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Penn Center for Molecular Discovery

Director: Diamond, Scott L., PhD


The Penn Center for Molecular Discovery (PCMD), founded at the University of Pennsylvania, is a multi-disciplinary center that screens small molecules from around the world in search of new, potentially useful biologically effective agents. The Penn Center for Molecular Discovery is housed within the Institute for Medicine and Engineering.The Director of the Center is Scott Diamond, Ph.D., the Arthur E. Humphrey Professor of Chemical and Biomolecular Engineering. The PCMD contributes to a massive, public-domain database (PubChem) where interactions between the NIH repository and thousands of biological targets can be data-mined at an unprecedented level.





  • Chemical Genomics/Organism HTS ( Material analysis service )

    Model organisms such as C. elegans, drosophila, and zebrafish have helped define the underpinnings of modern biology with critical impact in neurology, stem cells, embryo development, morphogenesis, multi-organ physiology and toxicology, lifespan control and ageing. Organism-based chemical genomics allows unbiased genome wide exploration of target space. While the human genome is fully available, only half of human genes have any report whatsoever in the literature, and fewer than 2000 have five or more papers associated with them. This means that over half the target space for chemical discovery is largely untapped. Through the use of model organisms, the vision of the PCMD is to leverage organism-based, genome-wide research to bring new targets for chemical HTS. Special emphasis is placed on organism-based platforms that are HTS compatible. Central to this vision is the rapid translation of discovery in model organisms to mammalian HTS/probe production.

    Chemical Genomics:
    John Hogenesch, PhD
    The combination of genomic cell based screening approaches and chemical biology offers new opportunities to both define targets and their modulators. As part of the PCMD, we are taking these approaches to delineate important signal transduction pathways, determine new components, and perturbagens that modulate them.

    Viral Genome/Host Interactions:
    Sara Cherry, PhD

    C. Elegans Discovery:
    Todd Lamitina, PhD

    Zebrafish Discovery:
    Michael Pack, PhD

  • Cheminformatics and Computational Chemistry ( Material analysis service )

    Computational Strategies for Optimizing Biologically Active Leads in the PCMD

    Lead chemical series identified through automated screening of the MLSMR (Molecular Libraries Small Molecule Repository, NIH) can be further optimized using a variety of computational strategies. These include: 1.) substructure and similarity searching, 2.) virtual library enumeration around the active lead series followed by protein/ligand docking and scoring, 3.) quantitative structure activity relationship (QSAR) model derivation, and 4.) chemical reactivity estimations. These methodologies are currently used within the PCMD by a multi-disciplinary team of scientists in Medicinal Chemistry, Biochemistry/Bioengineering, and Computational Chemistry to understand the activities of chemical probes at the molecular level for various therapeutic targets.

    Software Tools
    Protein/ligand docking: several packages available from Schrodinger, Inc., including Glide for binding site docking (standard precision and extra precision modules), LigPrep for Glide small molecule database creation, and MMPBSA and MMGBSA to augment the SP and XP scoring schemes.

    Chemical reactivity estimates: PC Spartan (Wavefunction, Inc.), used for semi-empirical geometry optimizations prior to docking and pharmacophore searching, and also for calculations of electrostatic potentials for estimating electrophilicity at specific molecule centers (for ex., as applied to covalently bound protease inhibitors).

    QSAR model derivation: MOE software, available from Chemical Computing Group, CCG, used for descriptor calculation and model building. Linear models utilize PCR and PLS regression techniques. ADMETPredictor and ADMETModeler (available from SimulationsPlus, Inc.) used for descriptor calculations and non-linear regression model building.

    3D Pharmacophore Searching: Unity software, Tripos, Inc., and Phase software, Schrodinger, Inc. Databases available: LeadQuest, Zinc, and MLSMR (NIH). Flexible 3D pharmacophore searching can be carried out on the ‘ligand only’, or, if protein structural information is available, on the complementary ligand/protein interaction sites. When creating 3D pharmacophores, generalized chemical features such as H-bond donor and acceptor sites, as well as aromatic and hydrophobic functionalities, can be assigned to atomic positions in the lead molecular series. Explicit chemical functions can also be retained as pharmacophore features. Partial matching algorithms allow for various combinations of pharmacophore features to be selected from an initial large pool of many pharmacophore features. This is especially useful when there is limited SAR information available for the lead series.

  • High Throughput Screening (HTS) ( Material analysis service )

    Homogeneous and separation-free well plate biochemical assay platforms include proximity-based assays such as scintillation proximity assay (SPA), LOCI (luminescent oxygen channeling assay (i.e. alpha-screen), and fluorescent resonance energy transfer, FRET). FRET has advanced with time-resolved fluorescence (TRF) instruments to read the long-lived cryptate lanthanides (Eu, Tb) to resolve specific energy transfer signal above nonspecific autofluorescence.

    Fluorescence polarization (FP) anisotropy is valuable for detection of enzymatic or binding reactions that result in a change in molecular weight of small fluorescent reactants with consequent change in rotational diffusivity. To list only a few examples, kinase HTS assays can involve: (1) fluorescent binders or quenchers to fluorescent phosphorylated substrates {e.g. anti-phosphotyrosine antibodies, fluorescent PDZ domains, IQ, and IMAP reagents}, (2) protease treatment of quenched peptide detector whose cleavage is blocked by phosphorylation (e.g. Z-lyte assay), (3) FP detection of binders to phosphorylated peptides, (4) detection of phosphorylated iodinated-peptides captured on anti-phosphotyrosine-SPA beads, (5) phophopeptide mediated aggregation of alpha-screen beads coated with anti-phosphotyrosine, or (6) luciferase or enzyme amplification assay of ATP consumption. These formats have reduced cost and complexity beyond the classic radioisotope assay of capture of P32 or P33-labeled phosphorylated peptides, and top-count reading of washed 384-well plates or use of FlashPlate scintillation. Detection of direct compound binding to protein targets have relied upon plasmon resonance, thermal denaturation profiling, or capture of fluorescent protein targets to anchored phamacophores.

    Cell based assays often involved the detection of: marker gene (gfp, luc, beta-gal) induction, calcium mobilization, dye binding to activated membranes, rubidium efflux, or cell killing/lysis assays for the discovery of anticancer agents, anti-apoptotic agents, anti-microbial agents. Imaging based assays of intracellular fluorescence localization has proven powerful for analysis of nuclear localization of hormone receptors. High throughput ion channel recording remains a challenging area despite gains in nanofabricated surfaces for cell recording. Cellular assays can often enhance the discovery opportunity for compounds that are cell permeable and non-toxic. Protease assays typically involve fluorogenic peptides that have coumarin leaving groups. Alternatively, quenched peptides can be designed that dequench when cleaved.

  • Metallo-Organic Pharmacophores ( Material analysis service )

    Metallo-organic compounds have Lipinski properties that are entirely dependent upon the coordinated ligands. The metallo-chemical bond is as strong as a covalent bond; they do not "fall" apart. These compounds are designed to be hydrophobic and can pass through cellular membranes. The rigidity of metallo-organics is potentially the source of their amazing specificity. These ruthenium coordinated compounds behave like organic compounds in every respect but they open new opportunities for accessing rigid globular structures in an economical fashion. The metal center is not involved in any interactions with the environment. Space filling models demonstrate that the metal is not accessible and completely buried in the center of the molecule. In addition, rapid ligand scanning around a metal center can be used as a drug discovery tool. It allows the Penn Center for Molecular Discovery to quickly access unexplored chemical space and to rapidly identify new biological active and unique structures.

    Most of the metal complexes are very rigid and it can therefore be predicted with high confidence in what conformation they bind to the active site. Thus, these metal complexes can be considered imprints of the active site and are therefore useful starting points for cheminformatics and the design of related purely organic compounds. The Meggers Lab has recently demonstrated that properly designed ruthenium compounds can enter mammalian cells and inhibit the protein kinase GSK-3 without displaying cytotoxicity even at higher micromolar concentrations. Furthermore, the comparison of the potency in cell-based assays surpasses significantly the activity of know organic GSK-3 inhibitors (kenpaullone, 6-bromoindirubin-3’-oxime).

  • Microarray Screening ( Material analysis service )

    The Diamond laboratory has developed a methodology to screen chemical libraries on microarrays (Gosalia and Diamond, PNAS 2003). Chemical compounds within individual nanoliter droplets of glycerol can be microarrayed onto glass slides at 400 spots/cm2. Using aerosol deposition, subsequent reagents and water are metered into each reaction center in order to rapidly assemble diverse multicomponent reactions without cross-contamination or the need for surface linkage. This proteomics technique allows for the kinetic profiling of protease mixtures, protease – substrate interactions, and high throughput screening reactions.

    Exploiting the low volatility of glycerol droplets on glass, we have created discrete reaction volumes via contact printing. Each droplet had an average volume of 1.6 nL after microarraying. A 16x24 array of 200 um diameter spots with 500 µm center-to-center spacing, equivalent to a 384-well plate format, occupied less than 1-cm2. In kinetic applications on a microarray, the need to initiate tens of thousands of reactions at once is not easily accommodated by the use of piezo dispensing micropipettes or ink-jet engines that have exacting surface tension or viscosity requirements and are prone to clogging. To solve the problem of rapid sample delivery to these small nonspreading droplets, we deposited onto the arrays using aerosol deposition. In this approach, the aerosolization of the sample resulted in a fine mist with a median droplet diameter of 18 µm (~3 pL). Aerosol droplets deposited evenly on and around the glycerol spots and rapidly evaporated within 7 seconds without mixing between spots.

  • Protease Proteomics ( Material analysis service )

    Due to their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases also participate in cellular homeostasis, inflammation, tissue remodeling, and coagulation and play an important roles in the pathogenicity and progression of many diseases such as viral infection or replication. Proteases comprise one of the largest protein families in organisms from E. coli to humans. Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets.

    To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementarity of the enzyme active site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors. We have used solution-phase substrate nanodroplet microarrays, in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles. These arrays allow high throughput characterization of the preferred residues on the P side of the substrate in a highly parallel and miniaturized format. We have studied the use of these arrays here to map the substrate specificity of 24 serine and cysteine proteases in a rapid and efficient manner. The combination of substrate mapping and compound screening provides critical information for training of Structure-Activity Relation (SAR) models.

  • Synthetic Chemistry ( Material analysis service )

    The Hit-to-Probe process, designed to identify and develop useful chemical probes from high throughput screening, is a complex multiple disciplinary process that requires close interaction between key functions (below). The interdependent relationship between biology and chemistry is critical for the success of the overall process. The Synthesis Core will play key roles in the success of identifying probes with appropriate properties for further biological evaluation.

    The focus of the Synthesis Core will be to optimize the “hits/probes” as obtained from the HTS Core. It is likely however that the properties of the initially derived hit/probes will not be ideal as research tools. The first effort will be to validate the hit/probe by synthesis, assuming that the compound is not commercially available. Once confirmed as a true or tractable hit/probe candidate, the principal goal of the Synthesis Core will be to discover new and/or improved hits/probes possessing improved the biological/physicochemical properties vis-a-vis the initially derived hit/probes. Efforts here will focus on improving both affinity and selectivity as well as pharmacological properties such as solubility and bioavailability. Two tactics will be applied to achieve these goals. The first will involve application the rapid ligand scanning/probe production, exploiting the exciting new protocol currently being developed in the Meggers Laboratory. This tactic holds the promise for the discovery of completely new classis of hit/probes. When a pharmacophore has been identified, the chemical efforts will include optimization by exploration of structure-activity relationships (SAR), including directed library generation employing both parallel synthesis and structure-guided design.

  • Zebra Fish Screening ( Material analysis service )

    A great advantage of zebra fish is the ability to perform high throughput assays in a living organism. Although not as efficient as biochemical or cell-based HTS, these studies have the advantage of analyzing the function of genes and pharmacological agents in a specific biological context. These high throughput assays include both genetic and pharmacological (small molecule) screens to identify genes that regulate cell behavior, organ development, or pathogenesis. Furthermore, using the zebra fish, one can analyze mechanisms of gene function using embryological assays not amenable to other model systems.

    As an example of capability in the area of zebra fish genetics and assay development, Dr. Pack developed a strategy to identify genes and pharmacological compounds that regulate cancer progression using the zebra fish. In this mutagenesis screen, Dr. Pack identified a recessive lethal mutation, meltdown (mlt), which causes cystic expansion of the larval zebra fish intestine. Cyst formation in mlt mutants occurs as a result of stromal invasion of posterior intestinal epithelial cells. Epithelial invasion in mlt mutants is regulated by the ectopic expression of genes causally linked to human cancer progression. Inhibition of these cancer progression genes rescues mlt mutants.

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Last updated: 2019-12-10T11:29:43.289-05:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016