LabCourse/Praktikum
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Lab Course responsibilityDr. Sylvia Els-Heindl ([email protected])
Introduction to LabCourse: 18.11.2018, before the lecture, small lecture hall8.00-8.30 h Brü34
Prior to practical work, all participants have to demonstrate basic knowledge on solid phase peptide synthesis!
Lab Course: M.Sc. Biochemistry, Biology (part of module 0701)Block I: 3.12.2019 - 20.12.2019, 10:15-17:00, Saal6Br Labor, Block II: 06.01. - 24.01.2020, 10:15-17:00, Saal6Br Labor
Lab Course Module/Vertiefungspraktikum: M. Sc. Chem/Int. Master (10 ECTS)Module 121-111-11163 weeks in blocks I or II with biochemists/biologists for introduction, 3 weeks individually with supervising PhD studentgraded protocol
*Bachelor thesis in our lab in peptide synthesis will get different/individual topic
BioOrg5-2
PEPTIDE-SHUTTLE SYSTEMS
C1 C2 C3
NH3+
-OOC
E1 E2 E3
GDP
Biogene Aminesadrenaline, dopamine,histamine, 5-HT
Amino Acidsglutamate
Lipidsprostaglandinsthromboxanescannabinoids
Peptides and Proteinsangiotensin, NPFF, NPY, bradykinin, endothelin, chemokines, ghrelin
Nucleosides and Nucleotidesadenosine ATP, ADP, UTP
ion channels,PI3KPLC-adenylyl cyclases
adenylyl cyclases,inhibition of
cAMP production,ion channels,
phosphodiesterases,phospholipases
PLC-DAG, Ca2+,
PKC
iGTP
qGTP
sGTP
adenylyl cyclases,increased cAMP
concentration
TM
RhoGEFs,Rho
12GTP
EX
IN
HETEROTRIMERIC G PROTEIN PATHWAY
Venkatakrishnan et al. Nature (2013) 494, 187
INTERNALIZATION OF GPC-RECEPTORS
nucleus lysosome
early endosome
recycling endosome
Rab5EEA1
Rab5EEA1
Rab4
Rab7
Rab11
Rab11
G protein-mediatedsignaling
GRK
P P
βarr
βarrP P
βarrP P
E3
UbUb
Ub
P P
P PG protein
agonist
GPCR
Ph
βarrP P
late endosome
AP2 clathrin
α βα β
TYPE OF GPCR DRUGS
Agonists AntagonistsInverse agonists
Allostericmodulators
full biased
G-Protein arrestin
partial
AntagonistsInverse agonists
Agonists
TYPE OF GPCR BASED SHUTTLESYSTEMS
Allostericmodulators
full biased
G-Protein arrestin
partial
( g )
For targeting a drug to certain cell type but no intracellular action is required
(Diagnostics)
Agonists AntagonistsInverse agonists
Allostericmodulators
full biased
G-Protein arrestin
partial
( g )
For targeting a drug to certain cell type but no intracellular action is required
(Diagnostics)
For targeting a drug to certain cell type and intracellular action of a specific drug is
required(Therapeutic, Theranostic)
TYPE OF GPCR BASED SHUTTLESYSTEMS
Peptide
99mTc, 67/68Ga111In, 18F
OOCH3
OHNH2
O OH
OCH3 O OH
OHCH2R
O
receptor
Diagnosis Therapy
visualization treatment
CONCEPT
I. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.
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PEPTIDE RECEPTOR RADIATION THERAPY (PRRT)
Molecular biologic criteria
cancer expresses the corresponding peptide receptor in the primary tumor and in metastases
high receptor density
high binding affinity
internalization
Clinical criteria
Patients with cancer and multiple inoperable metastases
Palliative therapies (chemotherapy) with little
or no success
Maximum Intensity Projection of a 18F-FDG
wholebody PET acquisition (wikipedia)
FDG: fluoro deoxy glucosePET: positron emission tomography
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PEPTIDE RECEPTOR RADIATION THERAPY (PRRT)
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PEPTIDES AS TUMOR MARKERS
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RADIO-ISOTOPES
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RADIO-ISOTOPES
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CHELATORS
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CHELATORS
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LABELLING
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SOMATOSTATIN-DERIVATIVES
H OHHN
O NHO S
NH
O NH
O
NH
NH
O
NH2
HN
O
OHHNH
OS
HNOHH
OH
NH
O NHO S
NH
O NH
O
NH
NH
O
NH2
HN
O
OHH
NH
O
SHN
HO
H
HO
N
N
N
N
O
OOH
O
HO
OHO
Somatostatin-14 Octreotide DOTATOC
BioOrg5-18BioOrg5-18
BOMBESIN-DERIVATIVES
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NEUROPEPTIDE Y FAMILY
Constant positionsamong all species
NPY PPPYY
22 15 7
NPY
PP
PYY
5 12 27 353398
Y P S KP DNP GE DAP AE DMARY Y S AL RHY I NL I T RQRY
Y P I KP E AP GE DAS P E E L NRY Y AS L RHY L NL V T RQRY
AP L E P V Y P GDNAT P E QMAQY AADL RRY I NML T RP RY
BioOrg5-20BioOrg2- 20
NEUROPEPTIDE Y AND TUMOR ...
total binding of 125I PYY
tumor normal breast tissue
PYY3-36
[L,P] NPY
total binding of 125I PYY 3-36
total binding of 125I [L,P] NPY
Reubi et al., Cancer Res. 2001,61, 4636
Y2 Y1 change during neoplastic transformationn=95, 85 % primary tumor, 100% lymph node metastasis
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COEXPRESSION OF PEPTIDE RECEPTORS
Reubi et al., Eur. J. Nucl. Med. 2002, 29, 855
three different typesof breast cancer
Y1-receptors:high and homogenouslyexpressed
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BINDING OF NPY AT MCF7 CELLS
22
Y1 receptorsIC50: 1.3nM
Y 1-re
cept
or
GAP
DH
mRNA
-12 -10 -8 -6 -4
0
50
100
150 NPY[Phe7,Pro34]NPY[Ala31,Aib32]NPY
log cPeptide [M]
% B
indi
ng
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PRINCIPLES IN LABELLING
23
[Phe7,Pro34] NPY
N
N
N
COOH
N
N
N
N
CO2H
CO2H
HO2C
HO2C
111In
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PRINCIPLES IN LABELLING
[Phe7,Pro34] NPY
X
O
COOC
NNNH
HO
H
CH2-CO-
CO
NH-NPY
His(ac)X=Re, 99mTc
no uptake in breast ofhealthy volunteer
99mTc(core)3+-(NHis-ac)-[Phe7, Pro34]NPYI. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.
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99MTC LABELLED NPY PEPTIDE
99mTc-MDP99mTc(core)3+-(NHis-ac)-[Phe7, Pro34]NPY
MDP: MethylendiphosphonateI. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.
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COMPARISON OF NPY-PEPTIDE AND MDP-SCAN
I. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.
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LABELING WITH 18F FOR PET
+Cu+
peptide peptide< 30 min
4427.2
2214.2
0
1000
2000
3000
4000
Inte
nsity
[AU
]
1000 1500 2000 2500 3000 3500 4000 4500 5000m/z
MWcalc. Mono 4219.14 (educt) MWcalc. mono: 4426.15 (product)
-11 -10 -9 -8 -7 -6 -5
0
5000
10000
15000
20000
25000 [Pra4(F-Glc),F7,P34]-NPY
[F7,P34]-NPY
log cPeptide [M]
3 H-In
osito
lpho
spha
te [d
pm]
[Pra4(F-Glc),F7,P34]-NPY
hY1 receptor activation potency maintained!
F
Pra: propargyl-alanine, Glc: Glucose Hofmann S. et al. Mol Pharm. 2015 Apr 6;12(4):1121‐30.
BioOrg5-28Hofmann S. et al. Mol Pharm. 2015 Apr 6;12(4):1121‐30.
[18F]-GLC-FP-NPY IN MCF7 TUMORXENOCRAFTS
[Pra4(18F-Glc), F7,P34]NPY (3b)
[Pra4, F7,P34]NPY (3a)
Pra: propargyl-alanine, Glc: Glucose
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[18F]-GLC-FP-NPY IN MCF7 TUMORXENOCRAFT MICE: PET
3b: [Pra4(F-Glc), F7,P34]NPY and non binding 4b: [Pra4(F-Glc), F7,A33,P34Ala35]NPY a
Hofmann S. et al. Mol Pharm. 2015 Apr 6;12(4):1121‐30.
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Peptide
receptor
Diagnosis Therapy
99mTc, 67/68Ga111In, 18F
visualization treatment
188Re,10B
THERAPEUTIC APPROACHES
INTERNALIZATION OF NEUROPEPTIDE Y RECEPTORS
Y2-EYFP Y5-EYFPY1-EYFP Y4-EYFP
+NPY/PP 10 m
in30
min
60 m
in
AP2 - adaptor protein complex 2; βarr - beta arrestin; EEA1 - early endosome antigen 1; Erk1/2 - extracellular regulated kinase; GRK - GPCR kinase;
INTERNALIZATION OF GPC-RECEPTORS
recycling endosome
Rab4
Rab11
Rab11
G protein-mediatedsignaling
G protein
agonist
GPCR
lysosome
early endosome
Rab5EEA1
Rab5EEA1
Rab7βarrP P
E3
UbUb
Ub
P P
Ph
βarrP P
late endosome
AP2 clathrin
GRK
P P
βarr
βarrP P
P Pα βα β
5 minarrestin 3hY2mergearrestin 3hY2merge
unstim
LIGAND INDUCED YR INTERNALIZATION WITH TAMRA-NPY
hY1R: HEK293_ hY1_EYFP; hY2R:BHK_ hY2_EGFP; Bar: 10 µm; TAMRA: 5(6)-Carboxytetramethyl rhodamine
No stimulation NPY TAMRA-NPY merge TAMRA-NPY Receptor TAMRA-random NPY
hY1R
hY2R
TAMRA-NPY
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CONCEPT OF BNCT(BORON NEUTRON CAPTURE THERAPY)
Tjarks, W et al. (2007), Chem Commun (Camb)
• Carbaboranes: 10 B-atoms per molecule, 80 % 11B, 20 % 10B
• binary therapy• low cytotoxicity of thermal neutrons• low cytotoxicity of carbaboranes
• 4He2+: α particle• short range of absorption• highly ionizing
Collaboration withEva Hey-Hawkins, Leipzig University
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WHY BORON?
Boron tends to form polyhedral compounds (clusters) High boron content Facile integration into biological structures L-BPA and BSH have successfully been used in clinical
trials (skin, brain tumors and deep-seated tumors)
Sodium mercaptoundeca-hydro-closo-dodecaborate (BSH)
L-Dihydroxyboryl-phenylalanine (BPA)
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INCREASED WATERSOLUBILITY
X-RF-7
X-RF-13
X-RF-10
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TARGETED TUMOR THERAPY
[F7,P34]‐NPY as hY1R‐preferring peptide
NPY: neuropeptide Y, hY1R: human Y1 receptor; Söll et al., Eur. J. Biochem. 2001, 268, 2828‐2837. Böhme et al., J. Pept. Sci. 2015, 3, 186‐200.
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FLUORESCENT [F7,P34]-NPY ANALOGUES
amide ester disulfide enzymatic
100 nM peptide, 1 h stimulation, HEK293_hY1R_EYFP cells
D. Böhme et al., ChemMedChem 2015, May;10(5):804‐14.
0 1 2 3 4 5 60
25
50
75
100
amide ester disulfide enzymatic
time [h]
rela
tive
fluor
esce
nce
inte
nsity
[%]
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stability assay in human blood plasma
STABILITY OF FLUORESCENT [F7,P34]-NPY ANALOGUES
NPY: neuropeptide Y, hY1R: human Y1 receptor; D. Böhme et al., ChemMedChem 2015, May;10(5):804‐14.
0 4 8 12 16 20 240
20
40
60
80
100
amide ester disulfide enzymatic
time [h]
inta
ct p
eptid
e [%
]
peptide t1/2 [h]
amide 31 ± 4
ester 3 ± 0.2
disulfide 0.7 ± 0.2
enzymatic 57 ± 3
[K4, F7, P34]-NPY
OHN
GFLGHN
O
enzymatic linker ‐ extracellularly stable but intracellularly cleavable
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[K4, F7, P34]-NPY
OHN
GFLGHN
O
[K4, F7, P34]-NPY
OHN
O
O
TOXIC [F7,P34]-NPY ANALOGUES
amide ester disulfide disulfide/ester enzymatic
= MTX
fluorophore toxophore
NPY: neuropeptide Y, MTX: methotrexate; D. Böhme et al., ChemMedChem 2015, May;10(5):804‐14.
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BINDING AND SIGNAL TRANSDUCTIONPROPERTIES
Y1
Y1
Y2
Y2
peptides show internalization and signal transduction properties like [F7,P34]-NPY
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TOXIC [F7,P34]-NPY ANALOGUES
NPY: neuropeptide Y, MTX: methotrexate, hY1R: human Y1 receptor; D. Böhme et al., ChemMedChem 2015, May;10(5):804‐14.
- toxicity assay with hY1R-expressing MDA-MB-468:
MTX
amide
ester
disulfide
enzymatic
disulfide/ester
501051 5025101
c [µM]
survivingcells
[%]
0
20
40
60
80
100
120
5025101 5025101 5025101 5025101
ester, disulfide/ester, enzymatic more toxic than amide linker; hY1R‐mediated toxicity
enzymatic also extracellularly stable used for further developments
YPSK-[F7, P34]-NPY (5-36)
-12 -11 -10 -9 -8 -7 -6 -5 -4
-25
0
25
50
75
100
125
NPY
OC500
log cpeptide [M]
IP a
ccum
ulat
ion
[% o
f ful
l res
pons
e at
Y2R
]
OC500
hY1R hY2R
hY2R
-12 -11 -10 -9 -8 -7 -6 -5 -4
-25
0
25
50
75
100
125
OC500NPY
log cpeptide [M]
IP a
ccum
ulat
ion
[% o
f ful
l res
pons
e at
Y1R
]
THERAPEUTIC APPROACHES: TUBULYSINE
4427.92630.0[M+2H]2+
0
500
1000
1500
2000
2500
1000 2000 3000 4000 5000 6000m/z
Time [min]0 10 20 30 40 50Ab
sorp
tion
220
nm[m
AU]
0
200
400
600
800
5258.5[M+H]+
hY1R
OC500
TUBULYSINE- CONJUGATE: STABILITY IN BLOOD
Ahrens VM, Kostelnik KB, et al. J. Control Release, 2015 Jul 10;209:170‐8.
HEK293_hY1R_EYFP HEK293_hY2R_EYFP Cell viability after 72 h 24 h after initial[K(Cyto)4,F7,P34] NPY treatment
[K(C
yto)
4,F7
,P34
] NPY
Cyt
oTUBULYSINE –CONJUGATE: TOXICITY
Cationic Peptides
• cell penetrating peptides
• unspecific, non-receptor-mediateduptake in cell
• drug shuttles of the future
47
proteins
aptamers
siRNA / asRNA
vector-DNAchemotherapeutics
Plasmamembrane: not penetratable for polar molecules
48microinjection
particle bombardment
lipofection
electroporation
carrier peptides
Possible Applications
49
Carrier-Peptides
differ in size
amphipathic: weakly cationic, hydrophobic
Mechanisms: Endocytosis ordirect uptake
Penetratin, HIV-Tat, Oligoarginines
carrier peptides
50
various uptakemechanisms supposed
• various uptake characteristica• no common structure motifs internalization• cell line dependent
Penetratin RQIKIWFQNRRMKWKK-NH2
part of Antennapedia homeodomain from Drosophila melanogaster
Transportan GWTLNSAGYLLGKINLKALAALAKKIL-NH2
chimera of neuropeptide galanin and wasp venom peptide mastoparan
HIV-Tat peptides LGISYGRKKRRQRRRPPQ-NH2
part of Tat transcription factor of the HI virus
advantages: - easy application on living cells- less cytotoxic than other transfer methods- no restriction to a certain number of cells
... peptides, capable to translocate across cell membranes withoutfurther transporter or receptor molecules
51
52
chlorpromazine
Transferrin
amiloride (EIPA) nystatin methyl--cyclodextrin (MBC)
Cholera toxin B (CTXB)
Pagano et al. 2007, Nature Rev. 8, 603-612
Mechanisms
53Stewart, K. et al. Org. Biomol. Chem. 2008, 6, 2242.