Post on 10-Dec-2021
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
Monovalent Homobivalent
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
Monovalent Homobivalent
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
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
A2AR-A2AR-D2R-D2R heterotetravalent ligands
Antagonist D2R
Antagonist A2AR
35-atoms distanceTet-35
35-atoms distanceTet-25
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
A2AR-A2AR-D2R-D2R heterotetravalent ligands
Tetravalent ligandAntagonist A2AR
35-atoms distanceTet-35
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
A2AR-A2AR-D2R-D2R heterotetravalent ligands
Tetravalent ligandAntagonist A2AR
35-atoms distanceTet-35
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