Andrea Calderan - UOS di Padova

Receptor targeting with peptides for tumor imaging and therapy

Use of peptide receptor ligands for targeting to cancer cells

Selective detecting and killingtumour cells while sparing normaltissues have been the goal ofresearch in Oncology even since PaulEhrlich’s theory of the “magic bullet”.

The discovery that tumoursoverexpress peptide receptors ontheir surfaces has spurred thedevelopment of peptide analoguesdesigned to target tumours cells.

Paul Ehrlich(Nobel Prize 1908)

Development of new strategies relying on themultivalent or multireceptor targeting concepts

To improve the selectivity and the affinity of ligand peptides to cancercells multivalent and/or multireceptor approaches have been developed.These are characterised by the presence of ligands that simultaneouslybind to either multiple homo (multivalent) or hetero (multireceptor)receptors present on the surface of the same tumour cell.

Tumour-directed constructs radiopharmaceuticalsfor

Peptides … why?

optical imaging

drugs

In contrast to macromolecules, such as proteins,peptides have the advantage of being:

flexible messenger molecules

with high affinity for their receptors site (IC50 = <10 µM)

low immunogenicity

fast blood clearance

rapid tissues and tumour penetration which results inadequate target-to-not target ratios.

Bolzati C. et al. Current Medicinal Chemistry, 2010 Vol. 17, No. 24

Peptide Receptors Overexpressed in Tumours

MTC: medullary thyroid cancerSCLC: small-cell lung cancer

screen combinatorial peptidesor chemical libraries

Design of targeting peptides

to avoid short half-life inplasma due to rapiddegradation by endogenouspeptidases or proteases

N- and C-terminallyblocked peptides

spacer

to preclude interferencebetween these twoportions of the molecule

repeated sequence of β-Ala or 6-aminohexanoic acid or lowmolecular weight PEG molecules

to increase the affinitytowards receptorsexpressed on tumour cells

modify known ligandsagainst cancer-associatedreceptors

apply molecular modelling ifthe structure of the targetreceptor or related moleculeare known

peptides containingeither unnatural or D-amino acid residues

replacement of peptidebonds by amide bondsurrogates

cyclic peptides

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Radiolabeled peptides useful for the diagnosisand/or therapy of tumors, such as melanomas, that:

- overexpress MC1R (melanocyte-stimulating hormone receptor)

- overexpress integrin αVβ3 Arg-Gly-Asp

His-phe-Arg-Trp

Crystal structure of the extracellular segment of integrinαVβ3 in complex with an Arg-Gly-Asp (RGD) ligand.PDB structure 1L5G (J.P. Xiong et al. Science 296, 151-155, 2002).

NAP-NS1 H-Cys-Ahx-βAla-Nle-Asp-His-phe-Arg-Trp-Gly-NH2

NAP-NS1(-Cys) H-Ahx-βAla-Nle-Asp-His-phe-Arg-Trp-Gly-NH2

NAP-NS2 H-Cys-Ahx-βAla-c(Lys-Glu-His-phe-Arg-Trp-Glu)-Arg-Pro-Val-NH2

NAP-Cys3 H-Cys-Ahx-βAla-Cys-Cys-Glu-His-phe-Arg-Trp-Cys-Arg-Pro-Val-NH2

RGDfK-NS c[Arg-Gly-Asp-phe-Lys(Cys)]

Selective binding of NAP-NS1/2 to B16 murine melanoma cells was achieved

Peptides have been tested for affinitytowards MC1R receptors overexpressedon B16 murine melanoma cells.

SYNTHESIZED PEPTIDE SEQUENCES

-CH3

aminodiphosphine

ε β

NAP-NS1

RGDfK-NS

R = -CH3 or -CH2OH

R' = -CH3

Complexes evidenced a goodstability in rat and human seraas well as in rat liver and ratkidney homogenates.

Rat biodistribution studies wereperformed to investigate theorgan uptake and excretionpathways.

Complexes present an extremelyfast elimination from the bloodand from significant organs; therenal excretion was extremelyrapid and the activity wasmainly eliminated through theurinary tract.

PNP43: aminodiphosphinewith R = R' = -CH3

NOTA NAP-NS1

stable for 24 h

Cathepsin B is a cysteine proteasethat in tumor tissues is localized inboth acidic lysosomes andextracellular spaces.

It can catalyze the cleavage ofpeptide bonds by two mechanisms: endoproteolytic attack with a pH

optimum around 7.4; and exoproteolytic attack from

the C-terminus with a pHoptimum at 4.5-5.5.

Cathepsin B substrates containing the Arg-Arg sequence, a specific hallmark of this enzyme

The “targeting domain” is represented by anRGD-containing cyclic peptide, able to bindselectively to αVβ3 integrin, which is known tobe highly over-expressed by both metastatichuman melanoma cells, and endothelial cellsof tumor vessels.

The “linker” is a short peptide which isspecifically cleaved by cathepsin B, a proteasehighly up-regulated in malignant tumors.

Design and the synthesis of a tripartate paclitaxel(PTX)-peptideprodrug potentially useful in the treatment of human melanoma

NH

O

NHNH2

N

H

O

NH

OH

NH

O

O

O

NH

O

NH

NHN

H

N

H

N

H

N

H

N

H

N

H

NH

O

O

O

NH

O

NH

O

O

O

NHNH2

NH2NH

O

O

O NH O

OH

OH

OO

O

O

O

O

OO

O

A possible origin for the appearance of chemotherapeutic resistant tumor cells that nolonger respond appropriately to antineoplastic agents may be an increase in theexpression of total GST (glutathione transferase) activity in tumor cells.

Human GSTP1-1.In black the Tyr residue proposed as involvedin the activation of glutathione (GSH).

On the other hand, the overexpression of some GST enzymes by severaltumors makes them a promising target for the prodrug therapy of cancer.

GST-activated prodrugs are latent cytotoxic GST ligands, whichundergo GST-catalyzed and GSH-dependent or -independentbreakdown to release a cytotoxic species responsible for theiranticancer effect.

The tyrosinase activated melanomaprodrugs (4-methoxyphenol and N-acetyl-4-S-cysteaminylphenol) werelinked to GSH by an ethoxycarbonylmoiety, and the Cys sulfhydryl groupwas oxidized to sulfone to givecompounds 2 and 3.

In this way, the Tyr phenoxidepresent in the G-site of GST wouldbe able to abstract one of the acidicmethylene protons linked to thesulfone moiety, releasing thetyrosinase-activated melanomaprodrugs.

ethoxy

ethoxycarbonyl

STABLE

DEGRADED BYCYTOSOLIC GSTs

PRODRUGsRELEASE !

GSHMelanoma cellsare characterizedby high levels ofGSTP1-1

2011 - 2014 “Coniugati peptidici di farmaci e diagnostici per i tumori”Progetto di Ricerca PM.P01.021.002 nell’ambito della CommessaPM.P01.021 - Peptidi e peptidomimetici per la salute.

2012 “Synthesis and characterization of novel bioconjugates for activetargeting of potent antiproliferative phenylpyrroloquinolinones”Progetto di Ateneo CPDA113783, Università degli Studi di Padova.

2009 - 2011 PRIN 2008 nell’ambito del Progetto “Farmaci e diagnostici per iltargeting e il delivery selettivi in patologie tumorali” Unità di Ricerca CNR su“Targeting recettoriale con radio-peptidi per la diagnosi e la terapiatumorali”.

2006 - 2009 “Progettazione, sintesi e valutazione biologica di coniugatipeptidici del paclitaxel per la terapia del melanoma”Progetto di Ateneo CPDA064182, Università degli Studi di Padova.

Barbara BiondiAndrea CalderanPaolo RuzzaAnna MarchianiNicola AntoliniAlessio OslerAndrea GuiottoGianfranco Borin

Cristina BolzatiFiorenzo Refosco

Istituto di Chimica Biomolecolare del CNR

Università degli Studi di Padova

Antonio RosatoCarlo R. RossiMaura FloreaniLuigi QuintieriDavide CartaNicola SalvareseNicolò Morellato

Istituto per l'Energetica e le Interfasi del CNRex Istituto di Chimica Inorganica e delle Superfici

Institute of Radiopharmaceutical Cancer ResearchHelmholtz-Zentrum Dresden-Rossendorf, Germany

Christian JentschelStefan ThiemeRalf BergmannHans-Jurgen Pietzsch