PEPTIDI CON POTENZIALITA’ IN MEDICINA E NANOTECNOLOGIE

Marco Crisma

ICB-CNR, U.O.S. di Padova

PEPTIDES FOR DRUG TARGETING AND DELIVERY

PEPTIDE INHIBITORS OF ENZYMATIC ACTIVITY

Peptide receptors ligands

Cell penetrating peptides

Antamanide and analogs

FluoroPro and/or Hyp containing peptides

Aggregation inhibitors

Diagnosis / Therapy

Tyrosinase inhibitors / Cyclophilin inhibitors

SH3 (PTKs)

PEPTIDE – PROTEIN INTERACTIONS

α-synuclein

Drug delivery

PEPTIDES FROM Cα-TETRASUBSTITUED α-AMINO ACIDS

Conformational properties

Templates and spacers

Nanomaterials

Peptide antibiotics

Pharmaceuticals, 3, 1045 (2010)

Cell-Penetrating Peptides (CPPs): A Comparative Study on Lipid Affinity and Cargo Delivery Properties

The binding properties of six different CPPs were studied in absence or presence of the same cargo peptide (HS1pY, recognized by SH2 domain of PTKs). The phospholipid binding properties were correlated to the conformational and chemical characteristics of peptides, as well as to the cell penetrating properties of the CPP-cargo conjugates.

Confocal microscopy analysis of the HS1pY uptake on CHO cells incubated at 4 °C (A) or 37 °C (B, C and D) for 30 min with 12.5 μM concentration of either the cargo alone (A and C) or its conjugated to Tat (peptide 3a) (B) and to kFGF (peptide 1a) (D). While kFGF places the cargo on the cell surface (D), Tat is able to deliver it inside the cells (B).

PLoS ONE, 6, e16280 (2011)

The effect of AA on PTP opening was evaluated by means of Ca2+

retention capacity (CRC) assays on isolated mouse liver mitochondria and compared with that of PTP inhibitors CsA or Ubiquinone 0.

Antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D

Antamanide (AA) is a cyclic decapeptide derived from the fungus Amanita phalloides

(in collaboration with Dpt. of Biomedical Sciences, Univ. of Padova)

O

NH2

OH

OH

O

NH2

OH

OH

OH

O

NH2

OH

O

O

NH

COOH

OH

OHN

COOH

O

OH

Tyr DOPA DOPAquinone

leucoDOPAchromeDOPAchrome

tyrosinasetyrosinase

Antamanide analogs as potential inhibitors of tyrosinase

UV-Vis spectra of L-Tyr (2 mM) in phosphate buffer 50 mM, pH 6.8, alone or in presence of tyrosinase (1000 U/ml) at different time.

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

Abs

Wavelength (nm)

before mixing after mixing (1 min) after mixing (5 min) after mixing (9 min) after mixing (13 min) after mixing (17 min)

Products of tyrosine oxidation by the tyrosinase/O2 oxidizing system

0 5 10 15 20

0.0

0.3

0.6

Abs

@ 4

80 n

mTime (min)

Tyr alone Tyr + AG6 Tyr + AG9 Tyr + AOG9 Tyr + DHB

Time-course of tyrosine oxidation by the tyrosinase/O2 oxidizing system in presence of peptide inhibitors (0.2 µM). The AG9 peptide shows an 85% inhibitory efficiency relative to DHB.

Model of Gly9-Antamanide/Tyrosinase interaction

(in collaboration with ICB-SS) Amino Acids, in preparation (2014)

Binding equilibrium involving a conformational manifold of the HPK1 peptide P2. The association process involves the redistribution of the conformational ensemble from the binding-incompetent (left) to the binding-competent (PPII) species, and then the interaction of the latter with the SH3 domain.

Electron-withdrawing substituent in the L-Pro pyrrolidine ring: effects on conformation and binding of HPK1 P2 to the SH3 domain

Sequences of HPK1 P2 Proline-rich peptide analogs. Residue position nomenclature according to Lim et al. (1994).

Amino Acids, 44, 607 (2013)

The stereochemistry of the electron-withdrawing substituent at position 4 controls the puckering of the L-Pro pyrrolidine ring.

0 10 20 30 40 50

-1

0

1

2

[Θ] 1

0-3 d

eg d

mol

-1cm

2

H3 F3 H2 F2 H5 F5

P2

Temperature (°C)

The results of the present study suggest that the ability of 4-(R)-electron-withdrawing group to stabilize the PPII-like conformation is strictly related to the residue position in addition to the configuration of the γ-substituent.

200 210 220 230 240 250-24

-20

-16

-12

-8

-4

0

4

5°C 15°C 25°C 35°C 45°C

[Θ] 1

0-3 d

eg d

mol

-1cm

2

Wavelength (nm )

Far-UV CD spectra of the H3 peptide in 20 mM Tris-HCl, pH 7.5, buffer solution as a function of temperature.

Amino Acids, 44, 607 (2013)

Electron-withdrawing substituent in the L-Pro pyrrolidine ring: effects on conformation and binding of HPK1 P2 to the SH3 domain

Temperature dependence on the maxima in the CD spectra for the P2 peptide and its Hyp or FPro analogs in 20 mM Tris-HCl, pH 7.5, buffer solution.

260 280 300 320 340

-6.0x10-4

-4.0x10-4

-2.0x10-4

0.0

280 285 290 295 300 305-2.0x10-4

-1.5x10-4

-1.0x10-4

-5.0x10-5

0.0

5.0x10-5

∆A (A

L - A

R)

Wavelength (nm)

∆A (A

L - A

R)

Wavelength (nm)

Near-UV CD spectra for the binding of H3 to the GST-SH3m-cort domain 20 mM Tris-HCl, pH 7.5 buffer solution at room temperature. The inset highlights the Trp CD region. The ΔA values were measured as a function of increasing the GST-SH3/peptide molar ratio.

The binding affinity of the proline-rich peptides to both SH3m-cort and SH3HS1 domains is the result of a delicate balance of mutually compensating contributions that play a role in the binding energetic: the PPII helix conformation that promotes aromatic-proline interactions, and the ionic interaction involving the Lys-5 residue outside the classical class II binding motif.

Amino Acids, 42, 1361 (2012) Biopolymers (Pept. Sci.), 94, 298 (2010)

(in collaboration with G. Siligardi, Diamond Light Source, Didcot, UK and M. Spaller, Wayne State Univ., Detroit)

Electron-withdrawing substituent in the L-Pro pyrrolidine ring: effects on conformation and binding of HPK1 P2 to the SH3 domain

Peptides as modulators of α-synuclein aggregation

Table 1. Peptide sequences. The substituent at position 4 is indicated in bold.

Peptide Sequencea Phe4 H-Arg-Lys-Val-Phe-Tyr-Thr-Trp-NH2

DPhe4 H-arg-lys-val-phe-tyr-thr-trp-NH2

MePhe4 H-Arg-Lys-Val-MePhe-Tyr-Thr-Trp-NH2

DMePhe4 H-Arg-Lys-Val-mephe-Tyr-Thr-Trp-NH2

Tic4 H-Arg-Lys-Val-Tic-Tyr-Thr-Trp-NH2

DTic4 H-Arg-Lys-Val-tic-Tyr-Thr-Trp-NH2

Ala4 H-Arg-Lys-Val-Ala-Tyr-Thr-Trp-NH2

alower-case first letter of an amino acid indicates a residue of the D-series.

Structure of conformationally restricted analogs of the phenylalanine residue (A) and Newman projections of the three low-energy staggered conformations in L-amino acids (B).

A

NH

Hβ3

Hβ2

Hα

R

O

Hβ3

R

NH

Hβ2

HαO Hβ2

NH

Hβ3

Hα

R

O

gauche (-) trans gauche (+)

N

O

R1

H

R

CH3

N

O

R1

H

R

L-MePheL-Tic

NH

O

R1

H

R

L-Phe

B

The capability of the heptapeptide MePhe4, an inhibitor of Aβ-peptide aggregation, and related analogs to interact with α-synuclein, interfering with amyloid fibril formation was investigated.

The aggregation of α-synuclein to form insoluble fibrils is related to pathological conditions such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy

Protein and Peptide Letters, submitted (2014)

Fluorescence intensity of Thioflavin-T (a marker for amyloid aggregation) at 480 nm. Th-T was added to a small aliquot of an AS solution and incubated at 37°C and 500 rpm up to 11 days, either alone or in the presence of 3 eq. of each peptide.

ASL-P

heD-Phe

L-MeP

he

D-MeP

heL-T

icD-Ti

c Ala0

20

40

60

80

100

120

Th-T

fluo

resc

ence

inte

nsity

0 1 2 3 4 7 9 11

All peptides investigated are able to accelerate the aggregation of AS, thus decreasing the lifetime of the more toxic protofibrils. Our findings might pave the way toward peptidomimetic drugs effective against the related neurodegenerative diseases.

Far-UV CD spectra of α-synuclein (AS) in the presence of increasing amounts of Phe4 peptide. Peptide/AS molar ratios are indicated.

200 220 240-4

-3

-2

-1

0

∆A =

(AL -

A R)10

-4

Wavelength (nm)

0.0 0.3 1.5 2.0 3.0 5.0

Protein and Peptide Letters, submitted (2014)

Peptides as modulators of α-synuclein aggregation

Cα-TETRASUBSTITUED α-AMINO ACIDS: CONFORMATIONAL PROPERTIES

α-Aminoisobutyric acid, 1-aminocycloalkane-1-carboxylic acids, Cα-methyl analogs of protein amino acids, …

310-HELIX

Cα-Ethylated residues and symmetrically Cα- disubstituted glycines with both side chains longer than methyl

2.05-HELIX (fully-extended)

Mixed sequences (proteinogenic and Cα-tetrasubstituted residues)

α-HELIX

Reviews: Conformational preferences of quaternary amino acids: Biopolymers (Pept. Sci.), 60, 396 (2001) The fully-extended conformation: Biopolymers (Pept. Sci.), 100, 621 (2013) Helix screw-sense preferences of chiral quaternary amino acids: Biopolymers (Pept. Sci.), 102, in press (2014)

CONH

H2C (CH2)n-2

CONH

CH3CH3

CONH

RCH3

Aib(αMe)Ala

(αMe)AAs (R = CH3)

Acnc(n = 3-12)

Deg

H2C

COHN

CH2

CH3H3C

Dbzg

H2C

COHN

CH2

*

H2C

COHN

(CH2)4

CH3H3C

Epg

310-HELIX

i → i+3 C=O … H-N H-bonds (C10) φ,ψ = -57

,-30

3.2 residues per turn Rise per residue 1.9 Å

2.05-HELIX

i → i C=O … H-N H-bonds (C5) φ,ψ = 180

,180

2.0 residues per turn Rise per residue 3.7 Å

Biopolymers (Pept. Sci.), 84, 3 (2006).

TOWARD A PEPTIDE-BASED MOLECULAR SPRING

310-HELIX

FULLY-EXTENDED

A 2D-NMR method has been developed, able to discriminate between the fully-extended and the 310-helical conformations in solution Tetrahedron, 68, 4429 (2012)

Fully-extended - 310-helix equilibria have been investigated by time-resolved fuorescence and molecular mechanics on Deg homo-peptides carrying an electron trasfer donor – acceptor pair as the terminal groups Biopolymers (Pept. Sci.), 100, 51 (2013) (with M. Venanzi, Univ. Rome Tor Vergata) THE CONFORMATION OF DEG HOMO-PEPTIDES CAN BE CONTROLLED BY SOLVENT POLARITY Chloroform → fully-extended Acetonitrile → 310-helix

THE 310-HELIX AS A TEMPLATE AND SPACER FOR PHYSICOCHEMICAL INVESTIGATIONS

New bis-ferrocenyl end-capped peptides: synthesis and charge-transfer properties Biopolymers (Pept. Sci.), 100, 14 (2013) (with S. Santi, Univ. Padova)

Energy transport in peptide helices Proc. Natl. Acad. Sci. USA, 104, 12749 (2007) (with P. Hamm, Univ. Zürich)

Aib-based peptides give rise to 310-helices of high population (88% and almost 100% for the homo-hexamer and octamer, respectively, in CDCl3 solution) which do not unfold even at 150

C in DMSO. They represent an attractive system for the study of distance-dependent phenomena.

Boc-Ala-(R)-Aic(NN)-Ala-OMe

Aic(NN)

(nitronyl nitroxide 2-aminoindan-2-carboxylic acid)

• Conformationally restricted (turn/helix promoter)

• Stable free radical: EPR probe

• Potentially exploitable for the development of peptide-based magnetic materials

Eur. J. Org. Chem., 1741 (2014)

NOVEL Cα-TETRASUBSTITUED α-AMINO ACIDS

(in collaboration with K. Wright, CNRS – Univ. Versailles, France)

IN SEARCH FOR THE ELUSIVE (2.27-) γ-HELIX PEPTIDE CONFORMATION

Tfa-(Adm)2-NHiPr

(Adm, 2-aminoadamantane-2-carboxylic acid)

Boc-[L-(αMe)Aze-L-Ala]2-OMe

[(αMe)Aze, 2-methyl-azetidine-2-carboxylic acid]

i-1 → i+1 C=O … H-N H-bonds (C7) φ,ψ = -70

,+70

Rise per residue 2.8 Å

PRELIMINARY RESULTS (X-ray diffraction structures)

New J. Chem., in press (2014)

← 2.2 nm →

Z-(Ala)3-(Aib-L-Ala)4-OH

Z-(Ala)3-(Aib-L-Ala)4-OMe in water (conc. > 15 mM) self-assemble in vescicles up to 100 nm in diameter

Vescicles are able to encapsulate Au nanoparticles (∅ 6 nm)

Mechanism ? Polymer J., 45, 516 (2013)

PEPTIDE-BASED NANOMATERIALS Hydrophobic Aib/Ala peptides solubilize in water through formation of supramolecular assemblies

Bis(azobenzene)-Based Photoswitchable, Prochiral, Cα-Tetrasubstituted α-Amino Acids for Nanomaterials Applications

Magnetic susceptibility of Au NP can be reversibly controlled by irradiation

Chem. Eur. J., 17, 12606 (2011) (in collaboration with C. Cativiela, Univ. Zaragoza)

A Poly(γ-benzyl-L-glutamate) - azobenzene hybrid Supramolecular vesicles formation – phototriggered disassembly

Macromolecules, in press (2014) http://pubs.acs.org/doi/abs/10.1021/ma501601r

ANTIBIOTICS - RESISTANCE

• Use / misuse of antibiotics has led to the emergence of pharmacoresistant bacterial strains

• Antibiotics of current use are progessively becoming ineffective • Antimicrobial resistance is responsible for about 25,000 deaths every year in the EU,

with annual treatment and social costs estimated to be € 1.5 bn • Novel antibacterial molecules, able to act through mechanisms different from those

exerted by current antibiotics, are needed • Research to combat the growing problem of antimicrobial drug resistance has been a

high priority on the agendas of World Health Organization and EU Framework Programmes ever since the Microbial Threat Conference in Copenhagen (1998)

• Pharma companies are resisting the call to invest in research on new antibiotics as they are expensive to develop but are used briefly by most patients

NATURAL DEFENSES AGAINST INFECTIONS

• Higher organisms (vertebrates): adaptive immune system • All organisms: innate immunity → antimicrobial peptides (AMP)

ANTIMICROBIAL PEPTIDES • from a variety of sources (bacteria, fungi, plants, insects, amphibian skin, human cells) • usually: 10 - 50 amino acids cationic - a few positively charged side chains (Lys, Arg) high content in hydrophobic amino acids • secondary structure: i) α-helical, ii) β-stranded (2 or more disulfide bonds), iii) β-hairpin or

loop (1 disulfide bond and/or cyclization) • amphiphilic

PEPTIDE-INDUCED MEMBRANE PERMEABILIZATION

a) Barrel-stave pore. Peptides insert perpendicularly in the bilayer, associate and form a pore. The peptides line the pore lumen in a parallel direction relative to the phospholipid chains, which remain perpendicular to the bilayer plane.

b) Carpet mechanism. Peptides adsorb parallel to the bilayer and, after reaching sufficient coverage, produce a detergent-like effect that disintegrates the membrane.

c) Toroidal pore. As for the barrel-stave pore, peptides insert perpendicularly in the bilayer, but instead of packing parallel to the phospholipid chains, induce a local membrane curvature in such a way that the pore lumen is lined partly by peptides and partly by phospholipid head groups. A continuity between inner and outer leaflets is established.

d) Disordered toroidal pore. A recent modification to the toroidal pore proposes that less-rigid peptide conformations and orientations are formed; the pore lumen is lined by the phospholipid head groups.

N.M. Melo et al., Nature Reviews Microbiology, 7, 245 (2009)

PEPTAIBOL ANTIBIOTICS / PEPTAIBIOTICS

Peptides, produced by various fungi, rich in α-aminoisobutyric acid (Aib), with a C-terminal 1,2-aminoalcohol and an N-terminal acyl group

ALAMETHICIN Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phol TRICHORZIANIN Ac-Aib-Ala-Ala-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Leu-Aib-Ile-Gln-Gln-Wol ZERVAMICIN Ac-Trp-Ile-Glu-Iva-Val-Thr-Aib-Leu-Aib-Hyp-Gln-Aib-Hyp-Aib-Pro-Phol HEPTAIBIN Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-Hyp-Gln-Aib-Hyp-Aib-Phol TYLOPEPTIN B Ac-Trp-Val-Aib-Aib-Ala-Gln-Ala-Aib-Ser-Aib-Ala-Leu-Aib-Gln-Lol TRICHOVIRIN Ac-Aib-Asn-Leu-Aib-Pro-Ser-Val-Aib-Pro-Aib-Leu-Aib-Pro-Lol HARZIANIN Ac-Aib-Asn-Leu-Ile-Aib-Pro-Iva-Leu-Aib-Pro-Lol PEPTAIBOLIN Ac-Leu-Aib-Leu-Aib-Phol Lipopeptaibols TRICHOGIN A IV Oct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Lol TRIKONINGIN KB I Oct-Aib-Gly-Val-Aib-Gly-Gly-Val-Aib-Gly-Ile-Lol TRICHODECENIN Dec-Gly-Gly-Leu-Aib-Gly-Ile-Lol HALOVIR A Myr-Aib-Hyp-Leu-Val-Gln-Lol

More than 1000 sequences in the Comprehensive Peptaibiotics Database: https://peptaibiotics-database.boku.ac.at/ Chem. Biodivers., 10, 734 (2013)

Active on biomembranes by altering their permeability “long” peptaibols → ion channels

CNH CO

H3C CH3

A i b

LONG AND MEDIUM-SIZE PEPTAIBIOTICS

Alamethicin F50/5 - Heptaibin - Tylopeptin B

Studies on peptide-membrane interaction (conformation, orientation, aggregation, mode of action) Analogs

N CH3

CH3

H3CH3C

H2N

O

O

OHESR probe (TOAC) Yu.D. Tsvetkov (Novosibirsk, Russia)

fluorophores (dansyl, Fmc, antAib) L. Stella, M. Venanzi (Rome)

isotopic labels for NMR investigations (19F) B. Bechinger (Strasbourg, France)

IR absorption probes (-CN or -NO2) L. Stella (Rome)

Chem. Biodivers., 10, 887 (2013) Chem. Biodivers., 10, 904 (2013) J. Struct. Chem., 54, S73 (2013) Chem. Biodivers., 11, 1069 (2014) Chem. Biodivers., 11, 1163 (2014)

J. Pept. Sci., 17, 585 (2011) J. Phys. Chem. B, 116, 5653, (2012) Biochemistry, 51, 10124 (2012) BBA-Biomembranes, 1828, 1013 (2013) Appl. Magn. Reson., 44, 495 (2013) J. Pept. Sci., 19, 246 (2013)

First lipopeptaibol isolated from Trichoderma longibrachiatum [C. Auvin-Guette et al., JACS, 114, 2170 (1992)] active against Gram-positive bacteria

TRICHOGIN Oct-Aib-Gly-L-Leu-Aib-Gly-Gly-L-Leu-Aib-Gly-L-Ile-L-Lol (Oct, 1-octanoyl; Lol, leucinol).

mixed 310/ α-helical conformation

conformation retained in solvents of low polarity and membrane-mimetic environments

amphiphilic: Gly residues on the hydrophilic face

helix lenght 16 Ǻ

preferential orientation parallel to the membrane plane

Nature Struct. Biol., 1, 908 (1994) J. Pept. Sci., 9, 679 (2003)

Trichogin is effective against clinically isolated, antibiotic-resistant S. aureus strains Trichogin is significantly stable against enzymatic degradation, which is a common drawback for the exploitation of peptides as drugs. Therefore it can exert a long lasting action in vivo At bactericidal concentrations, trichogin has low hemolytic activity. To some extent it is able to discriminate between eukaryotic and prokaryotic cell membranes (in collaboration with Kyung-Soo Hahm, Chosun University, Korea)

Current developments • Deeper understanding of the stability against proteolytic enzymes • Factors affecting antibacterial / hemolytic balance. • Cationic analogs

Org. Biomol. Chem., 10, 1285 (2012)

TRICHOGIN GA IV: BIOACTIVITY

TRICHOGIN ANALOGS: AIB TO L-LEU SUBSTITUTIONS

Oct-Aib1-Gly-L-Leu-Aib4-Gly-Gly-L-Leu-Aib8-Gly-L-Ile-L-Lol (Trichogin) Oct-L-Leu1-Gly-L-Leu-Aib-Gly-Gly-L-Leu-Aib-Gly-L-Ile-L-Lol (L1)

Oct-Aib-Gly-L-Leu-L-Leu4-Gly-Gly-L-Leu-Aib-Gly-L-Ile-L-Lol (L4)

Oct-Aib-Gly-L-Leu-Aib-Gly-Gly-L-Leu-L-Leu8-Gly-L-Ile-L-Lol (L8)

Oct-L-Leu1-Gly-L-Leu-Aib-Gly-Gly-L-Leu-L-Leu8-Gly-L-Ile-L-Lol (L1,8)

The L1 and L8 analogs are more active than trichogin, whereas for L4 the activity is sharply reduced

TRICHOGIN ANALOGS: AIB TO L-LEU SUBSTITUTIONS

Amino Acids, 43, 1761 (2012)

AIB → LEU TRICHOGIN ANALOGS: CONFORMATION

2D-NMR / restrained molecular dynamics on the L1 analog in aqueous SDS: mixed 310-/α-helical conformation, similar to that of trichogin

X-Ray diffraction structure of the L4 analog: Helix-loop-helix motif, U-shaped backbone: devoid of amphiphilicity, hardly compatible with any mode of membrane insertion / perturbation

Amino Acids, 43, 1761 (2012)

ANTIMICROBIAL TEXTILES

to reduce the exposure to biological agents of the personnel in the healthcare sector and the

propagation of nosocomial infections

production of fabrics for sportswear, aimed at reducing the smell caused by sweat microorganisms

COVALENT BOND between the peptide and the cotton fibers makes the final antimicrobial textile extremely resistant to washing and use

(in collaboration with S. Oancea, University of Sibiu, Romania)

ICB - Padova Unit

CNR staff Barbara Biondi, Andrea Calderan, Marco Crisma, Paolo Ruzza

Lisa Bissacco (admin.)

University Associated (Department of Chemical Sciences, University of Padova)

Massimo Bellanda, Marina Gobbo, Fernando Formaggio, Stefano Mammi, Alessandro Moretto, Elisabetta Schievano, Claudio Toniolo