| Biodefense
Program
Biodefense and Emerging Pathogens Screening System
Robotic Drug Screening System
Biodefense
and Emerging Pathogens System
Yale
Pharmaceutical Research Institute (YalePharma) has the
expertise and facilities to perform projects which involve
emerging infectious diseases at the BSL2 and BSL3 level. With
capabilities of studying both viral and bacterial agents,
the laboratory is well positioned to work on critical issues
facing our nation and the world today. From environmental
monitoring, to in vitro assay development, to the performance
of research to monitor the effects of immunomodulatory and
anti-infective agents, to evaluations of drug sensitivity
and agent identification, the group is poised to perform research
with agents with biodefense and world health implications.
- Environmental Microbiology
The
Biodefense and Emerging Pathogens Program has extensive experience
and capabilities in the environmental microbiology arena.
Environmental
detection program
 |
liquid culture |
|
PCR confirmatory assays (or phage assay) |
|
Bacillus strain identification |
|
antibiotic sensitivity |
Environmental detection of other viral, bacterial and fungal
agents
- In
Vitro Evaluation of Emerging Pathogens and
Biodefense Agents
Numerous assays are readily available to evaluate and study
emerging pathogens and biodefense/biothreat agents.
The Emerging Pathogens Program at Yalepharma has extensive
experience with the many of these assays, some of which are
listed below.
In
Vitro Capabilities
|
Immunoassay:
design, development, optimization and validation
|
|
Molecular
diagnostics
|
|
ELISA:
design, development, optimization and validation
|
|
Cellular
and humoral immunity
|
|
Innate
immunity
|
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Cytokine
evaluation
|
|
Anti-infective
antibiotic/drug sensitivity evaluation |
Several
animal models are available for the evaluation of vaccines
and chemotherapeutics for efficacy against emerging pathogens
and biothreat agents. The Biodefense and Emerging Pathogens
Program has experience in several areas listed below; and
we have appropriate ABSL-2 and ABSL-3 animal facilities to
safely conduct studies in a time-efficient manner. Please
contact us to further discuss your research needs.
|
Rodent
models |
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Non-human primate models |
|
Evaluation of immunotherapeutics |
|
Evaluation of vaccines and biologics |
- Emerging
Pathogens Research Program
Current Capabilities
|
Agents
of interest: tularemia, Q fever, plague, EEE, VEE,
WEE, West Nile, Yellow Fever |
|
Evaluation of therapeutics: vaccines, drugs and other
biologics |
|
Antibiotic/anti-infective resistance studies to permit
rapid detection of wild type versus drug resistant
organisms |
|
Clinical
infectious agent characterization and quantification |
|
Bio-detector evaluation |
|
Aerosol
programs |
|
Infectious disease consulting services |
|
Research
and analytical services at BSL2 and/or BSL3 level |
Robotic
Drug Screening System
The
Robotic Drug Screening and Target Development program is designed
for rapid target-specific analyses utilizing robotics-based
evaluation of large compound libraries. This target-specific
assay development program utilizes both commercially available
and proprietary biochemical and cell-based screening assays.
Coordination of these diverse functions is achieved through
a computerized data management system that facilitates the
selection and tracking of compounds, including the analysis
of chemical diversity, primary and secondary screening, and
the subsequent optimization of lead compounds. In conjunction
with our ongoing robotics program, we are actively involved
in the identification of novel proprietary targets against
a variety of viral and cancer causing genes for integration
into our assay development/robotics evaluation program. This
program has already yielded a number of targets with a broad
range of potential therapeutic applications. The system is
designed to be compatible with the majority of our existing
in-house assays, yet contains a number of features that allow
for flexible and novel screening assay design. This flexibility
results from the technology embodied in the Beckman-Coulter
integrated robotics and liquid handling systems. A large collection
of primary and secondary assays is available to provide the
client with an early and comprehensive profile of a compound¡¯s
clinical potential.
Equipment
YalePharma
has multiple Beckman Coulter robotic platforms to support
large-scale screening projects.
These
high throughput systems automate the assay process from drug
preparation (initial plate setup, mother-daughter plate creation)
through assay completion. The liquid-handling stations have
single-, eight-, and 96-channel pipetting capabilities. Other
stations on the platforms allow bar code printing and reading,
plate and reservoir storage (ambient, cold-storage, and CO2
humidity-controlled), microplate delidding, plate shaking,
filtration, plate sealing, and continuous disposable tip supply.
Results from the assays are quantified using one of the various
plate readers. Our detections capabilities include fluorescence
(including time-resolved), luminescence, photometrics, alpha,
beta, and gamma labels, liquid scintillation and absorbance.
The
ORCA (Optimized Robot for Chemical Analysis) Arm serves as
the interface between the equipment on each platform. It carries
the transports (plates and reservoirs) and consumables (tips)
from station to station. All of the instruments are controlled
by the SAMI NT (SAGIAN Automated Method Interface) method
creation software and the SILAS (SAMI NT Software Integration
of Laboratory Automation Systems) router software. SILAS serves
as the underlying architecture for all of the equipment and
software communications. SAMI NT is the user-interface for
the programming of assays on the platform.
The
LIMS (Laboratory Information Management System) software allows
tracking of samples, plates and consumables from receipt to
long-term storage or disposal. The LIMS also serves as a data
analysis tool with the capability to generate charts, graphs,
tables and reports from the raw data.
Assay
Capabilities
High
Throughput CPE-Based Antiviral Assays: Virus-induced cytopathic
effects (CPE)-inhibition assays using Promega¡¯s CellTiter
reagent are employed to evaluate compounds for antiviral activity
against a wide variety of viruses. Antiviral assays are designed
to test single doses of twenty-eight compounds per plate against
the challenge virus in microtiter plate wells containing host
cells. Cell controls containing medium alone, virus-infected
controls containing medium and virus, drug cytotoxicity controls
containing medium and each drug concentration, reagent controls
containing culture medium only (no cells), and drug colorimetric
controls containing drug and medium (no cells) are run simultaneously
with the test samples. A computer program is utilized to calculate
the % CPE reduction in the virus-infected wells and the %
cell viability of uninfected drug control wells. Active compounds
are retested for verification of activity prior to the performance
of additional assays to generate dose-response curves.
Virus
systems currently in place are included in the following table.
Additional virus systems can be developed as requested.
Current
HTS Virus Systems:
-
| Virus |
Cell
Line |
Assay
Length |
| HIV-1 |
CEM-SS |
6
days |
| HSV-1 |
Vero |
5
days |
| HSV-2 |
Vero |
5
days |
| BVDV |
MDBK |
6
days |
| Coxsackie |
MRC-5 |
5
days |
| Echovirus |
MRC-5 |
4
days |
| Enterovirus |
MRC-5 |
4
days |
| Rhinovirus |
MRC-5 |
5
days |
| Adenovirus |
HeLa |
3-4
days |
| Influenza |
MDCK |
2-3
days |
| Parainfluenza |
Hep2 |
3
days |
| RSV |
Hep2 |
4
days |
Identification
of Inhibitors of Protein-Protein Interaction: Interactions
between proteins are the key to all cellular processes. Disease
develops when a mutation(s) in a protein leads to an upset
in the delicate balance of these interactions. Viruses also
require protein-protein interactions at all stages of their
replication cycles. Viral proteins interact with other viral
proteins as well as with countless cellular proteins. Identifying
inhibitors of these protein-protein interactions will lead
to the development of new therapies for all sorts of diseases.
Using this strategy, we have developed and are continuing
to develop numerous assays to screen for novel inhibitors
of HIV and herpesviruses. Integration of this approach with
our HTS capabilities allows us to screen large chemical libraries
in a minimum of time, resulting in the identification of new
disease therapies. Identification of Inhibitors of Protein-Protein
Interaction: Interactions between proteins are the key to
all cellular processes. Disease develops when a mutation(s)
in a protein leads to an upset in the delicate balance of
these interactions. Viruses also require protein-protein interactions
at all stages of their replication cycles. Viral proteins
interact with other viral proteins as well as with countless
cellular proteins. Identifying inhibitors of these protein-protein
interactions will lead to the development of new therapies
for all sorts of diseases. Using this strategy, we have developed
and are continuing to develop numerous assays to screen for
novel inhibitors of HIV and herpesviruses. Integration of
this approach with our HTS capabilities allows us to screen
large chemical libraries in a minimum of time, resulting in
the identification of new disease therapies.
Assay
Background: The CheckMate Mammalian Two-Hybrid System,
available from Promega, can be utilized to detect protein-protein
interactions in vitro using a mammalian cell line of
choice. This system utilizes two different types of luciferase,
Renilla and firefly, allowing use of Promega¡¯s Dual-Luciferase
Reporter Assay System for quantitation of the luciferase reporter
genes as a measure of these interactions. Once the suspected
interaction between two proteins of interest has been confirmed,
this system can be used to identify compounds capable of inhibiting
this specific protein-protein interaction. The luciferase
level expressed in treated samples is compared to the level
expressed in untreated samples, allowing for determination
of the inhibitory activity of the compound against the protein-protein
interaction. Assay Background: The CheckMate Mammalian Two-Hybrid
System, available from Promega, can be utilized to detect
protein-protein interactions in vitro using a mammalian
cell line of choice. This system utilizes two different types
of luciferase, Renilla and firefly, allowing use of Promega¡¯s
Dual-Luciferase Reporter Assay System for quantitation of
the luciferase reporter genes as a measure of these interactions.
Once the suspected interaction between two proteins of interest
has been confirmed, this system can be used to identify compounds
capable of inhibiting this specific protein-protein interaction.
The luciferase level expressed in treated samples is compared
to the level expressed in untreated samples, allowing for
determination of the inhibitory activity of the compound against
the protein-protein interaction. The CheckMate Mammalian Two-Hybrid
System, available from Promega, can be utilized to detect
protein-protein interactions in vitro using a mammalian
cell line of choice. This system utilizes two different types
of luciferase, Renilla and firefly, allowing use of Promega¡¯s
Dual-Luciferase Reporter Assay System for quantitation of
the luciferase reporter genes as a measure of these interactions.
Once the suspected interaction between two proteins of interest
has been confirmed, this system can be used to identify compounds
capable of inhibiting this specific protein-protein interaction.
The luciferase level expressed in treated samples is compared
to the level expressed in untreated samples, allowing for
determination of the inhibitory activity of the compound against
the protein-protein interaction.Assay Background: The CheckMate
Mammalian Two-Hybrid System, available from Promega, can be
utilized to detect protein-protein interactions in vitro
using a mammalian cell line of choice. This system utilizes
two different types of luciferase, Renilla and firefly, allowing
use of Promega¡¯s Dual-Luciferase Reporter Assay System for
quantitation of the luciferase reporter genes as a measure
of these interactions. Once the suspected interaction between
two proteins of interest has been confirmed, this system can
be used to identify compounds capable of inhibiting this specific
protein-protein interaction. The luciferase level expressed
in treated samples is compared to the level expressed in untreated
samples, allowing for determination of the inhibitory activity
of the compound against the protein-protein interaction. The
CheckMate Mammalian Two-Hybrid System, available from Promega,
can be utilized to detect protein-protein interactions in
vitro using a mammalian cell line of choice. This system
utilizes two different types of luciferase, Renilla and firefly,
allowing use of Promega¡¯s Dual-Luciferase Reporter Assay System
for quantitation of the luciferase reporter genes as a measure
of these interactions. Once the suspected interaction between
two proteins of interest has been confirmed, this system can
be used to identify compounds capable of inhibiting this specific
protein-protein interaction. The luciferase level expressed
in treated samples is compared to the level expressed in untreated
samples, allowing for determination of the inhibitory activity
of the compound against the protein-protein interaction.Assay
Background: The CheckMate Mammalian Two-Hybrid System, available
from Promega, can be utilized to detect protein-protein interactions
in vitro using a mammalian cell line of choice. This
system utilizes two different types of luciferase, Renilla
and firefly, allowing use of Promega¡¯s Dual-Luciferase Reporter
Assay System for quantitation of the luciferase reporter genes
as a measure of these interactions. Once the suspected interaction
between two proteins of interest has been confirmed, this
system can be used to identify compounds capable of inhibiting
this specific protein-protein interaction. The luciferase
level expressed in treated samples is compared to the level
expressed in untreated samples, allowing for determination
of the inhibitory activity of the compound against the protein-protein
interaction. The CheckMate Mammalian Two-Hybrid System, available
from Promega, can be utilized to detect protein-protein interactions
in vitro using a mammalian cell line of choice. This
system utilizes two different types of luciferase, Renilla
and firefly, allowing use of Promega¡¯s Dual-Luciferase Reporter
Assay System for quantitation of the luciferase reporter genes
as a measure of these interactions. Once the suspected interaction
between two proteins of interest has been confirmed, this
system can be used to identify compounds capable of inhibiting
this specific protein-protein interaction. The luciferase
level expressed in treated samples is compared to the level
expressed in untreated samples, allowing for determination
of the inhibitory activity of the compound against the protein-protein
interaction.
Assay
Development and Optimization: Assay
Development and Optimization: DNA sequences from the proteins
of interest are cloned into the pACT and pBIND vectors of
the CheckMate system. Positive clones are sequenced to verify
that an intact reading frame has been maintained. Each cDNA
of interest is cloned into both vectors to allow for determination
of the stronger interaction based on the vector context for
the inserts. To determine the optimum combination of vectors,
the pACT and pBIND constructs with the cDNA inserts of interest
are transiently transfected into mammalian cells, and luciferase
expression from each combination is evaluated using the Dual-Luciferase
Reporter Assay System. The assay is then optimized and validated
on the core system using the appropriate vector combination.
Assay Development and Optimization: DNA sequences from the
proteins of interest are cloned into the pACT and pBIND vectors
of the CheckMate system. Positive clones are sequenced to
verify that an intact reading frame has been maintained. Each
cDNA of interest is cloned into both vectors to allow for
determination of the stronger interaction based on the vector
context for the inserts. To determine the optimum combination
of vectors, the pACT and pBIND constructs with the cDNA inserts
of interest are transiently transfected into mammalian cells,
and luciferase expression from each combination is evaluated
using the Dual-Luciferase Reporter Assay System. The assay
is then optimized and validated on the core system using the
appropriate vector combination.
Compound
Screening: Compound Screening:
Compound libraries are screened for their ability to inhibit
the specific protein-protein interaction in the test system.
Following incubation, luciferase expression in each well is
determined using the Dual-Luciferase Reporter Assay System
from Promega. Inhibitors of protein-protein interaction are
identified as those that cause lower amounts of luciferase
to be produced than untreated controls. A software program
determines the % inhibition for each compound using the firefly
luciferase measurement and the Renilla luciferase measurement
is used to determine compound toxicity. Active compounds are
retested for verification of activity prior to the performance
of additional assays to generate dose-response curves.
Assay
Design and Optimization: Assay Design and Optimization:
Our laboratory is experienced in the design and development
of new assays and the adaptation of current assay systems
to high throughput, automated screening systems. Our core
assay system is equipped with various readers that enable
us to develop assays based on your choice of endpoint detection
methods. More specifically, we are set up to support your
drug development in the following ways: Assay Design and Optimization:
Our laboratory is experienced in the design and development
of new assays and the adaptation of current assay systems
to high throughput, automated screening systems. Our core
assay system is equipped with various readers that enable
us to develop assays based on your choice of endpoint detection
methods. More specifically, we are set up to support your
drug development in the following ways:
-
Design
of assay systems for your target of interest
-
Development
and optimization of high throughput screens
-
Adaptation
and optimization of existing assays to high throughput screens
-
Technical
support for your assay development personnel
We
can determine the most cost-effective method for evaluating
compound libraries without compromising assay sensitivity.
High
Throughput Screening: Once an assay has been designed
and optimized, the HTS group at Southern Research has complete
capabilities for performing high throughput screening of your
compound library. We provide quality results with a rapid
turnaround time. This allows you to devote more time and resources
to the other aspects of drug development.
Support
for our antiviral high throughput evaluations and target selection
program has been obtained from both government and commercial
contracts and grants. With all of the resources available
at our facility, Yalepharma provides a competitive and extremely
versatile service to the research community.
Targets
HIV
Targets
|
Enzymes:
RT/RNaseH, PR, IN |
|
Structural
Proteins: MA, CA, NC, (p6) |
|
Glycoproteins:
gp120, gp41, HIV-1 coreceptor |
|
Regulatory
Proteins: Tat,
Rev,
Nef,
Vif,
Vpu,
Vpr |
|
Others:
Ribosomal Frameshifting, packaging signals, etc. |
Herpesvirus
Targets
| |
HHV-8 DNA
polymerase and processivity factor |
| |
HHV-8 cyclin
|
| |
HHV-8 GPCR |
Client-Specified
Targets
We
custom-design assays for client-specified targets.
|
