Breaching Biological Barriers: The Synthesis of Probes and QuickTime and a TIFF (Uncompressed) decompressor

  • View
    1

  • Download
    0

Embed Size (px)

Text of Breaching Biological Barriers: The Synthesis of Probes and QuickTime and a TIFF (Uncompressed)...

  • Medicinal Chemistry

    Breaching Biological Barriers:

    The Synthesis of Probes and

    Development of New Methods

    for Biological Release

  • Electronic access to lectures:

    http://lcbim.epfl.ch/teaching

    The password for open the documents: cbsm2012

  • Overview

    • Introduction: Molecular Transporters

    • Intracellular Cargo Delivery by Cell Penetrating

    Peptides Adapted to Target Cancer Cells through

    Cell Surface Protease Activation

    • Releasable Luciferin-Transporter Conjugates: Real-

    time Analysis of Uptake and Bioactivatable Release

    in Cells and Transgenic Reporter Mice

    • Applications of this technology for delivery of real

    therapeutics

  • QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

    Celia Henry, C&EN, August 25, 2003

    Function Oriented Synthesis • June 26, 2000 human genome

    • Proteomics (targets)

    • Systems Biology (pathways)

    • Combinatorial Chemistry (ligands) LIFE DEPENDS ON BARRIERS…

    TISSUE

    BARRIERS

    CELL

    BARRIERS

    THERAPY DEPENDS ON

    BREACHING BARRIERS…

    DRUG / PROBE

    TARGET

    X

    TRANSPORTER

    Varmus, Klausner, et al. “Grand Challenges in Global Health” Science, 2003, 398-399.

    Jain, R.K. “The Next Frontier of Molecular Medicine: Delivery of Therapeutics” Nature Med. 1998,

  • Breaching Biological Barriers

    POLAR

    DRUGS NON-POLAR

    DRUGS

    Log P Box

    Log P

    MOST

    DRUGS

    Physical Property Control

    siRNA TAXOL

    http://www.youtube.com/watch?feature=player_detailpage&v=JShwXBWGMyY#t=7

    http://www.youtube.com/watch?feature=player_detailpage&v=kfy92hdaAH0

    http://www.youtube.com/watch?feature=player_detailpage&v=JShwXBWGMyY#t=7 http://www.youtube.com/watch?feature=player_detailpage&v=JShwXBWGMyY#t=7 http://www.youtube.com/watch?feature=player_detailpage&v=kfy92hdaAH0 http://www.youtube.com/watch?feature=player_detailpage&v=kfy92hdaAH0

  • CELL

    DRUG

    PROBE

    LEAD

    Facilitated Uptake

    POLAR

    DRUGS NON-POLAR

    DRUGS

    Log P Box

    Log P

    MOST

    DRUGS

    X X

    DRUG

    PROBE

    LEAD

    Molecular

    Transporters

    TRANSPORTER

    Breaching Biological Barriers

  • Molecular Transporters: Opportunities

    •ENHANCE BIOAVAILABILITY OF EXISTING DRUGS ($15-50 BILLION) •CHANGE EFFICACY EXISTING DRUGS: SIDE EFFECTS, SELECTIVITY. •IMPROVE FORMULATION/ADMINISTRATION AND UPTAKE •ENABLE DELIVERY OF NEW CARGOES (DNA, siRNA, PEPTIDES, etc) •ACCESS TO DIFFICULT SITES (e.g., BBB, eye, etc) •SELECTIVITY: ADMINISTRATION WHERE & WHEN NEEDED •AVOID FIRST PASS METABOLISM/SYSTEMIC TOXICITY •MINIMIZE CANDIDATE OPTIMIZATION TIME •LIBRARY HIT ENHANCEMENT •IMAGING •TARGET VALIDATION

    *THERAPY

    *DIAGNOSTICS

    *IMAGING

    *DRUG DISCOVERY

    Recent review with lead references to other groups: Goun, E.A. et. al. ChemBioChem 2006, 1497.

  • *HIV tat and Antennapedia are transcription factors that cross biological membranes

    MEPVDPRLEPWKHPGSQPKTACTTCYCKKCCFHCQVCFTTKALGISYGRKKRRQRRRPPQGS QTHQVSLSKQPTSQPRGDPTGPKE*KKKVERETETDPFD

    Natural Chemical Codes for Facilitated Cellular Uptake

    *Evolutionary pressures have produced mechanisms to prevent and promote uptake

    H2N

    H N

    N H

    H N

    N H

    H N

    N H

    H N

    N H

    X

    O

    O

    O

    O

    O

    O

    O

    O

    O

    HN

    NH3

    NH2

    HN H2N O

    H2N NH2

    HN

    H3N NH2

    NH

    H3N NH2

    HN

    H3N NH2

    H3N NH2

    NH

    H3N NH2

    *HIV tat 49-57 is required for translocation (Frankel, Pabo 1988) SIGNIFICANTLY, IT IS CHARGED BUT EXHIBITS FACILITATED UPTAKE

    arginine-lysine-lysine-arginine-arginine-glutamine-arginine-arginine-arginine

    49 57

    *This works for the HIV tat protein, the “gold standard” in research but - it would have limited use in therapy (cost of goods, metabolism, etc) and

    - it is not necessarily an optimized system

    *Design a better transporter; what’s required in tat?

    “The bilayer…serves as a relatively impermeable barrier to the flow of water soluble

    molecules” …most standard textbooks

  • Assay Protocol for Cellular Uptake

    Uptake of Peptide-aca-FITC conjugate (25 mM) into Human Jurkat T Cells

    Wender, Mitchell, Pattabiraman, Pelkey, Steinman, Rothbard Proc. Natl. Acad. Sci. USA , 2000, 13003;

    Rothbard, Garlington, Lin, Kirschberg, Kreider, McGrane, Wender, Khavari, Nature Medicine 2000, 1253.

    57 49 SPACER PROBE TRANSPORTER

    N H

    H N

    N H

    H N

    N H

    H N

    N H

    H N

    N H

    X

    O

    O

    O

    O

    O

    O

    O

    O

    O

    HN

    NH3

    NH3

    HN H2N O

    H2N NH2 HN

    H2N NH2

    NH

    H2N NH2

    HN

    H2N NH2

    H2N NH2

    NH

    H2N NH2

    O

    N H

    N H

    S

    O

    O

    HO

    COOH

    CELLS TREATED WITH

    FLUORESCEIN ALONE

    CELLS TREATED WITH

    FLUORESCEIN-TAT

    SOME UPTAKE NO UPTAKE

    PROTOCOL: EXPOSE CELLS TO CONJUGATE (MINUTES); WASH; CONFOCAL MICROSCOPY & FACS ANALYSIS

    CONTROLS: FLUORESCEIN ITSELF DOES NOT ENTER CELLS; AZIDE PRE-TREATMENT BLOCKS ENTRY

    M e

    a n

    F

    lu o

    re s

    c e

    n c e

    Tat

    49-57

    0

    300

    100

    200

    Fl

    alone

    Tat

    49-57

    +N3-

  • Chemical Codes For Cellular Uptake

    Uptake of Peptide & Peptoid-aca-FITC conjugates into Human Jurkat T Cells

    Proc. Natl. Acad. Sci. USA , 2000, 97, 13003; Nature Medicine 2000, 6, 1253-1257; J. Pept. Res. 2000, 56(5), 318-325. ; J. Med. Chem.

    2002, in press. Organic Letters 2001, 322

    R5 R6 R7 R8 R15 R20 R25 R30 R9

    CONFOCAL ANALYSIS

    HEAD GROUP TYPE &

    TRANSPORTER LENGTH

    N N

    N NN

    O

    O

    O

    O

    O

    N O

    O

    OR N

    O N H

    N H

    S

    O

    O

    HO

    COOH n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    n

    HN

    H2N NH2

    PEPTOID TRANSPORTER

    N-

    hex7

    N-

    hex8

    N-

    hex9

    SUPERIOR SYSTEMS

    AND

    COST OF GOODS*

    M e a n

    F lu

    o re

    s c e n

    c e

    0

    1000

    K9

    NOT SIMPLY

    CHARGE

    Tat

    49-57

    UPTAKE

    BACKBONE STEREOCHEMISTRY

    & POSITION (PEPTOIDS)

    Tat

    49-57

    R7 R9 r7 r9

    M e a n

    F lu

    o re

    s c e n

    c e

    0

    L

    D

    UPTAKE

  • Molecular Transporters for Cellular Uptake

    RXRXRXRXRXRXR

    SPACED

    ARG-TRANSPORTERS J. Med. Chem. 2002, 3612

    NH2O

    O N H

    NH

    n

    TFA NHH2N

    R

    OLIGOCARBAMATE

    TRANSPORTERS J. Am. Chem. Soc. 2002 13382

    ARBOREAL DENDRIMER

    TRANSPORTERS Organic Lett. 2005, 4815

    Guanidine is required for activity

    OUT

    (polar)

    CYTOSOL

    (polar)

    Non-polar

    ADAPTIVE

    TRANSLOCATION

    Na+

    K+ N3

    -:

    ADP->ATP

    1

    2

    3

    4

  • OUT

    (polar)

    CYTOSOL

    (polar)

    Non-polar

    J. Am. Chem. Soc. 2004, ASAP ; Proc. Natl. Acad. Sci. USA , 2000, 97, 13003; Nature Medicine 2000, 1253

    Octanol

    Water

    Fl-O8 Fl-R8 Fl-O8 NaCO2R

    Fl-R8 NaCO2R

    H

    N

    RH

    H N

    HH

    N

    N

    H H

    RH

    O

    P

    O

    XX

    RR'

    O

    P

    O

    XX

    RR'

    N

    HH

    N

    N

    H H

    RH

    H

    N

    RH

    H

    Na+

    K+ N3

    -:

    ADP->ATP

    Mechanism of Uptake for Guanidinium Transporters

    1

    2

    3

    4

  • Alkylated Guanidine Transporters: Inhibition of Uptake

    0

    200

    400

    600

    800

    M e

    a n

    F lu

    o re

    s c

    e n

    c e

    N H

    NH2

    O

    N

    NN

    8

    H N

    O

    H

    H

    5

    H N

    S

    O

    OH

    O

    H

    R2

    R1

    TFA

    CO2H

    N

    N N H

    H

    H

    H

    HR

    O O P

    N

    N N H

    Me

    H

    H