Therapeutic Development and Drug Discovery

In vivo biology and testing

In vivo biology and testing section (IVBDT) of the CBT assists investigators with the development of cell- and animal-based strategies for ascertaining target engagement, disease modification and toxicity.  The IVBDT works closely with the Target Validation and Screening section (TVS) to develop screening assays that are translatable to in vivo models. Capabilities within the IVBDT include in vitro verification of ADME properties (metabolic stability, plasma protein binding, permeability/efflux, HepG2, TK6, phospholipidosis, hERG channel activity, and more); in vivo pharmacokinetic analyses (MTD, bioavailability, metabolite identification); as well as project specific pharmacodynamic outcomes for assessing target engagement, disease modification and biomarker development (in vivo imaging, behavioral studies, and more).  The IVBDT currently maintains mouse models for neurological diseases, cancer and a variety of signal transduction pathways.  Expertise, reagents and equipment are also available for generating new animal models from concept to founder:  design/generation/ purification of targeting constructs and transgenes; ES cell targeting/identification; and a dedicated room for radiolabeling of nucleic acids.  The laboratory is equipped for harvesting/processing specimens for chemical (PK), molecular biology (RNA/DNA), biochemical (protein) and cell/tissue structure analyses (paraffin embedding, histochemical staining, immunohistochemical/ immunofluorescence staining).  The IVBDT's tissue culture suite is equipped with biosafety cabinets, CO2 incubators, inverted phase microscope, liquid nitrogen storage units, and a cryogenic freezer.

Genomic and bioinformatics

The Center for Biomolecular Theraputics works directly with The Institute for Genome Sciences (IGS) for its bioinformatics and genomics work.  Such work involves identifying mutations and biological pathways that are “drivers” for disease progression.  Such analyses are used to generate experimental protocols/methods for identifying/targeting pathogens and/or testing the targeting of potential targets from the human genome for drug development.  The relevance of these targets in disease are then tested directly in the Target Validation and Screening (TVS) section.  The IGS was established within the University of Maryland, School of Medicine on May 1, 2007, houses an inter-disciplinary, multi-departmental team of collaborative investigators with a broad spectrum of research programs related to the genomics of infectious disease agents, human microbial metagenomics, and bioinformatics. The Institute is led by Claire M. Fraser-Liggett, Ph.D., one of the world’s preeminent genome scientists and previously the Director and President of The Institute for Genomic Research (TIGR).  IGS is currently located at the UMB BioPark, a biomedical research park located adjacent to the University of Maryland Medical Research Complex.

      IGS Investigators are supported by two resource centers: the Genomics Resource Center (GRC) and the Informatics Resource Center (IRC). The Genomics Resource Center is a high-throughput core laboratory and data analysis section using state-of-the-art technology to generate high quality genomic data in a cost effective manner. The GRC staff consists of scientists, bioinformatics software engineers, bioinformatics analysts and research specialists to support these activities. The Informatics Resource Center (IRC), under the direction of Owen White, Ph.D., provides genome annotation and analysis services as well as IT infrastructure, web, and database services to IGS Investigators. The IRC is staffed by scientists, computational biologists, software engineers, and IT specialists to support these activities. The IRC has built a state-of-the-art computational infrastructure that includes a computational grid, an internal 10-gigabit network, clustered database servers, and a hierarchical storage management system.

Medicinal and synthetic chemistry

The Medicinal Chemistry section (MC) is part of The University of Maryland Center for Biomolecular Therapeutics (CBT) that conducts innovative, multidisciplinary and collaborative research programs to discover and develop new drugs for improving human health. The MC section is dedicated to hit to lead medicinal chemistry and lead optimization. The MC section occupies modern laboratory space within the CBT and the Institute for Biosciences and Biotechnology Research (IBBR) at the University of Maryland College Park, Shady Grove campus in Rockville, Maryland. Our laboratories are fully equipped to meet our synthesis and analysis need for a robust drug discovery and development program. The laboratory contains, amongst others, rotary evaporators, vacuum pumps, chromatographic equipment, infrared spectrometers, Parr hydrogenators, analytical and large-scale balances. Major equipment include: 400 and 600 MHz NMR, with multinuclear probe capability, LC/MS and LC/MS/MS, microwave synthesizer (Milestone START), fully automated HPLC systems (analytical and preparative), CombiFlash Rf-200 automated Flash Chromatography System, Autopol III – dual wavelength automatic polarimeter, FTIR Infrared spectrometer (Perkin Elmer 1600 series) and high resolution mass spectrophotometer (MS/HRMS). We also have access to other equipment at National Institute of Standards and Technology located in the Shady Grove Campus.  In addition, the medicinal chemists within the CBT work together with the Computer Aided Drug Design (CADD) center and other Medicinal Chemists within the School of Pharmacy (SOP).  Thus the efficient design of inhibitors, searches of structurally related compounds (SAR by catalogue), and virtual screening capabilities are done via CADD in conjunction with syntheses efforts from the MC section.

Target validation and screening

The goal of the Target Validation & Screening (TVS) section in the CBT is to provide investigators access to automated liquid handling equipment, sensitive high throughput detectors, and diverse collections of screening libraries to facilitate the identification of therapeutic targets and screen for chemical perturbagens as a step towards developing new drugs and therapies. The TVS program provides the expertise in cell-based or in vitro assay development and will assist the adaptation of an investigator’s existing assay into higher throughput formats. The TVS can train users on the operation of the automated liquid handling workstations and the multimode microplate readers, or the TVS personnel can be contracted to perform all aspects of a screening project. The TVS section has dedicated tissue culture facilities and is capable of maintaining cell lines, creating new genetically altered lines to overexpress, knockdown via shRNA/siRNA, or knockout genes via targeted homologous recombination. The program regularly performs molecular biology techniques (RNA/DNA purification, RT-PCR, cloning, stable and transient gene expression/knockdown in mammalian cells) for characterization of cells lines. It works closely with the Protein Production & Biophysics (PPB) program to make recombinant proteins for in vitro assays and in assay development using biochemical techniques (FPLC, recombinant protein expression, chemical labeling, SDS-PAGE).

            The TVS regularly performs a wide range of in vitro protein-protein (fluorescene polarization competition assays, FRET, TR-FRET, AlphaScreens, AlphaLISA) and protein-ligand (lanthanide metal competition assay) assays and cell based assays include mammalian cell viability, chemiluminescence gene reporter assays, yeast and bacterial growth, and cell based ELISAs for screening small drug like compounds. The center currently has 60,781 small drug like compounds available for screening that are maintained by the TVS personnel to assure the highest purity and maintain control of compound inventory using ChemBioFinder Software (ChembridgeSoft). In addition, the TVS program has access to a Bruker 800 MHz NMR equipped with a 60-sample auto-loader to run NMR based screens in conjunction with the Structural Biology (SB) section that allows for direct binding of ligands to proteins via NMR experiments. The NMR is particular useful in fragment-based lead discovery a new lead discovery approach in which much lower molecular weight (100-300Da) compounds are screened relative to traditional drug screening campaigns. Fragment-based hits are typically weak inhibitors and therefore need to be screened at higher concentration using very sensitive biophysical detection techniques such as protein crystallography and NMR as the primary screening techniques.  While theses fragments are simpler, less functionalized compounds with correspondingly lower affinity, the final goal is the chemical crosslinking of multiple fragments increasing their affinity and specificity so the new compound is suitable for optimization into clinical candidates with good drug-like properties.

TVS has two automated liquid handling stations. The Biomek FX with a 96-well head capable of rapid liquid transfers in 96, 384, and 1536-well plates and large reservoirs and moving plates and pipette tip boxes within the 23 deck positions. The Biomek FX has been upgraded with a magnetically mounted V&P Scientific 96-pin-tool for transfer of nanoliter volumes that will eliminate dilution steps saving time and resources.  The Biomek NXp is equipped with an 8-span head that has 8 independent pipette tips and 11 positions on the deck.  While slower at some task than the Biomek FX it is capable of manipulations and reformatting in vials, reservoirs, tubes, and multitude of assay plates including 6, 12, 24, 48, 96, and 384-well plates.  The three BMG multimode plate readers equipped with autoloaders, (1) BMG Optima POLARstar (2) BMG PHERAstar Plus, and (3) BMG PHERAstar FS, that together have the ability to read assays in 96, 384, 1536, and even 3456-well plates in several detection modes including fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer (FRET), time resolved fluorescence (TR), luminescence, UV/Vis absorbance spectra. 

            The TVS has 60,781 small drug like compounds available:  (1) The ChemDiv 40K collection is composed of 40,000 diverse chemical compounds purchased from Chemical Diversity Labs (ChemDiv).  (2) The Maybridge HitFinder Collection is a collection of 14,400 drug like compounds.  (3) MicroSource Spectrum Collection is made up of 2,000 compounds, 50% drugs, 30% natural products, and 20% other bioactive components.  (4) The SBi Collection is a collection of 4,381 compounds collected from several commercial sources plus 67 compounds synthesized by Coop Lab in UM School of Pharmacy).  The TVS has just purchased the Maybridge Ro3 Fragment a collection of 1,000 compounds meeting the Rule-of-3 Fragment Library offers keys features necessary and available in many fragment libraries while guaranteeing solubility of the compounds at 200 mM in DMSO and having chemically linkable analogs available for future linking of fragments into higher affinity compounds.

Computer-aided drug design

The CBT is a partner with the Computer-Aided Drug Design Center in the School of Pharmacy, led by Dr Alexander MacKerell.  Advances in structural biology have allowed for the determination of the 3-dimensional (3D) structures of biological target molecules via the techniques of nuclear magnetic resonance (NMR) or X-ray crystallography, with over 100,000 3D structures currently available in the Protein Data Bank. Computer-aided drug design (CADD) approaches can use the information in the 3D structures of biological target molecules to identify chemicals with a high potential for binding to the biological target molecules. These chemicals may then be obtained and experimentally assayed to select those with the desired biological activity. The selected compounds are referred to as lead compounds and may then be subjected to additional structural optimization via structural biology, CADD and novel organic synthetic methods to obtain compounds with improved activities. Both the lead compounds and their optimized analogs represent chemical entities with a high probability of being developed into therapeutic agents and, therefore, are of great interest to pharmaceutical companies.

   The University of Maryland, Baltimore, including the School of Pharmacy, contains a collection of scientists of varied backgrounds, including computational chemistry, structural biology, biochemistry, molecular biology and cellular biology that, in combination, represent the expertise required for CADD based studies. The CADD Center in partnership with the Center for Biomolecular Therapeutics (CBT) provides collaborative opportunities for biologists to apply CADD approaches to their research programs. These efforts focus on 1) the identification of chemical compounds with the desired biological activity and 2) structural optimization of the identified compounds to enhance their desired biological activity.

Protein production and biophysics

The goal of the Protein Production and Biophysics Section (PPB) in the CBT is to produce and characterize purified proteins for investigators including to those in other CBT programs and for internal studies by the PPB section.  The PPB has assembled the expertise to provide a wide range of methods for studying the biophysical properties of proteins including protein-ligand and protein-protein interactions.

The facility is capable of cloning and expressing tagged and untagged proteins in a wide range of scales from flasks to bioreactors in bacteria, yeast, baculovirus, or mammalian systems.  In addition, uniform stable isotope (2H, 15N, 13C) and selective methyl labeling of proteins is regularly produced for NMR studies.  The section is skilled in protein purification methods needed for tagged and untagged proteins including HPLC/FPLC techniques. The PPB section regularly performs molecular biology techniques (RNA/DNA purification, RT-PCR, cloning, stable and transient protein expression in cells).

The program has expertise in the characterization of the proteins including stability, structure/dynamic properties, size, covalent modification (i.e. phosphorylation, ubiquitination, methylation, glycosylation etc).  Services for the identification of unknown proteins are available using a multitude of biophysical/biochemical techniques including denaturing polyacrylamide gel electrophoresis (SDS-PAGE), FPLC, mass spectrometry (MS), isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, UV/Vis, DLS, analytical ultracentrifugation (AUC), kinetic stop-flow/quench-flow, and surface plasmon resonance (SPR) techniques.  Expertise & equipment for measuring enzyme kinetics, binding kinetics, and thermodynamic properties of a enzyme/proteins are also available including monitoring binding to substrates, other proteins, ligands, and/or oligomerization properties.