sistemas multivalentes para nanomedicina multivalent

Sistemas Multivalentes para Nanomedicina

Multivalent Systems for Nanomedicine

VII Encuentro de Dendrímeros, EDEN-7

Málaga, 13-14 de Febrero 2020

Biocompatible multivalent polymers,

as polyglutamic acid (PGA):

Drug delivery systems for cancer therapy

Homo- and heterobivalent ligands:

Pharmacological tools to study GPCRs clusterization

Dendrimers and dendritic platforms:

Drug delivery systems for cancer therapy

Imaging therapeutic systems

Biomaterials for tissue regeneration

Multivalent chemical platforms

N N

NH

O

N

NH

O

H2N

N

O

N

NH

O

N

NH

O

NH

O

NH2

R2R2 R2

R1R1 R1

=R1 R2 =R1 R2

Multifunctional oligomers,

as g-peptides:

Cell-penetrating peptides

Transporters

Antialzheimer drugs

Multivalent self-assembled systems, as micelles or lipid nanovesicles:

Drug delivery systems for cancer and lysosomal diseases

DPTA/NTA-OEG dendrimers: versatile platforms for biomedicineapplications

Dendrimers

Oligoethylene glycol (OEG) moieties as branches

Develop a synthetic methodology that allows the

incorporation of branches of distinct natures: different

sized OEGs, aliphatic chains, exchange OEGs by

peptides or fluorophores,…)

Different surface groups can be incorporated (NH2, SH, COOH, N3, maleimide…)

Develop multimodal dendrimers with differentiated surface positions that can be generated

precisely in terms of type, number and distribution.

DTPA/NTA-like derivative as the core

(Diethylenetriaminepentaacetic acid Nitriloacetic acid

OEG based dendrimers are non-toxic, hemocompatible, non-immunogenic

DPTA/NTA-OEG dendrimers

Organic and Biomolecular Chemistry 2013, 11, 4109-4121.Macromolecules 2014, 47, 2585–259.

Amide network

Triazole network

Amidedendrons

Triazoledendrons

Cytotoxicity

Hemolysis

Blood cellmorphology

Number /sizedistribution of

cells

Complementsystem activation

Hemeostasiscontrol

[G1 & G2(NH2, NHAc)]

• Non cytotoxic (only G2-NH2 at ↑conc.)

• Hemocompatible

DPTA-OEG dendrimers

Multimodal DPTA-OEG dendrimer platforms

Organic Letters 2014, 16, 1318−1321.

• Simple and robust chemical reactions:

- Selective removal of protecting groups (compatible PG)

- Amide bond formation:

• Easy purification: Aqueous extractions (basic and/or acidic)

+

Precipitation protocols

Cyclic anhydride (desymmetrization)

PyBOP (or other acylating agents

Imaging therapeutic systems

DPTA-OEG dendrimer platform applications

Biofunctionalization of hydrogels for musculoskeletal tissue regeneration therapy

Acta Biomaterialia 2014, 10, 4340-4350.

Macromolecular Bioscience 2015, 15. 1035-1044.

Carbosilane-DPTA-OEG Janus dendrimers for HIV

gene therapy

European Journal of Medicinal Chemistry 2014, 31, 43-52

The size matters?

The effect of the branches lenght

Targeting peptide: CF-Cys-Ahx-AKRGARSTA-NH2 (LinTT1, ligand of p32 receptor)

Imaging agent: Cyanine 7.5

Targeted dendrons

Targeted imaging dendrons

PEG8-dendron

Targeted imaging dendrons: G8-dendron(LinTT1)4-Cy 7.5

Synthesis (purification by Amicon® centrifugal filter (MWCO 3000)) HPLC-MS characterization

PEG8-dendron(LinTT1)4-Cy 7.5

Targeted imaging dendrons: PEG27-dendron(LinTT1)4-Cy 7.5

PEG27-dendron(LinTT1)4-Cy 7.5

Synthesis (purification by Amicon® centrifugal filter (MWCO 3000)) HPLC-MS characterization

1 h 4 h

24 h

PEG8 PEG27 PEG8 PEG27

Fluorescence in vivo

Female balb/c mouse; orthotopic 4T1 tumor (TNBC)

IVIS imaging 1 h, 4 h, 24 h (cyanine 7.5 dye)

In both cases (PEG8 & PEG27) tumor

accumulation is detected at 4h and is increased

after 24 h

Biodistribution seems dependent of PEG size

(differences in circulation time??, less excretion of

PEG 27??, higher liver accumulation of PEG27??)

Targeted imaging dendrons: In vivo biodistribution

Fluorescence ex vivo

Targeted imaging dendrons: Ex vivo biodistribution

PEG8-LinTT1 PEG27-LinTT1 PEG8-LinTT1 PEG27-LinTT1

4 hours 24 hours

Brain

Liver

Lung

Tumor

Spleen

Kidney

Heart

Tumor homing in both PEG conjugates at 4 h and 24 h

In case of PEG8, tumor accumulation seems higher at

24 h

PEG27 accumulates more in liver in spleen than PEG8

Targeted imaging dendrons: Ex vivo biodistribution

It seems there is a better biodistribution and tumor accumulation profiles of the PEG8 derivative

Fluorescence ex vivo

15

Monomers One receptor (protomer)… one function

Modulation of receptor function

Biochemical / functional properties

different from monomers

Potential novel therapeutic targets

Homomers

(homodimer)

Higher-order

oligomers

Heteromers

(heterodimer)

Bivalent and multivalent ligands to study GPCR oligomerization

Selective chemical molecules to study GPCR oligomers

Bivalent ligand

Bivalent and multivalent ligands to study GPCR oligomerization

Bivalent ligands single chemical entities composed of two pharmacophore units covalently linked by an appropriate linker/spacer,

designed to interact simultaneously with the orthosteric sites of a (homo/hetero) GPCR dimer.

Linker/Spacer length is a key factor in these ligands

• Interfaces (TM) forming the dimer

• Structure of the pharmacophores

• Attachment point (pharmacophore-bivalent system)

Tipical design high number of compounds experimental optimization for “n”

Bitopic ligand Dual-acting ligand Bivalent ligand

n

D2R and A2AR bivalent ligands

Rational Design of Bivalent Ligand for the dopamine D2R

and A2AR receptor homo- and heterodimers

J. Med. Chem. 2018, 61, 9335-9346 Bioinformatics, 2018, 1–7

n

Heterobivalent ligand vs two different receptors

n

Heterobivalent ligand vs two equivalent receptors (agonist-antagonist)

Homobivalent ligand vs two equal receptors

n

Multivalent ligand vs four receptors

D2R and A2AR bivalent ligands design

Nitrilotriacetic acid derivative (NTA)

Pharmacophore unit: a ligand that binds the orthosteric binding site with high affinity (antagonist)

Spacer: appropriate length to cover the distance between both protomers

Different length oligoethylene glycol (OEG)

Scaffold: at least two chemical functionalities that can be properly derivatized

J. Med. Chem. 2018, 61, 9335-9346

D2R and A2AR bivalent ligand synthesis

Homobivalent ligands

Heterobivalent ligands

n

n

vs two different receptors

n

vs two equivalent receptors (agonist-antagonist)

Monovalent ligands

n

Pharmacophore + spacer

D2R-D2R homobivalent ligands

J. Med. Chem. 2018, 61, 9335-9346

35-atoms distance

25-atoms distance

Monovalent Homobivalent

Table 1. Affinity constants (KD) of the D2R ligands 7, 12-15

Compound KD (nM) + TM6 D2R + TM6 A2AR

7 0.70±0.06

12 0.07±0.03*###

13 0.021±0.003**###

1.1±0.3 ^^

0.05±0.01

14 1.5±0.6*

15 0.77±0.04 0.8±0.2 0.8±0.2

Values are mean ±SEM from 3-10 determinations. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s post hoc test.

*p<0.05,

**p<0.01 compared with 7.

###p<0.001 compared with the

corresponding monovalent ligand. ^^

p<0.01 compared with the respective control.

Bivalent (35-atoms)

Bivalent (25-atoms)

Monovalent (35-atoms)

Monovalent (25-atoms)

( Radioligand competition-binding assays )

Pharmacophore

Homobivalent ligand

Bivalent compounds 12 and 13 enhance significantly the binding

affinity relative to monovalent compounds 14 and 15 (21-fold and

38-fold respectively).

Compound 13 is the best ligand for D2R (3.5-fold higher affinity

relative to 12)

Monovalent ligand

12 or HomoBD2-35

13 or HomoBD2-25

14 or MD2-35

15 or MD2-25

D2R D2R

D2R D2R

D2R-D2R homobivalent ligands

J. Med. Chem. 2018, 61, 9335-9346

25-atoms distance


No TM peptide

TM6 of A2AR (negative control)

TM6 of D2R

Table 1. Affinity constants (KD) of the D2R ligands 7, 12-15

Compound KD (nM) + TM6 D2R + TM6 A2AR

7 0.70±0.06

12 0.07±0.03*###

13 0.021±0.003**###

1.1±0.3 ^^

0.05±0.01

14 1.5±0.6*

15 0.77±0.04 0.8±0.2 0.8±0.2

Values are mean ±SEM from 3-10 determinations. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s post hoc test.

*p<0.05,

**p<0.01 compared with 7.

###p<0.001 compared with the

corresponding monovalent ligand. ^^

p<0.01 compared with the respective control.

13 or HomoBD2-25 15 or MD2-25

Homobivalent ligand

Monovalent ligand

“Disturber” peptide: TM6 D2R

Monovalent 15 Homobivalent 13

D2R D2R

D2R D2R

When the D2R-D2R homomer is disturbethe affinity of compound13 decreases 50 times.

A2AR-A2AR homobivalent ligands

Homobivalent ligands two equal receptors: A2AR homomers

Homobivalent ligand

A2ARA2AR

Monovalent ligand

A2ARA2AR

25-atoms distance

HomoBA2A-35

HomoBA2A-25

MA2A-25

35-atoms distance


MA2A-35

Compound KD1 A2AR (nM) KD2 A2AR (nM)

Pharmacophore A2AR 7±1 400±100 MA2A-25 40±10 MA2A-35 170±30 HomoBA2A-25 1.8±0.4 100±30 HomoBA2A-35 1.05±0.05 290±20

Antagonist A2AR

Antagonist A2AR

A2AR-D2R heterobivalent ligands

Antagonist D2R Antagonist D2R

35-atoms distance 25-atoms distance

Antagonist A2AR Antagonist A2AR

n vs two different receptors: A2AR and D2R

Heterobivalent ligand

Monovalent ligand

HetB-35 HetB-25

Monovalent ligands MA2A-35 (A2AR) , MD2-35 (D2R) MA2A-25 (A2AR) , MD2-25(D2R)

Compound KD1 A2AR (nM) KD2 A2AR (nM) KD D2R (nM)

Pharmacophore A2AR 7±1 400±100 MA2A-25 40±10 MA2A-35 170±10 HetB-25 5±1 0.7±03 HetB-35 7±1 1.2±0.2 MD2-25 0.77±0.04 MD2-35 1.5±0.6 Pharmacophore D2R 0.7±0.6

D2R

D2R

A2AR

A2AR

A2AR-A2AR-D2R-D2R heterotetravalent ligands

A2A and D2 receptors form high-order oligomers composed by D2R-D2R and A2A-A2AR homomers

Tetravalent ligand vs four receptorsHeterobivalent ligands vs two different receptorsHomobivalent ligands two equal receptors

D2RD2R

A2ARA2AR


A2A and D2 receptors form high-order oligomers composed by D2R-D2R and A2A-A2AR homomers

Tetravalent ligand vs four receptorsHeterobivalent ligands vs two different receptorsHomobivalent ligands two equal receptors


Antagonist D2R

Antagonist A2AR

35-atoms distanceTet-35


Compound KD1 A2AR (nM) KD2 A2AR (nM) KD D2R (nM)

Pharmacophore A2AR 7±1 400±100 MA2A-25 40±10 MA2A-35 170±10 HomoBA2A-25 1.8±0.4 100±30 HomoBA2A-35 1.05±0.05 290±20 HetB-25 5±1 0.7±03 HetB-35 7±1 1.2±0.2 Tet-25 1.6±0.12 0.031±0.04 Tet-35 0.2±0.1 0.05±0.02 HomoBD2-25 0.021±0.003 HomoBD2-35 0.07±0.03 MD2-25 0.77±0.04 MD2-35 1.5±0.6 Pharmacophore D2R 0.7±0.6

Tetravalent ligand

D2RD2R

A2ARA2AR


Tetravalent ligandAntagonist A2AR


Antagonist D2R

“Disturber” peptide: TM5 A2ARaffects A2AR-D2R heteromer

“Disturber” peptide: TM6 A2ARaffects A2AR-A2AR homomer

D2RD2R

A2ARA2AR

D2RD2R

A2ARA2AR

D2RD2R

A2ARA2AR

“Disturber” peptide: TM2 A2ARdo not be located at the contactinterface between receptors

The A2AR affinity of Tet-35 only decreases substantially when the heteromer A2AR-D2R is disturbed.

A2AR affinity

Compound KD A2AR (nM) KDcompound/KDTet-35

Tet-35 0.36±0.05 1.0 Tet-35+TM5 A2AR 4±2 11 Tet-35+TM6 A2AR 0.24±0.06 0.7 Tet-35+TM2 A2AR 0.1 0.2


Tetravalent ligandAntagonist A2AR


Antagonist D2R“Disturber” peptide: TM5 D2Raffects A2AR-D2R heteromer

“Disturber” peptide: TM6 D2Raffects D2R-D2R homomer

D2RD2R

A2ARA2AR

D2RD2R

A2ARA2AR

D2RD2R

A2ARA2AR

“Disturber” peptide: TM2 D2Rdo not be located at the contactinterface between receptors

A2AR affinity

D2R affinity

The affinity of Tet-35 for D2R decreases substantially when A2AR-D2R heteromers (50 times) and D2R-D2R homomers (13 times) are disturbed. The tetravalent ligand (Tet-35) seems only interact with three centers (two D2R and one A2AR).

Compound KD A2AR (nM) KDcompound/KDTet-35

Tet-35 0.36±0.05 1.0 Tet-35+TM5 A2AR 4±2 11 Tet-35+TM6 A2AR 0.24±0.06 0.7 Tet-35+TM2 A2AR 0.1 0.2

Compound KD D2R (nM) KDcompound/KDTet-35

Tet-35 0.052±0.005 1.0 Tet-35+TM5 D2R 2.9±06 56 Tet-35+TM6 D2R 0.7±0.3 13 Tet-35+TM2 D2R 0.17 3.3

Multifunctional oligomers: g-peptides as cell-penetrating peptides and blood brain barrier transporters

N N

NH

O

N

NH

O

H2N

N

O

N

NH

O

N

NH

O

NH

O

NH2

R2R2 R2

R1R1 R1

=R1 R2 =R1 R2

g-peptides

HN

O

OH

H2N

(2S, 4S)-4-amino-pyrrolidin

carboxylic acid

Moderate cell-uptake properties (~40%)

Low cytoxicity in HELA and COS-1 cells

High stability to proteases

Medium solubility in water

NMR studies of some of these g-peptides oligomers showed indications that form a ribbon C9.

J. Am. Chem. Soc., 2005, 127,9459-9468J. Am. Chem. Soc., 2004, 126, 6048-6057

g-peptide oligomer library

N N

NH

O

N

NH

O

H2N

N

O

N

NH

O

N

NH

O

NH

O

NH2

R2 R2 R2R1

R1 R1

=

N

NH

O

Rn

N

NH

O

Rm O

N

NH

O

Rn

R1 R2

=R1 R2

O

H2N HN

NH

H2N

O

NH2

O

NH

HN

NH2

NH

NH

O O

N

NH

O

NH

N

O

O

H2N

O

HO

O

NH

HN

NH2

O O

O

NH2

O

NH2

O

NH

O

O

NH

HN

NH2

O

N

Aim: Improve solubility in water, cell-uptake propertiesand study their sub-cellular localization.Establish structure-activity relationship.

g-peptide oligomer library synthesis

A library of 53 g-peptides and their corresponding

carboxyfluoresceinated version was synthesized following an

orthogonal protecting scheme that combines Fmoc/Boc/Alloc

protecting groups.

g-peptide oligomer library synthesis

NH

HN

OAcylations

R-COOH /DIC / HOBt (5eq)

DMF N

NH

O

O

R

N

NH

OGuanidinylations

NH2

NHBocBocN

TfmsN

DIEA (10 eq) / DCM

(5eq)

N

NH

O

HNNH

H2N

1)

2) 40% TFA / DCM

Alkylations

NH

NH

O

1) R-CHO (5eq) DMF

2) AcOH (cat)3) NaBH3CN (5eq) / MeOH

N

NH

O

R

N

NH

O

R

Methylations

MeI (20eq)

DIEA (5eq)

N

NH

O

NH2

N

NH

O

R

N

NH

O

N

N

O

H2N

R

5(6)-Carboxyfluorescein (5eq)

PyAOP / HOAt / DIEA (5/5/10) N

O

CFHN

R

Fluorescent tag

106 peptides were synthesized. Purities of the g-peptide crudes were ranged from 65%

to 90% by HPLC at 220nm.

g-peptides were purified (≥90%).

g-peptide oligomer applications

-Low Toxicity -High cell-uptake efficiency

-Stable to proteases -Soluble in water

-Cell type selectivity. Higher uptake in biological barrier model

cells (ie: Caco-2)

-Preferential accumulation in lysosomes. Lysosomotropic.

Lysosome drivers on enzyme

replacement therapy (Fabry disease).

Cell-penetrating peptide conjugated

to dual imaging tool (fluorescence

and MRI)

New transporters for

antileishmania drugs

g-peptide oligomers as transporters trough the BBB

The blood brain barrier (BBB) is a highly selective

permeability barrier that separates the circulating

blood from the brain extracellular fluid in the central

nervous system (CNS).

One strategy to overcome the BBB is tagging drugs or

drug delivery systems with ligands of receptor mediated

transcellular transport or compounds with BBB crossing

abilities. Some of these ligands are peptides.

Drosophila melanogaster (Dm) has been described as an

complementary model of mammalian BBB due to homologies detected

between Dm and mammalian components:

-Homologies between proteins (ie: claudins) located at the Dm septate

junction and mammalian tight junction (required for paracellular barrier

function).

-Dm also possesses a full array of xenobiotic transporters from both,

ATP binding cassette (ABC) and solute carrier families that participate

in active drug flux between biological compartments.

All these findings strongly indicate that there is solid evolutionaryconservatism between these two barrier systems.

Drosophila melanogaster as animal model for BBB crossing experiments

1) Peptide solution injection in Drosophila melanogaster abdomen. Two Dm strains used:

w-iso (BBB functional) restricted entry

moody null (BBB disfunctional) not restricted entry

2) Retina observation after a determined post-injection time (2h). Epifluorescence experiments .

3) Confocal laser scanning microscopy experiments

Peptide ligand controls

RGV peptide -YTIWMPENPRPGTPCDIFTNSRGKRASNG-NH2

Transferrin receptor peptide binders:

pTf-1 -HAIYPRH-NH2

pTf-2 -THRPPMWSPVWP-NH2

Angiopep-2 -TFFYGGSRGKRNNFKTEEY-NH2

Angiopep-7 -TFFYGGSRGRRNNFRTEEY-NH2

Tat -RRRQRRKKRG-NH2

Penetratin (pAntp) -RQIKIWFQNRRMKWKK-NH2

CF

CF

CF

CF

CF

CF

CF

26

20

34

76

90

Peptides selected to be evaluated their BBB crossing abilities

g-peptides selected

C

moodynull

w-isomoody null

w-iso

CF CF-pTf-2 9088

CF

CF CF-pTf-2 88 90

CFA.

B.

Epifluorescence experiments

g-peptide90

TR-dextran

moodynull

w-iso

Exclusion

line

Exclusion

line

Merged

Retinas of moody null y w-iso flies inoculated with peptide 90 and TR-dextran 10000 kDa.

Confocal laser scanning microscopy experiments

Cornea

Pseudocon

Pigment cells

Rabdomers

R1-R7

A. B.

C.

Peptide 90 and TR-dextran 10000 kDa co-injected in moody

null fly.

Fluorescence intensity quantification in a fixed volume.

Fly retina 3D reconstruction

moodynull

w-iso

moodynull

w-iso

CF CF-RGV CF-pTf-1 CF-pTf-2 CF-Angiopep-2 CF-Angiopep-7

CF-pAntp CF-Tat

Cornea

Pseudocon

Pigment cells

Rabdomers

R1-R7

Confocal images of control peptides

moodynull

w-iso

34 was evaluated in a Human BBB Cellular Model (a monolayer (in a

transwell) of human endothelial cells derived from pluripotent stem

cells co-cultured with bovine pericytes).

76( CF-Gp-9) 88( CF-Gp-10) 90( CF-Gp-11)CF

g-peptides images and BBB crossing capacity estimation

g-peptides images and BBB crossing capacity estimation

A. Lamina

B. Medulla

Barrier

moody-Gal4; UAS-cd8:GFP

Atto 565-90

A

B

moody-Gal4; UAS-cd8:GFP strain genetically modified with Moddy protein GFP labeled. Moddy protein is a

specific protein of the barrier.

A. Lamina B. Medulla. Axial projections

Atto 565-90 peptide (red) Fluorescence label emitted in red.

Acknowledgements

Dr. Daniel Pulido

Prof. Fernando Albericio

Dr. Ginevra Berardi

Dr. Marta Melgarejo

Dr. Daniel Carbajo

Dr. Josep Farrera-Sinfreu

Dr. Lorena Simón

Dr. Peter Fransen

Dr. Ximena Pulido

José Juan Jara

Julia Gutiérrez

Francesca di Angelis

Raffaella Giordano

Dr. Vicent Casadó

Dr. Estefania Moreno

Dr. Antoni Cortés

Dr. Verònica Casadó

FUNDING

Dr. Yolanda Fernández

Dr. Vanessa Díaz-Riscos

Dr. Ibane Abasolo

Dr. Simó Schwartz Jr.

SAF2011-30508-C02-01, SAF2014-60138-R and RTI2018-093831-B-I00

Dr. Elena Rebollo

Dr. Leonardo Pardo

Dr. Arnau Cordomí

Dr. Laura López

Dr. Laura Pérez-Benito

Dr. Sergi Ferré

Dr. Luís Javier Cruz Ricondo

Marieke Stammes

Chantal Sevrin

Prof. Christian Grandfils

Prof. Rafael Gómez

Prof. F. Javier de la Mata

Dr. Javier Sánchez-Nieves

Dr. Raquel Lorente

Dr. Ryan J. Seelbach

Dr. David Eglin

Dr. Mauro Allini

Dr. Luis Rivas

Prof. Tambet Tesaalu

Dr. Lorena Simón

Dr. Alvaro Mata

sistemas multivalentes para nanomedicina multivalent

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