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.     

Rapid assembly and in situ screening of bidentate inhibitors of protein tyrosine phosphatases

[reaction: see text] We have successfully designed and synthesized a small library of protein tyrosine phosphatase (PTP) inhibitors, in which the so-called "click chemistry" or Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction was carried out for rapid assembly of 66 different bidentate compounds. Subsequent in situ enzymatic screening revealed a potential PTP1B inhibitor (IC(50) = 4.7 microM) which is 10-100 fold more potent than other PTPs.     

Sol-Gel Derived Thin Films for Hydrogen Sulphide Gas Sensing

Utilizing the sol-gel fabrication route, we have successfully modified and tailored the optical absorption in the visible spectrum of thionine dye trapped in an MTMS gel-matrix to coincide with the emission of a red diode laser operating at 660 nm. These thionine-doped MTMS thin films coated onto transparent substrates have shown a remarkable change in optical absorption in the presence of gaseous hydrogen sulphide (H₂S) diluted in air and in the absence of any buffer gas. The rapid response, sensitivity, reversibility and durability shown by this material can be exploited in developing absorption-based optical H₂S sensors in either an integrated optics or all-optical fiber approach using a red diode laser source.     

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.     

Inhibitor fingerprinting of matrix metalloproteases using a combinatorial peptide hydroxamate library

We report the inhibitor fingerprints of seven matrix metalloproteases, representing all five established families of this important class of enzymes, against a highly diversified small-molecule library. A total of 1400 peptide hydroxamates were individually prepared by systematically permuting both natural and unnatural amino acids across the P1', P2', and P3' positions, thereby generating an inhibitor library with three-pronged structural diversity. High-throughput screenings were efficiently conducted in microtiter plate format, providing a rapid and quantitative determination of inhibitor potency across the panel of enzymes. Despite similarities in substrate preferences and structural homologies within this class of enzymes, our findings revealed distinct patterns of inhibition for each MMP against varied chemical scaffolds. The resulting inhibitor fingerprints readily facilitated the identification of inhibitors with good potency as well as desirable selectivity, potentially minimizing adverse effects when developing such leads into candidate drugs. The strategy also offers a novel method for the functional classification of matrix metalloproteases, on the basis of the characteristic profiles obtained using the diverse set of inhibitors. This approach thus paves the way forward in lead identification by providing a rapid and quantitative method for selectivity screening at the outset of the drug discovery process.     

Alternative fiber-optic backreflectance sensor for measurement of the effective focal lengths of optical elements and distances

An alternative and simple fiber-optic backreflectance sensor method for accurate measurement of either effective focal lengths of optical elements and short distances using a single-mode optical fiber and cw laser is presented. The method is based on the intensity sensing of focused backreflectance laser emission and the spatial location of three specific points: the focal point of the focusing optical element and two object points. The single-mode fiber is a key optical element and serves simultaneously as a point laser source for testing, an object for projecting and a highly sensitive point receiver of the focused backreflectance emission. The experimental and analytical results demonstrate the potential of the method for locating the spatial points and determining the effective focal length with accuracies exceeding 1 μm and 0.5%, respectively, as well as for short distance measurement with submicron accuracy.     

Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases

Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P(1) position. It also allows efficient synthesis of vinyl sulfone-containing, activity-based probes of cysteine proteases used in a proteomic experiment.