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BIOCIUS RapidFire Management Team Publications: Can Özbal


Development of a High-Throughput Online Solid-Phase Extraction/Tandem Mass Spectrometry Method for Cytochrome P450 Inhibition Screening
J Biomol Screen. 2010 Mar ; 5 
Kheng B. Lim, Dr., Can C. Özbal, and Daniel B. Kassel

A high-throughput online solid-phase extraction/tandem mass spectrometry (online SPE/MS/MS) system has been developed to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of 8 test concentrations and against a panel of 6 cytochrome P450 (CYP) enzymes, 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The new online SPE/MS/MS system has reduced the analysis time to less than 15 min per 96-well plate, translating to a 15-fold time savings compared to the 2-min LC/MS/MS method.

Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay.
J Biomol Screen. 2010 Jan ; 15(1): 52-61 
Langsdorf EF, Malikzay A, Lamarr WA, Daubaras D, Kravec C, Zhang R, Hart R, Monsma F, Black T, Ozbal CC, Miesel L, Lunn CA

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described ... The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.
Assay Drug Dev Technol. 2009 Oct ; 7(5): 495-506
Holt TG, Choi BK, Geoghagen NS, Jensen KK, Luo Q, LaMarr WA, Makara GM, Malkowitz L, Ozbal CC, Xiong Y, Dufresne C, Luo MJ

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. ... Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.
Anal Chim Acta. 2008 Oct 3; 627(1): 105-11
Soulard P, McLaughlin M, Stevens J, Connolly B, Coli R, Wang L, Moore J, Kuo MS, LaMarr WA, Ozbal CC, Bhat BG

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues ... High-throughput mass spec screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable ... The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1.

Back to basics: label-free technologies for small molecule screening.
Comb Chem High Throughput Screen. 2008 Mar ; 11(3): 231-7
Shiau AK, Massari ME, Ozbal CC

Small molecule high-throughput screening in drug discovery today is dominated by techniques which are dependent upon artificial labels or reporter systems. While effective, these approaches can be affected by certain experimental limitations, such as conformational restrictions imposed by the selected label or compound fluorescence/quenching. Label-free approaches potentially address many of these issues by allowing researchers to investigate more native systems without fluorescence- or luminescence-based readouts. However, due to throughput and expense constraints, label-free methods have been largely relegated to a supporting role as the basis of secondary assays. In this review, we describe recent improvements in impedance-based, optical biosensor-based, automated patch clamp and mass spectrometry technologies that have enhanced their ease of use and throughput and, hence, their utility for primary screening of small- to medium-sized compound libraries. The ultimate maturation of these techniques will enable drug discovery researchers to screen large chemical libraries against minimally manipulated biological systems.

High-throughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase.
J Biomol Screen. 2007 Aug ; 12(5): 628-34
Forbes CD, Toth JG, Ozbal CC, Lamarr WA, Pendleton JA, Rocks S, Gedrich RW, Osterman DG, Landro JA, Lumb KJ

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFiretrade mark platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target.

High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha.
J Biomol Screen. 2007 Jun ; 12(4): 473-80
Quercia AK, LaMarr WA, Myung J, Ozbal CC, Landro JA, Lumb KJ

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited less than or = 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selection of compounds is expected to identify common actives, common inactives, false positives, and false negatives. Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation.

High throughput screening via mass spectrometry: a case study using acetylcholinesterase.
Assay Drug Dev Technol. 2004 Aug ; 2(4): 373-81
Ozbal CC, LaMarr WA, Linton JR, Green DF, Katz A, Morrison TB, Brenan CJ

Mass spectrometry-based screening can be applied to a wide range of targets, including those intractable targets that use substrates such as lipids, fatty acids, phospholipids, steroids, prostaglandins, and other compounds not generally amenable to conventional screening techniques. The major limitation to this approach is throughput, making HTS via mass spectrometry impractical. We present a mass spectrometry-based technique and hardware for lead discovery applications. Mass spectrometry enables the design of label-free assays using biologically native substrates for a wide range of enzymatic targets.