Chenggang Zhu

Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity

Poly(ADP-ribose) polymerases (PARPs) are ADP-ribosylating enzymes and play important roles in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, a poly-ADP-ribose transferase, and suffer from poor selectivity. PARP16, a mono-ADP-ribose transferase, has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Here we newly characterized epigallocatechin-3-gallate (EGCG) as a potential inhibitor of PARP16. We found that EGCG was associated with PARP16 and dramatically inhibited its activity in vitro. Moreover, EGCG suppressed the ER stress-induced phosphorylation of PERK and the transcription of unfolded protein response-related genes, leading to dramatically increase of cancer cells apoptosis under ER stress conditions, which was dependent on PARP16. These findings newly characterized EGCG as a potential inhibitor of PARP16, which can enhance the ER stress-induced cancer cell apoptosis, suggesting that a combination of EGCG and ER stress-induced agents might represent a novel approach for cancer therapy or chemoprevention.

Characterization of protein expression levels with label-free detected reverse phase protein arrays

In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies.

Calibration of oblique-incidence reflectivity difference for label-free detection of a molecular layer

Oblique-incidence reflectivity difference (OI-RD) is a form of polarization-modulation ellipsometry that measures properties of thin films on a solid surface through the change in polarization state of light upon reflection from the surface. The measurement accuracy depends on the precision of the phase modulation amplitude and azimuthal alignments of key polarizing optical elements and, thus, requires careful calibration. In the present work, we describe robust methods of such calibrations that enable precise determination of the modulation amplitude and static retardation of a phase modulator and azimuths of key polarizing optics in an OI-RD system.

Detecting Signals With Direct Fast Fourier Transform for Microarray Data Collection

Small signals are routinely detected at a frequency away from the 1/f noise. Typically, the signal source is modulated at a suitable frequency, and the harmonics of the modulation frequency in the signal are analyzed with phase sensitive detectors such as Lock-in amplifiers. Yet, the high cost and poor portability of stand-alone Lock-in amplifiers are obstacles to making such an approach of harmonic detection into a widely usable instrumentation. In this letter, we show that for many applications where signals are not too small (e.g., more than tens of micro-volts) the direct fast Fourier transform using a commercially available, general-purpose data acquisition board serves as a cost-effective alternative to Lock-in amplifiers without sacrificing the signal-to-noise ratio.

Fast Focal Point Correction in Prism-Coupled Total Internal Reflection Scanning Imager Using an Electronically Tunable Lens

Total internal reflection (TIR) is useful for interrogating physical and chemical processes that occur at the interface between two transparent media. Yet prism-coupled TIR imaging microscopes suffer from limited sensing areas due to the fact that the interface (the object plane) is not perpendicular to the optical axis of the microscope. In this paper, we show that an electrically tunable lens can be used to rapidly and reproducibly correct the focal length of an oblique-incidence scanning microscope (OI-RD) in a prism-coupled TIR geometry. We demonstrate the performance of such a correction by acquiring an image of a protein microarray over a scan area of 4 cm2 with an effective resolution of less than 20 microns. The electronic focal length tuning eliminates the mechanical movement of the illumination lens in the scanning microscope and in turn the noise and background drift associated with the motion.

Label-free Microarray-based Binding Affinity Constant Measurement with Modified Fluidic Arrangement

The oblique-incidence reflectivity difference (OI-RD) scanning microscopy has the capability of simultaneously measuring binding curves of a protein probe with tens of thousands molecular targets in a microarray and yielding reaction rate constants. However, the quality of reaction rate constants is influenced by the fluidic system. To improve the quality of reaction rate constant measurement over the entire microarray, we demonstrate a fluidic chamber that allows the fluid to flow from the bottom to the top uniformly across the microarray and thus provides more uniform and accurate measurements of reaction rate constants with simplified fluidic design.

High-throughput screening based on label-free detection of small molecule microarrays

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

High-throughput screening based on small-molecule microarrays and OI-RD microscope

We developed a novel high-throughput screening platform based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) microscope. OI-RD microscope is able to monitor tens of thousands of biomolecular interaction in a single experiment without the need to label any biomolecule. We also developed surface chemistry to prepare SMMs which is able to immobilize compounds with any nucleophilic residues on phenyl-isocyanate functionalized glass slides with high efficiencies. By using OI-RD microscope and SMMs with 3,375 bioactive compounds, we screened target proteins in high-throughput and label-free mode and we found hits for respective target protein. The novel high-throughput screening platform enables target proteins with unknown structure and/or unknown function to be effectively screened.