microarray hits into a probe, putative protein markers were identified and pulled down from within apoptotic Hela lysates, demonstrating the potential of target validation and discovery. Taken together, these results demonstrate the utility of targeted SMMs in dissecting cellular biology in complex proteomic samples.

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs).

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.

Visual SNP genotyping using asymmetric PCR and split DNA enzymes.

Harnessing and applying genomic technologies in resource limited environments demand a next generation of platforms, which are convenient, quick and easy to apply. We describe here a visual colour change assay that can be applied to SNP genotyping, which unlike traditional methods, does not adopt complicated procedures or expensive instrumentation, desirable features in bringing genetic capabilities outside the laboratory. Our strategy involved a two-step method that first enriched target genomic regions using asymmetric PCR, followed by direct in situ application of split DNA probes. In the presence of target sequences that perfectly matched the complementary probes, the split probes reassembled active DNAzymes, which catalysed a colour change reaction. A single-base mismatch (indicative of a polymorphism) prevented this reassembly and colour change, providing the means for accurate SNP calling. This is the first report, to our knowledge, that demonstrates successful visual SNP genotyping of actual human DNA samples using DNAzymes.

Plasma cytokine profiles in preprotachykinin-A knockout mice subjected to polymicrobial sepsis.

During the course of polymicrobial sepsis, a range of pro- and antiinflammatory cytokines are produced by the host immune system. Successful recovery from sepsis involves striking a balance between these counteracting cytokines. We herein investigated the circulating cytokine profiles in preprotachykinin-A knockout (PPTA(-/-)) mice, which have been found to be protected significantly against microbial sepsis, by employing multiplexed bead-based suspension arrays for the measurement of 18 plasma cytokines. Four sets of PPTA(-/-) and wild-type mice, each with six mice, were subjected to cecal ligation and puncture-induced sepsis or a sham procedure and were killed at 1, 5, 8 and 24 h post surgery. The cytokine profiles revealed, rather interestingly, that both pro- and antiinflammatory cytokines were elevated in the knockout group in response to a septic challenge. The higher systemic levels of both pro- and antiinflammatory cytokines in PPTA(-/-) septic mice was similar to the increase that we observed earlier in lung tissue of PPTA(-/-) mice after induction of sepsis. Thus, elevated levels of both pro- and antiinflammatory mediators may act simultaneously and help to resolve the infectious assault at the early stages of sepsis without excessively damaging the host tissue in PPTA(-/-) mice. In addition, our results underline the importance of comprehensive clinical analysis of multiple biomarkers to provide a better prognostic tool.

Next generation chemical proteomic tools for rapid enzyme profiling.

Sequencing of the human genome provided a wealth of information about the genomic blueprint of a cell. But genes do not tell the entire story of life and living processes; identifying the roles of enzymes and mapping out their interactions is also crucial. Enzymes catalyze virtually every cellular process and metabolic exchange. They not only are instrumental in sustaining life but also are required for its regulation and diversification. Diseases such as cancer can be caused by minor changes in enzyme activities. In addition, the unique enzymes of pathogenic organisms are ripe targets for combating infections. Consequently, nearly one-third of all current drug targets are enzymes. An estimated 18-29% of eukaryotic genes encode enzymes, but only a limited proportion of enzymes have thus far been characterized. Therefore, little is understood about the physiological roles, substrate specificity, and downstream targets of the vast majority of these important proteins. A key step toward the biological characterization of enzymes, as well as their adoption as drug targets, is the development of global solutions that bridge the gap in understanding these proteins and their interactions. We herein present technological advances that facilitate the study of enzymes and their properties in a high-throughput manner. Over the years, our group has introduced and developed a variety of such enabling platforms for many classes of enzymes, including kinases, phosphatases, and proteases. For each of these different types of enzymes, specific design considerations are required to develop the appropriate chemical tools to characterize each class. These tools include activity-based probes and chemical compound libraries, which are rapidly assembled using efficient combinatorial synthesis or "click chemistry" strategies. The resulting molecular assortments may then be screened against the target enzymes in high-throughput using microplates or microarrays. These techniques offer powerful means to study, profile, and discover potent small molecules that can modulate enzyme activity. This Account will describe the concepts involved in designing chemical probes and libraries for comparative enzyme screening and drug discovery applications, as well as highlight how these technologies are changing the way in which enzymes may be rapidly profiled and characterized.

Metabolic Profiling of a Porcine Combat Trauma-Injury Model Using NMR and Multi-Mode LC-MS Metabolomics-A Preliminary Study.

Profiles of combat injuries worldwide have shown that penetrating trauma is one of the most common injuries sustained during battle. This is usually accompanied by severe bleeding or hemorrhage. If the soldier does not bleed to death, he may eventually succumb to complications arising from trauma hemorrhagic shock (THS). THS occurs when there is a deficiency of oxygen reaching the organs due to excessive blood loss. It can trigger massive metabolic derangements and an overwhelming inflammatory response, which can subsequently lead to the failure of organs and possibly death. A better understanding of the acute metabolic changes occurring after THS can help in the development of interventional strategies, as well as lead to the identification of potential biomarkers for rapid diagnosis of hemorrhagic shock and organ failure. In this preliminary study, a metabolomic approach using the complementary platforms of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS) was used to determine the metabolic changes occurring in a porcine model of combat trauma injury comprising of penetrating trauma to a limb with hemorrhagic shock. Several metabolites associated with the acute-phase reaction, inflammation, energy depletion, oxidative stress, and possible renal dysfunction were identified to be significantly changed after a thirty-minute shock period.

Applications of microarrays in pathogen detection and biodefence.

The microarray is a platform with wide-ranging potential in biodefence. Owing to the high level of throughput attainable through miniaturization, microarrays have accelerated the ability to respond in an epidemic or crisis. Extending beyond diagnostics, recent studies have applied microarrays as a research tool towards understanding the etiology and pathogenicity of dangerous pathogens, as well as in vaccine development. The original emphasis was on DNA microarrays, but the range now includes protein, antibody and carbohydrate microarrays, and research groups have exploited this diversity to further extend microarray applications in the area of biodefence. Here, we discuss the impact and contributions of the growing range of microarrays and emphasize the concepts that might shape the future of biodefence research.

Activity-based protein profiling: new developments and directions in functional proteomics.

Proteomic screening has become increasingly insightful with the availability of novel analytical tools and technologies. Detailed analysis and integration of the profound datasets attained from comprehensive profiling studies are enabling researchers to dig deeper into the foundations of genomic and proteomic networks, towards a clearer understanding of the intricate cellular circuitries they manifest. The major difficulty often lies in correlating the patho/physiological state presented with the underlying biological mechanisms; therefore, identification of causal variants as therapeutic targets is extremely important. Herein, we will describe methods that address this challenge through activity-based protein profiling, which applies chemical probes to the comparison and monitoring of protein dynamics across complex proteomes. Over recent years such activity-based probes have been creatively augmented with applications in gel-based separations, microarrays and in vivo imaging. These developments offer a newfound ability to characterise and differentiate cells, tissues and proteomes through activity-dependent signatures; this has expanded the scope and impact of activity-based probes in biomedical research.

Activity-based fingerprinting and inhibitor discovery of cysteine proteases in a microarray.

A panel of 20 peptide vinyl sulfone probes has been synthesized and used to generate activity-based fingerprinting profiles of cysteine proteases in both gel- and microarray-based formats; the inhibitor fingerprints of representative small molecule inhibitors targeted against 4 cysteine proteases were also obtained, in high-throughput, using the same protein microarray platform.

The Expanding World of Small Molecule Microarrays.

Speed and throughput are vital ingredients for discovery driven, "-omics" research. The small molecule microarray (SMM) succeeds at delivering phenomenal screening throughput and versatility. The concept at the heart of the technology is elegant, yet simple: by presenting large collections of molecules in high density on a flat surface, one is able to interrogate all possible interactions with desired targets, in just a single step. SMMs have become established as the choice platform for screening, lead discovery, and molecular characterization. This introduction describes the principles governing microarray construction and use, focusing on practical challenges faced when conducting SMM experiments. It will explain the key design considerations and lay the foundation for the chapters that follow. (An earlier version of this chapter appeared in Small Molecule Microarrays: Methods and Protocols, published in 2010.).

Array-on-Array Strategy For Activity-Based Enzyme Profiling.

We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.

Protein-Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

Small molecule microarrays: recent advances and applications.

Directed or exploratory drug development programs constantly seek robust screening platforms for the high fidelity identification and validation of potential targets. Small-molecule microarrays (SMMs) have risen to this call by elegantly forging the capability of combinatorial chemistry in producing myriad compounds with the powerful throughput afforded by microarrays. This synergism offers scientists a versatile tool for rapid compound analysis and discovery. Microarrays of small molecules have already been successfully applied in important areas ranging from protein profiling to the discovery of therapeutic leads. Recent interesting developments towards improved immobilization strategies and library creation methods, together with novel advances herein described, have set the stage for SMMs to take on wider and more routine applications in academia and industry. As a rapidly maturing technology, SMMs pave the way forward in high-throughput exploration, both in the identification of biologically significant natural and synthetic small molecules and in harnessing their vast potential towards medicinal and diagnostic applications.