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
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
HN
NH
HN
NH
HN
NH
HN
NH
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
NH
HN
NH
HN
NH
HN
NH
HN
NH
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
NH
NH
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
Me
an
F
luo
res
ce
nce
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
NN
NNN
O
O
O
O
O
NO
O
ORN
ONH
NH
S
O
O
HO
COOHn
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*
Mean
Flu
ore
scen
ce
0
1000
K9
NOT SIMPLY
CHARGE
Tat
49-57
UPTAKE
BACKBONE STEREOCHEMISTRY
& POSITION (PEPTOIDS)
Tat
49-57
R7 R9 r7 r9
Mean
Flu
ore
scen
ce
0
L
D
UPTAKE
Molecular Transporters for Cellular Uptake
RXRXRXRXRXRXR
SPACED
ARG-TRANSPORTERS J. Med. Chem. 2002, 3612
NH2O
ONH
NH
n
TFANHH2N
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
Me
an
Flu
ore
sc
en
ce
NH
NH2
O
N
NN
8
HN
O
H
H
5
HN
S
O
OH
O
H
R2
R1
TFA
CO2H
N
N NH
H
H
H
HR
O OP
N
N NH
Me
H
H
HR
OOP
N
N NMe
Me
H
H
HR
OOP
N
NN
Me
H
H
H
HRN
NN
Me
Me
H
H
HRN
NN
H
H
H
H
R H
Monomethylation partially inhibits uptake
Dimethylation completely inhibits uptake
Bidentate hydrogen bonding essential for uptake
The Mechanism of Guanidine-Rich Molecular Transporters
Adsorption
Ionic attraction with the cell surface
Guanidine - phosphate H-bond strength increases
Entropically favored ion exchange
Internalization
Lipids invert solubility of
oligoguanidines
Guanidine - phosphate H-bond in
lipid: 6-10 kcal/mol
Methylation prohibits lipid
complementation
Microscopic Reversibility
Emergence on inner leaflet of
plasma membrane
Diffusion into the cytosol
Uptake is energy dependent
“Single Molecule” Mechanistic Studies (@10-23 M)
S
NHAc
O
HN
(R)8CONH2
+ 8 TFA
O
NCCN
NC
N
O
N
O
O
ENVIRONMENTAL SENSORS
ICE H2Oliq
Stanford w/ Professor W.E. Moerner
UPTAKE MECHANISM OF A SINGLE MOLECULE:
Glass Fibronectin
Arg-DCDHF
Objective
Top
Bottom
Of the cells
0
2
4
6
8
10
12
0 2 4 6 8 10
0
10
20
30
40
50
0 0.2 0.4 0.6 0.8 1
0
20
40
60
80
100
120
0 0.2 0.4 0.6 0.8 1
Diffusion coefficient (mm2/sec)
2V-12
Transferrin-
Alexa594 2V-arg8
Lipid (fast) Protein (slow) Arg8 (mix)
New Drug Delivery Systems: Molecular Transporters
Image Min = -267.3 Max = 42271
counts
40000
30000
20000
10000
Color Bar Min = 933
Max = 42271
bkg sub
cosmic
flat-fielded
Human Gene Therapy, 2003, 1225.
PLASMID DNA
INTO PARASITES: Proc. Natl. Acad. Sci.
USA , 2003, 14281 DAPI
Triclosan_r8
C =12 μM
Triclosan_r8
O
O
HN
O
NH FITC
NH
NH2Cl
Cl Cl
O
NH
HN NH2
O 8
Toxoplasma gondii tachyzoites
Transporter-Peptide Cargo Uptake Into Heart Tissue
PEPTIDE DELIVERY TO PREVENT ISCHEMIC DAMAGE
PRECONDITIONING : BRIEF EPISODES OF
ISCHEMIA DECREASE NECROSIS DURING
PROLONGED ISCHEMIA
RACK PEPTIDES SIMULATE PRECONDITIONING
WHEN INJECTED INTO CELLS BUT CANNOT
ENTER BY DIFFUSION
HO2C-DYGIPDAHC-SS-CR7-CONH2
RACK-TRANSPORTER CONJUGATE
HO2C-DYGIPDAHC-SH
RACK PEPTIDE
(“DRUG”)
HS-CR7-CONH2
TRANSPORTER
+
•THE RACK PEPTIDE ITSELF
MUST BE MICROINJECTED
INTO CELLS
•TRANSPORTER RACK
CONJUGATE WORKS
WITHOUT INJECTION
RACKCS-SCTat
RACKCS-SCR7
50%
78%
NOTRMT
CREATINEPHOSPHOKINASE
RELEASE
DAMAGE
PROTECTION
BASIS FOR NEW
COMPANY, 2003
L. Chen, L. Wright, C-H. Chen, S. F. Oliver, P. A. Wender,* D. Mochly-Rosen*
CHEMISTRY & BIOLOGY, 2001, 1123
Cyclosporin A
HO
N
N
NO
N
O HN
O
NH
O N
HN
O
N
ON
HN
OO
O
O
O
__________________________________________________
-selective immunosuppressive agent
-prevent rejection of a transplanted
organs
- treats severe cases of psoriasis and
rheumatoid arthritis
Problems: High blood pressure; kidney problems
Bleeding, tender, or enlarged gums
Convulsions; fever or chills ; frequent urge to urinate; vomiting
http://www.youtube.com/watch?feature=player_detailpage&v=5fjynugHBXs#t=40
Cyclosporin A drug interaction __________________________________________________
• Amiloride (e.g., Midamor) or
• Spironolactone (e.g., Aldactone) or
• Triamterene (e.g., Dyrenium)—Since both cyclosporine and these medicines increase the amount of potassium in the body, potassium levels could become too high
• Allopurinol (e.g., Zyloprim) or
• Androgens (male hormones) or
• Bromocriptine (e.g., Parlodel) or
• Cimetidine (e.g., Tagamet) or
• Clarithromycin (e.g., Biaxin) or
• Danazol (e.g., Danocrine) or
• Diltiazem (e.g., Cardizem) or
• Erythromycins (medicine for infection) or
• Estrogens (female hormones) or
• Fluconazole (e.g., Diflucan) or
• Human immunodeficiency virus (HIV) protease inhibitors (e.g., Crixivan, Fortovase, Invirase, Norvir, Viracept) or
• Itraconazole (e.g., Sporanox) or
• Ketoconazole (e.g., Nizoral) or
• Nefazodone (e.g., Serzone) or
• Nicardipine (e.g., Cardene) or
• Verapamil (e.g., Calan, Covera-HS, Isoptin,
Verelan)
• Azathioprine (e.g., Imuran) or
• Chlorambucil (e.g., Leukeran) or
• Corticosteroids (cortisone-like medicine) or
• Cyclophosphamide (e.g., Cytoxan) or
• Mercaptopurine (e.g., Purinethol) or
• Muromonab-CD3 (monoclonal antibody)
Coal tar (e.g., Balnetar, Zetar) or
• Methoxsalen (e.g., Oxsoralen) or
• Radiation therapy or
• Trioxsalen (e.g., Trisoralen)—There may be
increased risk of some skin cancers
• Lovastatin (e.g., Mevacor)
• Simvastatin (e.g., Zocor)—
CsA - octaarginine conjugate
Biotin - biomolecule
Fluorescent - biomolecule
Fluorescent –
strepavidin
Transporter Enabled Drug Uptake in Human Skin Proc. Natl. Acad. Sci. USA , 2000, 97, 13003-13008; Nature Medicine 2000, 6, 1253-1257
HUMAN TRIALS ESTABLISHED SAFETY AND THERAPEUTIC LEVELS OF CONJUGATE:
T-L-CsA -> T-L + CsA
UPTAKE OF BIOTINYLATED CYCLOSPORIN (A) AND BIOTINYLATED CYCLOSPORIN TRANSPORTER CONJUGATE (C) IN
HUMAN SKIN GRAFTED ON IMMUNE DEFICIENT MICE. THE SKIN WAS EXCISED, FROZEN, SECTIONED AND STAINED WITH
FLUORESCEIN STREPTAVIDIN. PANELS B AND D ARE CONTROLS USING PROPIDIUM IODIDE TO VISUALIZE ALL CELLS.
HO
N
N
NO
N
O HN
O
NH
O N
HN
O
N
ON
HN
OO
O
O
O
CsA
New Barrier Problems: TISSUE UPTAKE Fluorescence Propidium Iodide Control
COLON
ORAL
LUNG
Intracellular Cargo Delivery by an
Octaarginine Transporter Adapted to
Target Cancer Cells through Cell
Surface Protease Activation
Why is it important?
• Targeted delivery systems are critical for refining effective molecular imaging approaches and enhancing therapeutic intervention
• Currently, there is no treatment that significantly prolongs survival in the setting of metastatic prostate cancer and the mortality rate from prostate cancer continues to increase.
• No single imaging strategy has been identified as ideal for prostate cancer at this time.
• Exploitation of cell-selective enzymes such as PSA in combination with CPP technology can open the door to unique mechanisms of delivery.
Proposed Mechanism for Selective Uptake in PSA-Expressing Prostate Cancer Cells
Library of synthesized peptides
#
Peptide
Sequence
MW, calcul. w/o count. ions
MW, found w/o count. ions
# Peptide Sequence MW, calcul. w/o count. ions
MW, found w/o count.
ions
1 r8-K-FITC C75H123N35O14S
1771.07 1770.11 11 CDGGDGGDGGD C36H48N14O21S2
(M+H) 1076.98 1076.69
2 r7-K-FITC C69H111N31O13S
1614.88 1615.87 12 CHSSKLQG-r8-K-FITC C111H180N48O26S2
(M+H) 2667.05 2667.10
3 r5-K-FITC C57H87N23O11S
1302.51 1302.01 13 DCCHSSKLQG-r8-K-FITC C118H191N51O30S3
(M+H) 2900.30 2893.29
4 r4-K-FITC C51H75N19O10S
1146.33 1145.29 14 DDCCHSSKLQG-r8-K-FITC C122H196N52O33S3
(M+H) 3015.39 3014.96
5 CD C12H15N5O6S2,
(M+H) 389.41 390.46 15 DGDCCHSSKLQG-r8-K-FITC C124H199N53O34S3
(M+H) 3072.44 3072.90
6 CDD C16H20N6O9S2,
(M+H) 504.49 505.26 16 DGGDCCHSSKLQG-r8-K-FITC C126H202N54O35S3
(M+H) 3129.49 3129.91
7 CDGD C18H23N7O10S2,
(M+H) 561.55 562.79 17 DGGGDCCHSSKLQG-r8-K-FITC C128H205N55O36S3
(M+H) 3186.54 3187.19
8 CDGGD C20H6N8O11S2,
(M+H) 618.60 620.03 18 DGGDGGDCCHSSKLQG-r8-K-FITC C134H213N57O40S3
(M+H) 3358.68 3359.76
9 CDGGGD C22H29N9O12S2,
(M+H) 675.65 675.90 19 DGGDGGDGGDCCHSSKLQG-r8-K-
FITC
C143H236N59O45S3
(M+H) 3597.96 3597.12
10 CDGGDGGD C28H37N11O16S2,
(M+H) 847.79 848.93 - - -
* FITC = fluorescein isothiocyanate; r8 = arginine octamer; all other letters correspond to standard single-letter abbreviations for
amino acids in the order from C to N terminal.
**3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine has been used for synthesis of peptides 5-11.
Goun, E.A. et. al. Bioconjugate Chem.(2006)17, 787 -796.
Comparison of cell uptake of ACPP with and
without prior PSA-mediated peptide cleavage
r8-K(FITC)
r4-K(FITC)
DGGDGGDGGDCCHSSKLQG-r8-
K(FITC)
DGGDGGDGGDCCHSSKLQG-r8-
K(FITC) + PSA enzyme
_________________________________________
PC3M cells were incubated with each conjugate solution for 15 min and washed twice.
The image was obtained using an IVIS 50 imaging system (Xenogen Corp.)
Bar representation of uptake of fluorescently
labeled PSA-targeting peptide with and
without precleavage by PSA
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
1.40E+09
1.60E+09
1.80E+09
r8 K
(FIT
C)
r4 K
(FIT
C)
pept. 19
pept.19 +
enzy
me
Ph
oto
n f
lux
PC3M prostate cancer cells
Conclusions
• The presented work demonstrates the feasibility of selective delivery of therapeutics or imaging agents to the intracellular compartments of cancer cells.
• This concept can be further adapted to other activation strategies (other locally restricted enzymes, light, or pH gradients).
http://www.youtube.com/watch?feature=player_detailpage&v=r0UU7tiMDbI
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