The Laboratory Robotics Interest Group
Mid Atlantic Chapter
November 2003 Meeting
ADME and Compound Profiling
Meeting & Poster Session
Date: Wednesday, November 5, 2003
Place: Somerset Marriott Hotel, 110 Davidson Ave., Somerset, NJ 08873
Phone: 732-560-0500, Fax: 732-560-3669
Itinerary: Poster Session & Social Period - 4:00 to
6:00 PM
Meeting & Presentations - 6:00 to
8:30 PM
Registration: REQUESTED, not required. Registering will
allow us to more accurately gauge seating requirements and refreshment
needs. Register
on the web at
https://www.lab-robotics.org/member/meetings.asp?rid=1. There will be
drawings from the web registrants for LRIG
laser pointers, photon keyring lights and any other donated prizes.
Door Prizes:
Laser Pointers (LRIG)
Photon Keyring Lights (LRIG)
Door prizes for the drawings gratefully accepted - a great way to get your
name out!
Agenda:
This meeting will focus on automation of ADME and compound profiling. Come early for the
poster session and join the
community. There are cash awards for the best posters:
$500 - 1st Place,
$250 - 2nd Place, and
$100 - 3rd Place.
All cash prizes are in the form of AMEX gift certificates.
Food and refreshments will be available FREE OF CHARGE during the
Poster Session and Social Period.
There is always a Job posting board at the social. Please encourage your recruiters to
give you material to post and distribute. Openings may also be posted at
https://www.lab-robotics.org/forum/default.asp?CAT_ID=2.
There is no fee to attend the meeting.
Presentation:
Introduction and Overview to ADME and Compound Profiling
Mel Reichman, Ph.D.; Pharmacophore Discovery; West Chester, PA;
[email protected]
Presentation:
Automation in Pharmaceutical Profiling
Edward H. Kerns, Li Di, Susan Petusky, Susan Li and Donna Huryn Wyeth
Research, Chemical and Screening Sciences, Princeton, NJ
Selection and optimization of candidates for activity and selectivity have
traditionally played a dominant role in drug discovery. However, in recent years
the complimentary nature of pharmaceutical properties in candidate success has
been recognized. This has led to the implementation of pharmaceutical profiling
as part of the discovery process. Several objectives of drug discovery
pharmaceutical profiling can be met through strategies and tactics that involve
automation: 1) The need for more information on in vivo delivery of the
candidate to the therapeutic target has led to the implementation of assays that
model key in vivo ADME barriers; 2) The need to improve discovery efficiency has
led to accelerated analysis and early collection of data to avert ADMET problems
that cause failure at later stages; 3) The need to perform discovery research
more effectively has led to the application of pharmaceutical property
information to better plan and interpret discovery activity assays. These
applications require high capacity because of the large number of discovery
compounds and speed because discovery moves quickly. This presentation describes
the automation of pharmaceutical profiling assays for the properties: integrity,
lipophilicity, solubility, permeability, metabolic stability, and CYP450
inhibition. The management of the resulting data and its delivery to discovery
teams are also key elements of the pharmaceutical profiling workflow.
Presentation:
Rapid Estimation of LogP Values and Bioavailability with Biosomes?in Mix and
Read Assays
Brendan Lucey, M.S., Project Manger, ABS Inc., Wilmington, DE.
Bioavailability is an essential characteristic of any potential drug
candidate; however, in vivo measurements of bioavailability are prohibitively
expensive. Consequently, various in vitro measures such as determination of
octanol/water partition coefficients (LogP), permeability assays using
artificial membranes, and cell based assays (e.g., Caco-2 cells) have been used
to estimate bioavailability. All of these in vitro measures have significant
disadvantages ranging from difficulty of use, to low throughput, to expense. ABS
is developing a technology that has major advantages over existing methods in
both ease of use and cost. This technology utilizes solutions of highly stable
fluorescent liposomes (Biosomes?, which are compatible with a wide range of pHs
and standard laboratory reagents including DMSO. Biosomes?change fluorescence
as compounds are taken up by the biomimetic liposomes; this allows one-step
homogeneous assays. ABS is currently developing Biosomes?formulations which
change fluorescence in proportion to compound characteristics such as LogP
values and oral bioavailability. Formulations can be varied to mimic specific
tissues and cell types. These Biosomes?formulations permit simple mix and read
assays, without the need for any special equipment, to enable compound screening
in relatively inexpensive mHT assays.
Presentation: Accurate Lead Selection: Biological Screening for Human Drug Properties
Peter Bullock, Ph.D.; Director, Discovery Support, Purdue Pharma LP;
Ardsley NY
Historically, the rate of success of development candidates (DC) from
nomination to first-in-man studies has been approximately 10%. Many failures
have been associated with inadequate pharmacokinetic behavior in humans (e.g.,
variable bioavailability, poor dose-proportionality, rapid clearance) or a
propensity for participating in drug interactions (e.g., cisapride). In
addition, some new drugs have caused unexpected and unsafe side effects in the
post-market period (e.g., terfenadine and toursade de pointes). Currently,
chemical, pharmacological and pharmaceutical characterization occur nearly
simultaneously. The nature and quantity of information available with which to
select DC has changed substantially during this period. At Purdue Pharma, a
rapid cycle of synthesis, purification and re-synthesis to prepare homologous
series of new molecular entities (NME) has been integrated tightly with
high-throughput evaluation of pharmacological activity (e.g., potency and
efficacy) and moderate-throughput assessments of the pharmaceutical properties
of these compounds that could be associated with developmental liabilities
(e.g., aqueous solubility, metabolic clearance/inhibition, intestinal/CNS
permeability, interaction with drug transporters, cytotoxicity, interaction with
cardiac ion currents). Furthermore, it is now possible to investigate the
pharmaceutical properties of NME with human-derived biological tools (e.g.,
cryopreserved cells, transfected cell lines, recombinant proteins), thus
reducing our dependence on rodent-human extrapolations.
Posters
1. Automated Screening of Aqueous Compound Solubility in Drug Discovery
Thomas Onofrey*, Greg Kazan, Chris Barbagallo, Jason Blodgett, and Alan
Weiss, Millipore Corporation
It is desirable and increasingly more common in drug discovery to
characterize compound solubility prior to biological testing. A filter-based
method has been developed for both semi-quantitative and quantitative solubility
determinations. Compounds solubilized in water-miscible solvents are added to
buffer in a filter plate and incubated on a shaker deck. The filter plate is
then transferred to a vacuum manifold and samples are filtered. A fixed volume
of each filtrate is transferred to a UV-transparent analysis plate. Depending on
the type of analysis - semi-quantitative or quantitative - a single calibrator
or standard solutions are transferred to the plate and absorbance is measured.
Results from the filter-based method correlate well with values obtained using
standard methodology (shake flask). The filter-based assay can be fully
automated on a number of laboratory robots. Depending on calibration and
replicate number, the method is capable of producing results on hundreds of
samples per day.
2. Automated MultiScreen?PAMPA and Permeability Assay Systems
Thomas Onofrey, John Lynch, and Dan Schmidt, Millipore Corporation, Life
Sciences Division, Danvers, MA USA
The permeability assays described herein are non-cell based assays designed
to predict passive, transcellular permeability of drugs in early drug discovery.
The assays are carried out in one of two different 96-well MultiScreen?plates
that are specifically designed to measure the ability of compounds to diffuse
from a donor to an acceptor compartment that are separated by an artificial
membrane. The artificial membrane in the Permeability assay is comprised of
hexadecane on a polycarbonate membrane support. The Parallel Artificial Membrane
Permeation Assay (PAMPA) is based on the use of a 0.45mm PVDF membrane that
contains a much broader range of lipophilic constituents. These methods can also
be used to determine the effect of pH on compound permeability by adjusting the
pH of the solutions used in the analysis. Data for a number of drugs and methods
for each assay are presented that highlight automation compatibility, assay
reproducibility and correlation with passive human intestinal absorption.
3. Automated Method Development for Solid Phase Extraction in an Online
SPE/LC/MS system.
Steven Eendhuizen and Alex Berhitu, Spark Holland Inc. 666 Plainsboro Road,
Suite 1336, Plainsboro, NJ 08536 USA.
A scientific approach for developing SPE methods in a fully automated SPE/LC/MS
system has been introduced. Key in developing SPE methods is to find the best
suitable sorbent/solvents combination that provides the highest extraction of
the compounds of interest and the best clean-up from the matrix components in
the sample. There are endless combinations to try, therefore traditionally this
has been a lengthy and complex process. Secondly, the results traditionally
represented only the recovery on the extraction cartridge tested. In the
proposed setup two SPE cartridges will be placed in series and are eluted
individually. The Spark Holland SymbiosisTM system not only will perform fully
automated each of the chosen combination of SPE protocols; it will also present
results on recovery, breakthrough and tubing adsorption. The obtained additional
information and the automated extraction has lead to a significant reduction in
the time spend on Method Development.
4. Higher Throughput ADMET Profiling for Drug Discovery Support
Paul V. Kaplita, Ph.D., Hanbo Hu, Lisa Liu, Thomas M. Farrell, Monica Patel
and Denice M. Spero
Boehringer Ingelheim Pharmaceuticals, Inc., Drug Discovery
Support, LDT209, 900 Ridgebury Road, Ridgefield, CT 06877-0368
[email protected]
The evolving union of high throughput screening (HTS) and absorption,
distribution, metabolism, excretion and toxicology (ADMET) technologies in
recent years has transformed the hit-to-lead and lead optimization stages of
drug discovery. Pharmacokinetic, drug metabolism, pharmaceutics and toxicology
information on pharmacologically active compounds can now be acquired very
shortly after synthesis, or after selection from screening "hit sets" or
libraries. The process of identifying discovery compounds with desirable
"drug-like" properties has consequently become more data-driven than ever
before. Human tissue-based, in vitro ADMET assays can efficiently generate
reliable profiles for structure-activity or structure-property relationships. A
Caco-2 cell model, which has been automated on a bench-top workstation, may be
used to evaluate the intestinal absorption of drug candidates. Distribution
properties may be gauged by means of a high throughput equilibrium dialysis
technique for measuring plasma protein binding. Drug metabolism can be evaluated
via automated measurements of metabolic stability in liver microsomes. Drug-drug
interactions can be predicted with HTS techniques using recombinant hepatic
cytochrome P450 isoforms. Assays for hepatotoxicity with human hepatocytes can
serve as early, high throughput indicators of potential systemic drug toxicity.
Physical pharmaceutical properties, such as solubility, lipophilicity and pKa
can also be studied with high throughput workstations. The early availability of
ADMET profiling data can now enable discovery scientists to understand more
thoroughly the factors that influence in vitro and in vivo pharmacology.
5. Recent Developments in High Throughput, Turn-Key Solubility and
Permeability Compound Ranking Assays
Susan Murphy & Mike Duvinski, Molecular Devices Corporation, Sunnyvale, CA, Greg
Kazan and Tom Onofrey, Millipore Corporation, Danvers, MA
When combined with data from primary screening assays, measurement of the
physiochemical properties of new chemical entities can provide a basis for the
ranking of compounds based on their potential to be developed into viable drug
candidates. The strategy of ranking the developability of new chemical entities
(NCE) based on characterization by high throughput characterization of compound
solubility and permeability is increasingly being applied in the drug discovery
industry.
We have developed automatable, turn-key assays to measure solubility and
permeability that use Millipore membrane technology and a Molecular Devices
SpectraMax absorbance microplate reader. The benefits of this approach include
good correlation with low throughput, old standard?assays, no methods
development, automation friendliness, reduced need for instrument expertise, and
software flagging of data validity.
6. ADMET Data Management for Lead Optimization
Kim Norman, IDBS, Cambridge, MA
Traditional ADMET assays are the latest examples of analyses that are being
pushed into the higher throughput framework. These assays play a pivotal role in
the drug discovery cycle, and are used to identify leads from hits generated by
traditional single-point HTS assays.
Advances in assay technologies and instrumentation have enabled these more
complex, multi-parameter assays to become feasible within a high-density plate.
These assays can now be used much earlier in the discovery cycle and their
predictive nature allows for a more informed decision to "fail early / fail
cheap". The caveat to making this early-informed decision is of course the
ability to make sense of the large volumes of ADMET data now being generated.
In this poster we describe the data capture and analysis of a small sampling
of ADMET assays using the ActivityBase suite of products provided by IDBS to
combine and visualize data to maximize drug discovery lead optimization.
7. High-Resolution Screening Assays for Kinetic Characterization of Small
Molecules Provide Valuable Insight into Optimization of Candidate Drugs: Rapid
Characterization of Compounds Binding to Estrogen Receptors α and β
Jaymie DeWitt, Biacore, Inc.
Surface plasmon resonance (SPR) technology was used to screen and
characterize binding interactions of candidate drug compounds against different
target proteins. Over 200 compounds were screened for specific binding to
different isoforms of a receptor protein using Biacore?S51. To obtain
information about early ADME properties, the compounds were also evaluated for
binding to human serum albumin (HSA) and alpha 1-acid glycoprotein (AGP). The
data acquired from the screen was used to select compounds with specific binding
properties to the targets as well as predict binding to serum proteins. Selected
compounds were then assayed in a high-resolution mode to determine the
association and dissociation rate constants for binding to ER? and ER?. The
kinetic properties obtained from the assay were used to demonstrate how
different structural features influence association and dissociation rates for
the compounds. This structure activity relationship (SAR) analysis illustrates
the importance of using detailed kinetic data obtained from SPR assays in the
optimization of candidate drug compounds.
8. SPR Biosensor Assay for Early ADME Screening of Drug Membrane
Absorption
Helena Widegren,Lena Westerlund and 彎a Frostell Karlsson, Biacore AB,
Uppsala,Sweden
Surface plasmon resonance (SPR) biosensors enable the interactions between
small molecules and liposomes to be monitored directly using a non-label assay
format. In the SPR biosensor assay presented here we explore the use of
different liposome surfaces to obtain binding data that reflect different
membrane constituents. Responses from different types of liposomes are combined
to resolve low, medium and high level absorption of compounds.
9. HTS Assay for Adaptor Protein Interactions with Receptor Tyrosine
Kinases
Bradford O. Fanger, Ph.D.. Savitha Rao, Tisola Noel-Birdsong, Jane Zhang,
Malcom Smith and Tim Schaefer, IGEN International, Inc., Gaithersburg, MD
Activation of tyrosine kinase receptors generates phosphotyrosine residues
that recruit adaptor proteins, including Grb2 (growth factor receptor bound
protein 2), through specific binding of SH2 (Src homology 2) domains. Here we
describe the development of a novel 384-well based assay to screen for compounds
that inhibit phosphporylated epidermal growth factor receptor (pEGFR) binding to
Grb2. The assay employs ORIGEN technology and IGEN's M-SERIES 384 analyzer.
Glutathione coated paramagnetic beads were used to directly capture a GST-Grb2
fusion protein from bacterial lysates. The GST-Grb2 modified beads were
challenged with A431 cell lysates containing phosphorylated EGFR. The complex
was reported through the use of an anti- EGFR antibody modified with ORI-TAGPlus
label, an electrochemiluminescent label. The assay conserves biomaterial,
generates S/B over 10, Z'-scores > 0.6, and has a protocol easily adapted to
automation.
10. High Throughput Solubility- Kinetic vs. Thermodynamic
O. Tsinman, A. Avdeef, C. Berger, M. Holland (pION INC), Woburn, MA
Turbidity-based solubility determinations, spurred principally by the highly
publicized work of Pfizer's Chris Lipinski, have gained popularity in many drug
discovery companies. The turbidity based method, also known as "kinetic
solubility," is often compared to the tried and true method of "thermodynamic
solubility." Thermodynamic often conjures images of shake flasks and laborious
experiments, but new technology for both methods are gaining a foothold in the
industry.
This poster outlines technology available from pION capable of performing
both methods using its patented "modified shake flask" solubility protocol.
Results of assays performed on the same compounds but with different methods
will be compared with discussion on how and why each method fits into the drug
discovery process.
11. From Target to Screen in Two Weeks: NFκB Reporter Assay with Transient
Target Expression
Mark Federici, Roland Tacke, Peter Haytko, Susan Power, Mei Cong, Tom
Livelli and Zhong Zhong, Cell & Molecular Technologies, Inc. Phillipsburg, NJ
Reporter assays have been successfully scaled up for high-throughput
applications. As it is more desirable to use cell lines that have been validated
to work well in robotized assays, considerable time is currently spent on
setting up cell lines that harbor both the target receptor and a response
element reporter. We report here an efficient process whereby a stable cell line
containing a NF-kB-luciferase response element reporter and validated in HTS is
transiently transfected with target receptors to allow rapid establishment of
cells suitable for reporter assays. We demonstrated that transient transfection
can be scaled up to 2x109 cells per batch, enough to seed 500 384-well plates,
at 10,000 cells/well density. Five fold induction and Z?of >0.7 was achieved
for the reporter assay. In addition, the transfected cells can be frozen for
storage, then thawed and plated yielding high viability and a robust response in
the reporter assay.
12. Automated DNA Purification Systems ?From Mouse tails to BAC
Conor Mulrooney, Sarah Howe, Julie Robinson, Janet Sayle, Michael Burgess,
Steve Jones, Steve Dodsworth, John Oultram, Tepnel Life Sciences PLC,
Wythenshawe, Manchester, UK
Tepnel Life Sciences (TLS) has extensive experience in the development of
nucleic acid purification products. Utilising diverse enabling technologies TLS
has developed a range of manual kits and automated systems to extract DNA from
different biological materials. The new Nucleopure and Nucleoplex instruments
provide cutting edge solutions to the challenges of rapid cost effective
automated purification.
The extraction of high quality genomic DNA is the first and often limiting
step in the process of mouse genotyping and most chemistries and systems fail to
consistently produce sufficient DNA of an appropriate quality to meet the
requirements of both PCR?and Southern Blotting. Tepnel Nucleopure automation
platform, using the patented Nucleon chemistry, not only produces excellent
yields of highly intact and pure DNA from mouse tail, ear and toe samples but
also automates the process of PCR set-up. Extraction of 192 samples takes
approximately 2.5 hours and PCR set up can be customised to meet specific
customer needs.
Tepnel Nucleoplex has been designed to extract and purify BAC DNA and has
been developed to support Life Science research activities. The user-friendly
nature of the interface and control systems minimises the requirement for
training enabling a wide range of users to easily operate the instrument.
Installation and training can be completed within 2 hours allowing users to test
the Nucleoplex within their own facility before purchase.
13. SHOW, Sample Handling Operation Wizard
Ping Du, Livingston, NJ
Sample Handling Operation Wizard (SHOW) is a system developed to guide manual
sample handling in the research laboratory. It comprises a sample tray mounted
on a flat-panel computer monitor. Up to four transparent micro titer plates and
up to 8 reagent vials may be placed on the sample tray. Images of the wells of
the plates and vials are generated on the monitor and controlled by computer
software. These images are directly aligned with the positions of the physical
wells. By highlighting the wells or vials involved in a sample handling step of
a pre-defined protocol, manual operation can be performed with precise guidance
from the system. As a result, the risk of locating a wrong sample or placing a
sample at a wrong location can be minimized, and sample handling operations
become more efficient and less stressful.
14. ADMET Assays On Tecan LabCD-ADMET System: MDR-1/P-glycoprotein (PgP)
ATPase Assay
Shari Ommert, Theresa Towle, Howard Haspel, Hitesh Jindal, Lance Gleich, and
Praveen Bansal, Tecan US
Tecan LabCD-ADMET System is an automated platform which integrates the
centrifugally driven microfluidic LabCD disc with GENESIS liquid handling
workstation, ULTRA LabCD detection system and application-specific software. A
novel protocol for P-glycoprotein (Pgp)-ATPase assay using enzymatic coupling to
an absorbance-based detection system has been seamlessly integrated with the
LabCD system. This automated protocol, using baculovirus-insect cell expressed
Pgp, enables the simultaneous and continuous detection of both the direct
stimulation and inhibition of verapamil-stimulated Pgp-ATPase activity by drugs.
The use of LabCD (20 microliter assay) permitted approximately a 50-fold
reduction in enzyme usage. The results from testing 13 drugs with Tecan
protocol as well as those of existing end-point detection methods were compared
with other literature values. The results were internally consistent for both
detection methods and consistent with the literature for both stimulatory and
inhibitory effects. Thus, the LabCD-ADMET System provides a miniaturized,
high-throughput turnkey solution for screening Pgp-drug interactions.
15. Cloning and Expression of Thiolase from Sunflower Cotyledons
Patricia Minihan, James H. Dyer, Dept. of Chemistry and Biochemistry at
Montclair State University, Upper Montclair, New Jersey, Anke C. Schiedel, Silke
Oeljeklaus, Cell Biology, MSB, New York University Medical Center, New York, NY
Two separate thiolase activities have previously been isolated from the
glyoxysomal fraction of sunflower (Helianthus annuus L.) cotyledons. Thiolase I
shows activity only toward short chain acetoacetyl CoA, while thiolase II
exhibits activity toward both short and long-chain acetoacetyl CoA and 3-oxoacyl
CoAs. The purpose of this research was to clone these two thiolases from
sunflower and express the active enzyme. Primers were first generated to
conserved sequence regions from other known thiolases and were based on the
known sequence of 3-ketoacyl-CoA thiolase of Cucumis sativus. These
gene-specific primers were used in an RT-PCR protocol to generate an internal
fragment of the sunflower thiolase, using total RNA isolated from etiolated
cotyledons as the RT template. 5? and 3?RACE protocols were then used to
generate the ends. A full-length cDNA was obtained using an RT-PCR protocol with
sunflower thiolase-specific primers flanking the coding region. This sequence
shares 76% identity with other known thiolases at the amino acid level. The
full-length thiolase was cloned into a bacterial expression vector. Further work
is underway to express, purify and biochemically characterize the active enzyme.
16. High throughput mutational spectrometer (HTMS) for the discovery of
genetic variations in large populations
Chira Deka (1), Qingbo Li (2), Arthur W. Miller (1), Joseph M. Fallon (1),
Brian J. Glassner (1), Aoy Tomita-Mitchell (2), Xiao-Cheng Li-Sucholeiki (1),
Kevin Arnold (1), Thomas E. Kane (3), Songsan Zhou (3), Sean Connell (3), John
W. Best (3), John Kernan (3), William G. Thilly (4) and Barry L. Karger (5)
(1) Beckman Coulter Inc. Woburn MA (formerly Peoples Genetics Inc.) (2)
Children Hospital of Philadelphia, Philadelphia PA (3) SpectruMedix LLC, State
College PA (4) Biological Engineering Division, Massachusetts Institute of
Technology, Cambridge MA (5) Barnett Institute and Department of Chemistry,
Northeastern University, Boston MA
A fundamental goal in genomics is the discovery of genetic variation that
contributes to disease states or to differential drug responses. Typically,
current genetic analysis technologies are limited to study sizes of a few
hundred individuals per experiment due to cost considerations. These sample
sizes limit statistical power and observable allele frequencies. Peoples
Genetics Inc. employs a breakthrough genomics technology enabling the
comprehensive discovery of mutations in very large populations. The technology
couples Constant Denaturant Capillary Electrophoresis (CDCE) with high fidelity
PCR, and can simultaneously and economically scan DNA on a pooled basis from up
to 100,000 individuals from any population of interest, while maintaining a
process sensitivity limit of 5 copies per 100,000 alleles. In this presentation
we describe a high throughput mutational spectrometer based on CDCE that enables
practical implementation of this process. The instrument features high optical
sensitivity (~1 pM fluorescein), extremely precise and stable temperature
control (?0.01oC), and extensive automation for sample delivery, matrix
replacement and fraction collection. Fundamental features of this instrument and
key performance characteristics will be presented.
17. Drug-Likeness and Lead-Likeness: An Overview of Recent Studies
Zhengming (Jimmy) Chen, Purdue Pharma L.P., Cranbury, NJ
The distinction between drug-like and non drug-like molecules has been a hot
research topic in recent years. The most well known early study in this field is
the ipinski rule of five?which was derived empirically from the analysis of
the World Drug Index on the properties that maximize an oral drug candidate
probability of surviving clinical development: molecular weight (MW) < 500,
number of hydrogen bond donors < 5, number of hydrogen bond acceptors < 10, and
ClogP < 5. The rule of five is now widely used to filter out compounds likely to
have poor pharmacokinetic properties early on in drug discovery. Lead-likeness
(compounds?likelihood to be good lead candidates), as distinct from
drug-likeness, is a new concept that is gaining acceptance in recent years.
Lead-like molecules are generally smaller to allow for structural additions to
enhance effectiveness during lead optimization, and is being incorporated into
the library design and lead optimization processes. In this presentation, I am
going to present an overview of recent studies on the topic of drug-likeness and
lead-likeness. The presentation will provide a few intriguing insights into the
influence of molecular properties on the likelihood of progression through the
drug development process and trends in modern drug discovery.
18. A New High Throughput Approach for Detecting 'Promiscuous Inhibitors'
Joseph Goodwin, BD BioSciences, Bedford, MA
Recently, much attention has been given to the 'promiscuous inhibitor'
phenomenon that has been proposed as a common mechanism for false positives in
high throughput screens. Investigators have demonstrated the ability to detect
and characterize these aggregates using a variety of technologies (electron
microscopy, and light scatter techniques). One of the primary challenges
involved with aggregate detection is the throughput of the detection assays.
Light scattering techniques that accurately characterize particle size
distribution down to the low nm range often take considerable time and require
large amounts of compound. Moreover, many compounds being evaluated in HT
screens have significant aqueous solubility issues, resulting in precipitate
that may be confused with or complicate aggregate detection. The current
evaluation examines the possibility of detecting 'promiscuous inhibitors' using
a flow cytometer optimized for drug solubility testing. The proposed method
detects aggregate formation and distinguishes it from compound precipitation in
a high throughput manner.
19. Controlled, Convergent Shock Waves Applied to ADME Sample Preparation
Processes
James A. Laugharn, Jr., President, Covaris, Inc., Woburn, MA
The Covaris process uses non-linear, high intensity, convergent acoustic
shock waves with a computerized control system for disruption (primary
tissue/cell homogenization), dissociation (extraction) and dissolution
(reagents). The apparatus transmits focused, ultra-sonic waves such that samples
receive closely defined non-ionizing, acoustic energy. The process is
non-contact, removing cross-contamination and may be run with either open or
closed vessels. Computerized control enables many samples to be identically
treated without any human intervention that is inherent in traditional sample
preparation systems. Computer control enables many samples to receive individual
(different) treatments if desired. Temperature of the samples can be monitored
and controlled. For primary sample homogenization, the process is scalable from
yeast cultures to a gram of fibrous muscle tissue. Data from improved target
molecule recovery, sample-to-sample reproducibility, and dissolution rates will
be presented.
20. Parallel liquid chromatography for high throughput ADMET profiling
Chris Phillips, Paren Patel, Jeffrey Koehler, Steve Hobbs, and Hugh McManus
Abstract The Nanostream Veloce systemhich includes an instrument, software,
and replaceable microfluidic cartridgesncorporates pressure-driven flow to
achieve chromatograms comparable to conventional HPLC instrumentation while
offering a dramatic increase in sample analysis capacity. The system enables
parallel chromatographic separations and simultaneous, real-time UV detection
for rapid determination of ADMET properties. Each Nanostream Brio?cartridge,
made of polymeric materials, incorporates twenty-four columns packed with
standard (C-18) stationary phase material to achieve reverse phase separations.
Mixing and distribution of the mobile phase to each of the 24 columns is
precisely controlled in each cartridge. The system provides an ideal platform to
accelerate assessment of physicochemical properties (i.e., log P, CHI, etc.) for
a large number of compounds.
This poster demonstrates how the Veloce system offers specific benefits for
rapid assessment of ADMET parameters. The 24-fold increase in sample analysis
capacity allows standard curve generation and simultaneous analysis of multiple
replicates of samples in a single run. By accelerating access to high quality
data, the Veloce system enables scientists to make decisions about promising
leads earlier in the drug discovery process.
21. Rapid compound purity screening using the Nanostream Veloce system
Li Zhang, Paren Patel, Sergey Osechinskiy, Chris Phillips, Surekha Vajjhala
Over the past decade, advances in combinatorial chemistry and target
characterization have significantly increased the number of drug candidates and
targets available to drug discovery screeners. To ensure a meaningful screen, an
increasing number of pharmaceutical companies are now evaluating compound
purity. The Nanostream Veloce system, together with 24-column Brio cartridges,
offers a novel approach to micro parallel liquid chromatography. This system
allows users to achieve unprecedented throughput for standard assays while
matching the performance of conventional LC instrumentation, thus enabling a
cost effective way to routinely monitor compound purity with minimal
modification to current methods and work flow. This poster presents results of a
study of pharmaceutical compound library samples using the Veloce system.
Individual chromatograms, percent purity results, study duration and total
solvent consumption are compared to results obtained using conventional HPLC.
STILL ROOM FOR POSTERS!
Please submit posters for consideration by e-mailing Tim Blankenship <[email protected]>
the following information:
Presenting Author:
Organization:
Address:
Telephone:
Fax:
E-mail:
Co-author(s) and their affiliation(s), if different
Abstract Title
Abstract (250 word max.)
The number of posters are limited so if you do not have a full abstract you can
reserve your spot by signing up immediately with full contact information and
title.
Our meeting will focus on ADME topics but any posters related in some way to
Laboratory Robotics and Automation, HTS, Genomics, Combichem, Informatics, etc.
are welcome, as are posters already presented elsewhere. Space for the posters
is estimated to be 6x3 feet.
Academics and end-users have a free poster space. Vendor posters will be accepted but we do ask for a $100 donation to help
defer the cost of the poster setup.
Payment (for Vendor Posters only):
Multiple poster spaces may
be purchased. The fee for this poster session is $100 each space. The check should be made payable
to:
The Laboratory Robotics Interest Group
and sent to our treasurer.
Alternatively, the money may be wired directly to our bank account. For details,
contact Tony.
Our Federal Tax ID Number (TIN) is 26-6033993.
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Visit The Laboratory Robotics
Interest Group homepage at https://www.lab-robotics.org