Lead Discovery References Using RapidFire

A potent, small molecule inhibitor with a favorable pharmacokinetic profile to allow for sustained SCD inhibition in vivo was identified. Starting from a low MW acyl guanidine (5a), identified with a RapidFire HighThroughput Mass Spectrometry (RF-MS) assay, iterative library design was used to rapidly probe the amide and tail regions of the molecule. Singleton synthesis was used to probe core changes. Biological evaluation of a SCD inhibitor (5b) included in vitro potency at SCD-1 and in vivo modulation of the plasma desaturation index (DI) in rats on a low essential fatty acid (LEFA) diet. In addition to dose-dependent decrease in DI, effects on rodent ocular tissue were noted. Therefore, in rat, these SCD inhibitors only recapitulate a portion of phenotype exhibited by the SCD-1 knockout mouse. 2011 Journal of Biomolecular Screening Advances in Label-Free Screening Approaches for Studying Sirtuin-Mediated Deacetylation. Rye PT, Frick LE, Ozbal CC, Lamarr WA. J Biomol Screen. 2011 Sep 12. [Epub ahead of print]. Web link: http://jbx.sagepub.com/content/early/2011/09/10/1087057111420291.abstract Abstract: The sirtuin enzymes, a class of NAD+-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were applied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data, including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via Oacetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of wholehistone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins using more biologically relevant molecules. The sirtuin enzymes, a class of NAD+-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were applied to investigating the deacetylation of multiple-peptide substrates by multiple-sirtuin enzymes. Kinetic data, including binding constants, inhibition, and, in some cases, activation, are demonstrated to correlate well, both between the methodologies and with previous literature precedent. In addition, the ability to monitor sirtuin activity via Oacetyl-ADP-ribose production permits experimentation on whole-protein substrates. The deacetylation of wholehistone proteins by SIRT3, and inhibition thereof, is presented and demonstrates the feasibility of screening sirtuins using more biologically relevant molecules. 2011 Journal of Biomolecular Screening Advances in Label-Free Screening Approaches for Studying Histone Acetyltransferases. Rye PT, Frick LE, Ozbal CC, Lamarr WA. J Biomol Screen. 2011 Sep 9. [Epub ahead of print] Web link: http://jbx.sagepub.com/content/early/2011/09/08/1087057111418653.abstract Abstract: Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves monitoring all Last Updated August 22, 2012 11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com possible acetylation states of a peptide concurrently to measure HAT activity. The second approach measures acetylation reactions, on both peptides and whole protein substrates, via direct detection of the acetyl-coenzyme A cosubstrate and coenzyme A coproduct. Lastly, the authors demonstrate the ability to monitor directly the acetylation state of whole histone proteins in the same high-throughput manner using time-of-flight mass spectrometry. The generation of compound-mediated inhibition data using each of these techniques establishes mass spectrometry as a versatile, label-free, and biologically relevant screening approach to this challenging target class. 2011 Journal of Biomolecular Screening -Pfizer/BIOCIUS High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using RapidFire Mass Spectrometry Maureen K. Highkin, Matthew P. Yates, Olga V. Nemirovskiy, William A. Lamarr, Grace E. Munie, John W. Rains, Jaime L. Masferrer, Marek M. Nagiec J Biomol Screen February 4, 2011 vol. 16 no. 2 272-277 Web link: http://jbx.sagepub.com/content/16/2/272.abstract Note: This was also presented as a poster during APA 2010. Abstract: To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96and 384-well plates prior to analyses using the RapidFire® mass spectrometry system. To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96and 384-well plates prior to analyses using the RapidFire® mass spectrometry system. 2010 Journal of Biomolecular Screening – Schering Plough/BioTrove/Seton Hall Univ./Merck/Idaho National Laboratory Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-Nacetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay. Langsdorf EF, Malikzay A, Lamarr WA, Daubaras D, Kravec C, Zhang R, Hart R, Monsma F, Black T, Ozbal CC, Miesel L, Lunn CA J Biomol Screen. 2010 Jan ; 15(1): 52-61 Web link: http://jbx.sagepub.com/content/15/1/52.abstract Abstract: A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described.. The results show that mass spectrometrybased screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions. A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described.. The results show that mass spectrometrybased screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions. The Situation: Find a way to perform a label-free inhibition screen based on the native LpxC substrate in order to develop a better anti-bacterial drug. LpxC (a deacetylase essential for endotoxin biosynthesis in gram-negative bacteria) is a well-known anti-bacterial and anti-inflammatory drug target, but effective drugs based on inhibitors of this enzyme have been difficult to develop. Typically, scientists have used an optical assay based on a LpxC surrogate substrate to screen compound libraries for LpxC inhibitors. The surrogate substrate has a Km >100x that of the native substrate and this large disparity in Km may be the reason for so many past failures to develop an effective inhibitor. The Solution: Addressing the Challenges: In collaboration with RapidFire scientists, an assay was developed using the RapidFire automated SPE-based system in conjunction with mass spec analysis of the native LpxC substrate. The RapidFire team quickly performed primary screening analysis using this new LpxC assay on the client’s compressed compound library (~250,000 wells). Secondary screens were then used to confirm several new inhibitors. The Results: Three new patent applications and the potential of a new effective drug to treat gram-negative bacterial infections and their associated inflammatory disease. 2009 ASSAY and Drug Development Technologies – Merck/BioTrove Last Updated August 22, 2012 11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications. Holt TG, Choi BK, Geoghagen NS, Jensen KK, Luo Q, LaMarr WA, Makara GM, Malkowitz L, Ozbal CC, Xiong Y, Dufresne C, Luo MJ Assay Drug Dev Technol. 2009 Oct ; 7(5): 495-506 Web link: http://www.liebertonline.com/doi/abs/10.1089/adt.2009.0200 Abstract: 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. 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. 2009 Combinational Chemistry and High Throughput Screening A Case Study on Acetyl-Coenzyme A Carboxylase using RapidFire – Mass Spectrometry (RFMS) Maxine Jonas, William A. LaMarr and Can Özbal Abstract: In this review various technologies and approaches for the utilization of mass spectrometry in high throughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical libraries again stenzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase (ACC) assay is described. In this review various technologies and approaches for the utilization of mass spectrometry in high throughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical libraries again stenzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase (ACC) assay is described. 2008 Analytica Chimica Acta – Pfizer/BioTrove Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry. Soulard P, McLaughlin M, Stevens J, Connolly B, Coli R, Wang L, Moore J, Kuo MS, LaMarr WA, Ozbal CC, Bhat BG Anal Chim Acta. 2008 Oct 3; 627(1): 105-11 Web link: http://www.sciencedirect.com/science/article/pii/S0003267008006867 Abstract: 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. 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. 2008 Combinatorial Chemistry & High Throughput Screening – Kalypsys (Wako)/BioTrove Back to basics: label-free technologies for small molecule screening. Shiau AK, Massari ME, Ozbal CC Comb Chem High Throughput Screen. 2008 Mar ; 11(3): 231-7 Web link: http://www.benthamdirect.org/pages/content.php?CCHTS/2008/00000011/00000003/0006A.SGM Abstract: 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 fluorescenceor 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 smallto 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. 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 fluorescenceor 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 smallto 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. Last Updated August 22, 2012 11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com 2007 Journal of Biomolecular Screening – Bayer/BioTrove High-throughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase. Forbes CD, Toth JG, Ozbal CC, Lamarr WA, Pendleton JA, Rocks S, Gedrich RW, Osterman DG, Landro JA, Lumb KJ J Biomol Screen. 2007 Aug ; 12(5): 628-34 Web link: http://jbx.sagepub.com/content/12/5/628.abstract Abstract: 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 RapidFire trade 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. 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 RapidFire trade 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. 2007 Journal of Biomolecular Screening – Bayer/BioTrove High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha. Quercia AK, LaMarr WA, Myung J, Ozbal CC, Landro JA, Lumb KJ J Biomol Screen. 2007 Jun ; 12(4): 473-80 Web link: http://jbx.sagepub.com/content/12/4/473.abstract Abstract: 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. 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. 2004 ASSAY and Drug Development Technologies – BioTrove High Throughput Screening via Mass Spectrometry: A Case Study Using Acetylcholinesterase Can C. .zbal, William A. LaMarr, John R. Linton, Donald F. Green, Arrin Katz, Thomas B. Morrison, and Colin J.H. Brenan Abstract: 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. Massspectrometry enables the design of label-free assays using biologically native substrates for a widerange of enzymatic targets. This system can be used for the direct quantification of analytes incomplex reaction mixtures with typical throughputs of 4Ð5 s per sample. A mass spectrometrybasedassay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinicalimportance in AlzheimerÕs disease. The system was used to screen a small chemical library. Severalpotent inhibitors were identified, and the IC50 values of the inhibitors were determined. 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. Massspectrometry enables the design of label-free assays using biologically native substrates for a widerange of enzymatic targets. This system can be used for the direct quantification of analytes incomplex reaction mixtures with typical throughputs of 4Ð5 s per sample. A mass spectrometrybasedassay was developed to identify inhibitors of acetylcholinesterase, an enzyme with clinicalimportance in AlzheimerÕs disease. The system was used to screen a small chemical library. Severalpotent inhibitors were identified, and the IC50 values of the inhibitors were determined. Lead Discovery Posters and Presentations Last Updated August 22, 2012 11 Audubon Road I Wakefield, MA 01880 I T: 781.928.2700 I www.Agilent.com 2012 SLAS – Agilent Technologies (Poster) A Label-Free Screening Approach to Epigenetic Drug Discovery: Using Mass Spectrometry to Monitor Protein and Nucleic Acid Modification Events William A. LaMarr, Peter T. Rye, Lauren E. Frick, Agilent Technologies, Inc. Wakefield, MA 0188