Primary High-throughput Screening Research Articles

Development of an LC–MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-l-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic–MS (LC–MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC–MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (≥10 fold), lower limit of quantitation (LOQ) (0.02μM versus 1μM) and wider linear dynamic range (≥4 fold) than the MG assay. Good precision for the LC–MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC–MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.

Dow’s new hafnium polymerization catalyst

High-throughput primary synthesis and screening methods have been applied to the heterogeneously-catalyzed gas phase oxidation of ethane to acetic acid using mixed metal oxide catalysts. The discovery libraries consisted of 16×16 arrays of 256 catalysts on 3″×3″ quartz wafers. Catalysts were prepared using automated liquid-dispensing techniques and screened in parallel for catalytic activity in a Symyx Technologies 256-channel microfluidic reactor. Product detection was performed using parallel colorimetric techniques on products adsorbed on silica-coated glass TLC plates. This workflow allows the screening of more than 3000 samples per day. Promising leads were confirmed in focus libraries and are being optimized in secondary screening. MoV was identified as the most active binary of redox metals and was subsequently doped with main group, rare earth, and transition metals to form ternaries. Prior art MoVX (X=Nb, Ni, Sb) catalysts were successfully reproduced and it was shown that Pd doping significantly increases the catalytic activity of these systems. Three novel MoVY ternary systems were discovered.

Gas phase oxidation of ethane to acetic acid using high-throughput screening in a massively parallel microfluidic reactor system

High-throughput primary synthesis and screening methods have been applied to the heterogeneously-catalyzed gas phase oxidation of ethane to acetic acid using mixed metal oxide catalysts. The discovery libraries consisted of 16×16 arrays of 256 catalysts on 3″×3″ quartz wafers. Catalysts were prepared using automated liquid-dispensing techniques and screened in parallel for catalytic activity in a Symyx Technologies 256-channel microfluidic reactor. Product detection was performed using parallel colorimetric techniques on products adsorbed on silica-coated glass TLC plates. This workflow allows the screening of more than 3000 samples per day. Promising leads were confirmed in focus libraries and are being optimized in secondary screening. MoV was identified as the most active binary of redox metals and was subsequently doped with main group, rare earth, and transition metals to form ternaries. Prior art MoVX (X=Nb, Ni, Sb) catalysts were successfully reproduced and it was shown that Pd doping significantly increases the catalytic activity of these systems. Three novel MoVY ternary systems were discovered.

Discovery from combinatorial heterogeneous catalysis: A new class of catalyst for ethane oxidative dehydrogenation at low temperatures

Combinatorial methodologies are emerging as powerful tools in heterogeneous catalysis. Previously, we have established a strong correlation between catalysts prepared as thin films for high-throughput primary screening to those prepared in bulk for secondary screening in a multi-channel fixed bed (MCFB) microreactor for the ethane oxidative dehydrogenation process [Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 11077]. Numerous catalysts with improved performance over literature compositions were also reported. With this high-throughput technology, tens of thousands of new compositions have been synthesized and tested. In this paper, we describe in detail the discovery of a new class of catalysts with extraordinary performance improvements. These catalysts contain Ni, Nb, and Ta or Co mixed oxides. The respective ternary systems were first screened in a thin film form (∼200 μg catalyst loading). Then they were scaled up to 50 mg level and screened in an MCFB microreactor. The optimal compositions were Ni 0.62Ta 0.10Nb 0.28O x and Ni 0.62Ta 0.20Nb 0.18O x . They convert 20.5% of ethane with 86.2% selectivity to ethylene and 20.0% of ethane with 86.1% selectivity to ethylene at 300 °C, respectively, as compared to 3.3% conversion of ethane with 82.9% selectivity to ethylene for the state-of-the-art catalyst Mo 0.72V 0.26Nb 0.02O x under identical testing conditions. One of these compositions, Ni 0.62Ta 0.10Nb 0.28O x , was further scaled up to 5 g and tested in a conventional bench scale reactor. The performance improvement is consistent with measurements made on the MCFB microreactor.

A fully integrated high-throughput screening methodology for the discovery of new polyolefin catalysts: discovery of a new class of high temperature single-site group (IV) copolymerization catalysts.

For the first time, new catalysts for olefin polymerization have been discovered through the application of fully integrated high-throughput primary and secondary screening techniques supported by rapid polymer characterization methods. Microscale 1-octene primary screening polymerization experiments combining arrays of ligands with reactive metal complexes M(CH(2)Ph)(4) (M = Zr, Hf) and multiple activation conditions represent a new high-throughput technique for discovering novel group (IV) polymerization catalysts. The primary screening methods described here have been validated using a commercially relevant polyolefin catalyst, and implemented rapidly to discover the new amide-ether based hafnium catalyst [eta(2)-(N,O)[bond](2-MeO[bond]C(6)H(4))(2,4,6-Me(3)C(6)H(2))N]Hf(CH(2)Ph)(3) (1), which is capable of polymerizing 1-octene to high conversion. The molecular structure of 1 has been determined by X-ray diffraction. Larger scale secondary screening experiments performed on a focused 96-member amine-ether library demonstrated the versatile high temperature ethylene-1-octene copolymerization capabilities of this catalyst class, and led to significant performance improvements over the initial primary screening discovery. Conventional one gallon batch reactor copolymerizations performed using selected amide-ether hafnium compounds confirmed the performance features of this new catalyst class, serving to fully validate the experimental results from the high-throughput approaches described herein.

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system

Antagonists to the human metabotropic glutamate receptor subtype 5 a(mGluR 5a) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson’s disease. To discover novel antagonists to the mGluR 5a, a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.