LabCourse/Praktikum

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Lab Course responsibilityDr. Sylvia Els-Heindl (sylvia.els@uni-leipzig.de)

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

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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.