UPDDI Core Facilities and Equipment
The drug discovery laboratory occupies a total of 14,000 square feet of space on the 9th and 10th floors of the Biomedical Science Tower 3 (BST3) at the Oakland campus. The 11,600 square feet on the 10th floor is used primarily for exploratory research and assay development, and includes four fully functional tissue culture rooms, five procedure rooms, and two walk-in cold rooms.
Residing on the 9th floor are the high throughput screening (HTS) and High-Content Screening (HCS) facilities and includes a 1,200 square foot HTS laboratory, 412 square foot HCS laboratory, two fully functional tissue culture rooms, a 201 square foot HTS reagent preparation room, and an additional 414 square foot assay development laboratory.
Within the HTS laboratory is house a Matrical Ministore modular compound storage platform with 3 VLM modules and the capacity for 12,900 384-well plates or approximately 4.95 million compounds. This system is integrated with LIMS to manage compound storage and retrieval.
Also in the HTS facility is an array of robotic liquid handling systems for bulk delivery of reagents or cells (Titertek Zoom, Titertek MAP-C2, Thermo Scientific Multidrop Combi, Biotek Microflo, and Molecular Devices Aquamax DW4) as well as for compound dilution and transfer to assay or daughter plates (Perkin Elmer Multiprobe, Evolution EP3, and Janus MDT, and the Velocity 11 Bravo). Two BioTek plate washers are available for washing cells in 96- and 384-well plates.
A variety of detection instruments at the UPDDI enable every kind of assay to be able to be run from absorbance and luminescent assays to fluorescent assays involving fluorescent intensity, time-resolved fluorescence, fluorescence polarization, and high-content fluorescent imaging. Plate reader detection systems include Molecular Devices SpectraMax M5, M5E multi-mode readers, and FlexStation 3 kinetic reader, and the Perkin Elmer EnVision multilabel reader and Topcount scintillation and luminescent reader. There are four automated high-content imaging platforms, the ArrayScan II and ArrayScan VTi wide-field systems, and two Molecular Devices Image Xpress Ultra confocal systems.
Other instrumentation includes a GE Healthcare Storm Phosphorimager, a Biacore T100, Bio-Rad iQ5 Real-Time PCR system, InVivo2 400 hypoxic workstation with iSTAT blood gas analyzer, Waters Model 2690 HPLC with a 2487 spectrophotometer, a Waters Model 994 photodiode array detector, a GE Healthcare Aktä FPLC, two Olympus IX5 microscopes equipped with epiflouorescence and monochrome/color CCD cameras (video capable), and two Leica DM IL inverted phase contrast microscopes.
Data generated at the UPDDI is managed by a state-of-the-art network comprised of nine servers including a PE 2850 Oracle Database and IDBS ActivityBase™ LIMS server with 8 GB memory and two Dual Core CPUs, a Dell PE 2850 with 8 GB memory to house the database for the two ImageXpress imaging platforms, a Dell PE 2850 with 4GB memory attached to a 25 TB NAS to serve as a file-server for the IXUs, a Dell PE 2950 server with 16 GB memory and two Dual Core CPUs to house the ArrayScan® VTI Cellomics Store™ database, a PE 2950 server with 4GB memory and two Dual Core CPUs attached to a 5TB disk storage to house the a ArrayScan® IV Cellomics Store™ database, a Dell 2950 application server with four analysis engines and two high-speed dual quad-core CPUs to house the Definiens image intelligence suite, a PE 2850 Windows file server with 8 GB memory and two Dual Core CPUs, a PE R310 as Primary Domain Controller server and a PE 1850 Secondary Domain controller server. Data are securely stored and backed up using a EMC CX300 scalable Storage Area Network (SAN) as the central data warehouse with 31.5 TB formatted storage (upgradeable to 70 TB) and a Dell PV 124T Backup tape drive directly attached to one of the SAN servers and a Dell TL-4000 Backup tape drive directly attached to one of the SAN servers.
Analysis of large and complex data sets is handled through LIMS and HTS/HCS analysis software including two IDBS ActivityBase™ LIMS with XLfit , Chemistry Selection Assistants, and Protocol Transfer Assistants seats, seven IDBS SARview seats, two Leadscope Enterprise 2.4.6-1 Chemical Structure analysis software seats, two Cellomics platinum package of Bio-Application image analysis algorithms (V2, V3 and V4) for their ArrayScan® imaging platforms, four vHCS™ Discovery Toolbox licenses, four vHCS™ View licenses, ten Spotfire® data visualization licenses, MetaXpress and Acuity site licenses, and a Definiens image intelligence suite for custom image analysis including; one developer, one architect, and two Cellenger licenses with four data analysis engines.
Chemistry/Medicinal Chemistry Facility
The Chemistry and Medicinal Chemistry facility is located at three sites across the Oakland campus in BST3, and in the Chevron and Salk buildings. These laboratories are equipped with the state-of-the art analytical instrumentation including seven 400-600 MHz Bruker NMRs (Salk, Chevron, BST3), two ATR FTIRs (1 Chevron, 1 BST3), four LCMS systems (2 Chevron, 2 BST3), and a Circular Dichroism system (Chevron). In addition they house a Waters SFC purification instrument (BST3), four Isco Combiflash Purification Systems (3 Chevron, 1 BST3), and multiple prep HPLCs (Chevron, Salk). Finally, for compound synthesis and isolation two Emrys Microwave optimizers (1 Chevron, 1 BST3), two Par Hydrogenators (1 Chevron, 1 Salk), two Lyophilizers (1 Chevron, 1 Salk), a Flexicool cooling systems and a cold room for low temp reactions (Chevron), multiple IKA control heat stirrers, and two glove boxes for inert atmosphere reactions (Salk) are at the chemists disposal.
Computational Biology and Chemistry
An important aspect of the new vision for drug discovery at the UPDDI is the integration of computational tools in the analysis of drug-target interactions and in developing strategies to advance novel chemical series. The Department of Computational and Systems Biology is well equipped to meet the challenge. Available to the group are 864 CPU cores and a 100 node cluster with 1.4 TB memory and 16 TB storage, Infiniband interfaces with 63 nodes having 20 Gbps and 12 having 40 Gbps, and Windows and Linux servers for sharing files, printers, and other resources. Various modeling software includes CCDC GOLD, MOE, Chimera, VMD, NAMD, and OpenEye Applications and Toolkits: Tripos Sybyl. Research software tools include GNM, ANM, and PCA_NEST servers, ProDy, and several network accessible databases of biological research data are routinely employed. The computation capacity for in silico HTS is up to 16 M compounds per day in a pharmacophore based screen and 2 M compounds in a docking screen when using 184 CPUs. For molecular dynamics simulations the capacity is 2 to 10 ns per day depending on the protein size using 24 to 72 CPUs.
The University of Pittsburgh School of Medicine’s aquatics research facility is one of the largest in the world. It occupies 4,600 sq. ft. of floor space on the 5th floor of the BST3, holds 11,000 fish tanks in its main system and 480 tanks in its quarantine system, and can maintain 500,000 adult zebrafish. The main system consists of two independent 5,500-tank systems with automated water filtration. Three independent light control chambers enable alternative photoperiods. Automated monitoring equipment regulates pH, conductivity, and temperature of the water in the system. A 282 net sq. ft. injection room holds six injection apparati within the facility. This allows up to six researchers to simultaneously perform microinjection on zebrafish embryos, an important aspect in the establishment of transgenic lines. In addition, there are seven fluorescent and bright-field, high-end, stereomicroscopes for visualizing and documenting transgenic reporter expression, and for screening chemically treated embryos. All veterinary and husbandry care is provided by veterinarians and supporting personnel in the Division of Laboratory Animal Resources (DLAR), which is overseen by the University Institutional Animal Care and Use Committee, and complies with the Animal Welfare Act, NIH policies, and all other applicable federal, state, and local laws. Animal facilities at the University of Pittsburgh have been fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC) since 1971, and the International Association for the Assessment and Accreditation of Laboratory Animal Care.
Drs Eiseman and Beumer closely collaborate on numerous preclinical projects. The combined resources available for preclinical studies are listed below.
The equipment available for in vitro/ex vivo work includes: analytical balances, , microcentrifuges, table top centrifuges, (fluorescent) microscopes, laminar flow hoods, CO2 incubators, shaking water baths, liquid nitrogen storage for cells, refrigerators, freezers, gel electrophoresis, microplate readers, pH meters, Z1 Coulter counter, light box, and access to the shared use equipment within UPCI which includes: liquid scintillation counters, gamma counters, X-ray film developers, Molecular Dynamics Densitometer, ultracentrifuges, autoclaving and glass washing services, Tecan Safire and Licor Odyssey, etc.
The Division of Laboratory Animal Resources University of Pittsburgh maintains the animal care facilities at the Hillman Cancer Center, Basic Research Pavilion, and is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Within the Central Animal Facility, Dr Eiseman has a dedicated laboratory (G11.8) and fully equipeed animal procedure room (G11.9), as well as an animal housing room (G11.28a) capable of handling 1500 rodents at any time.
Analytical equipment available for preclinical research includes a Perkin-Elmer model AAnalyst 600 Atomic Absorption spectrometer, two regular HPLC UV systems with dual wavelength, fluorescence, one HPLC with UV and radioactivity detection, two Thermo Finnigan MSQ mass spectrometers, two Waters QuattroMicro triple stage mass spectrometers with UV, and one Applied Biosystems 4000Q hybrid triple-quadrupole, linear ion-trap witha diode array detector. All mass spectrometric detectors are supplied with high purity nitrogen generated by a Parker Balston nitrogen generator model 75-880, supplied with compressed air by a Copto air-compressor located in the basement of UPCI.
Chemical Biology Facility
The Chemical Biology Facility (ChBF) is a cancer-focused component of the University of Pittsburgh Drug Discovery Institute, and one of the National Cancer Institute (NCI)-supported Shared Resources of the University of Pittsburgh Cancer Institute (UPCI). In this capacity, ChBF faculty and staff collaborate with UPCI investigators to optimize the discovery and development of new molecular entities for cancer, providing expertise and guidance in the application of quantitative systems pharmacology and innovations in chemistry and biophysics as well as in the development of novel technical approaches and application of traditional methods for identifying new cancer therapeutics. Drawing from UPDDI’s many technical strengths, among the services available to support UPCI are development of HTS/HCS assays for robust screening campaigns, hit characterization and lead optimization; implementation of high throughput and follow-up screens; and informatics support for data analysis and interpretation including computational mining of public databases. The ChBF is also designed to play a vital role in numerous research projects in oncology by providing UPCI members with access to state-of-the-art reagents and instrumentation (see Facilities) that are not readily available elsewhere at the University of Pittsburgh or at UPCI.