Quantitative inhibitor fingerprinting of metalloproteases using small molecule microarrays

Current methods to identify interactions on small molecule microarrays (SMMs) introduce false positives that are difficult to dissect from the "real" binding events without tedious downstream re-evaluation. To specifically elucidate only activity-dependent ligand binding interactions, we have developed a technique that can be universally applied to present SMM systems. Our method makes use of a dual-color application strategy and is based on the simultaneous application of differentially treated samples. Overcoming the limitations of slide-to-slide variation, this method directly revealed activity-dependent interactions through a one-step application of protein samples on SMMs. Besides providing lead molecules for further development, the high-throughput screening results confer activity-dependent fingerprints for quantitative characterization and differentiation of proteins. The procedure was tested using a synthetic hydroxamate peptide library with 1400 discrete sequences permuted combinatorially across P1', P2', and P3' positions. Functional profiling across a panel of metalloproteases provided 44,800 datapoints within just eight SMM slides. These data were globally analyzed for activities, specificity, potency, and hierarchical clustering providing unique insights into inhibitor design and preference within this group of enzymes. Quantitative K(D) measurements performed on SMMs using one of the enzymes in the panel, Anthrax Lethal Factor, the toxic component of a notorious bioterror agent, unraveled several lead micromolar binders for further development. Overall, the effectiveness of the SMM platform is shown to be enhanced and extended using the strategy presented in this work.     

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.     

Small molecule microarrays: the first decade and beyond

The turn of the century witnessed the development of small molecule, protein, cell and tissue microarrays, heralding a new era of discovery-driven research using a host of different chemical libraries, biomolecules and tissue types. This highlight takes stock of this first decade of small molecule microarrays (SMMs) and describes how the technology has matured into a robust screening platform. We also highlight the many interesting and unique applications appearing using small molecule microarrays, in order to project forward to possible areas in which SMMs could contribute further to over the next five to ten years.     

Absorption line profile recovery based on TDLS and MEMS micro-mirror for photoacoustic gas sensing

A novel and efficient absorption line recovery technique is presented. A micro-electromechanical systems (MEMS) mirror driven by an electrothermal actuator is used to generate laser intensity modulation through the mirror reflection. Tunable diode laser spectroscopy (TDLS) and photoacoustic spectroscopy (PAS) are used to recover the target absorption line profile which is compared with the theoretical Voigt profile. The target gas is 0.01% acetylene (C2H2) in a nitrogen host gas. The laser diode wavelength is swept across the P17 absorption line of acetylene at 1535.4 nm by a current ramp, and an erbium-doped fibre amplifier (EDFA) is used to enhance the optical intensity and increase the signal-to-noise ratio (SNR). A SNR of about 35 is obtained with 100 mW laser power from the EDFA. Good agreement is achieved between the experimental results and the theoretical simulation for the P17 absorption line profile.     

Twin-Bladed Microelectro Mechanical Systems Variable Optical Attenuator

The design and evaluation of a microelectro mechanical systems (MEMS) based variable optical attenuator is reported. The device contains two blades, which are each driven by a separate electrostatic comb microactuator, and move independently to form a variable slit. This device has been fabricated in silicon-on-insulator material which has been back-etched. Electro-mechanical design considerations, including factors to minimise the side instability of the comb drive, are described. Finite element modelling (FEM) of the variable optical attenuator (VOA) is backed up by theoretical results, and the results from the theoretical work verify the findings from the FEM. Optical modelling of the VOA using near field diffraction theory is also reported. Experimentally, the device was driven from 0-34V DC to measure its static characteristics. For dynamic characterisation, the device was operated from 0-28 V AC and its fundamental resonant frequency was measured to be 3 kHz. Optical measurements including wavelength dependent attenuation are also presented.     

Activity-based high-throughput profiling of metalloprotease inhibitors using small molecule microarrays

We herein describe a high-throughput small molecule microarray (SMM) method that enables quick and cost-effective identification of potent inhibitors of metalloproteases in an activity-dependent manner, thereby offering a rapid means for inhibitor discovery and profiling.     

Titanium(IV) and zirconium(IV) derivatives of tetradentate schiff bases

Summary Titanium and zirconium isopropoxides react with the tetradentate Schiff bases, I)is-salicylaldehyde-o-phenylenediimine (SBH₂ ) and bis-salicylaldehyde-p-phenylencdiimine (SBH₂) in anhydrous benzene in 1: 1 and 1 :2 molar ratios to give almost quantitative yields of M(OPr-i)₂ (SB) and M(OPr-i)2(SB)(SBH), where M = Ti or Zr and [SB]² is the anion of the corresponding Schiff base, SBH₂. The i.r. spectra of the completes have been recorded and tentative assignments for C=N and C-O stretching frequencies made.     

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.