The Latest Discovery: Exploring the Potential of

Cell Penetrating Peptides for Effective Drug Delivery

Dr Sarah Jones

Molecular Pharmacology Research Group, University of

Wolverhampton

History and Background of CPPs

Hurdles and Progress

Where the field is at the moment

Targeting Protein-Protein Interactions, from Bioportides to Sperm

Overview

• Inert vectors for the delivery of bioactive cargoes into the intracellular milieu

• Intracellular delivery of Peptides, Proteins, Drugs, Oligonucleotides (siRNA, PNA), Plasmids

• Viable alternative to viral vectors and current non-viral intracellular delivery

Cell Penetrating Peptides (CPPs)

1988 - The TransActivator of Transcription protein (TAT) derived from the HIV virus entered cells

1997 - Tat48-60, a truncated arginine-rich sequence conferred cellular penetration.

CPP History: A Long Time Coming!

Tat AntHD43-58 Penetratin

1994- Penetratin –Helix 3 of the Antennapedia homeodomain

Classification of CPPs

Protein-derived

Chimeric

Synthetic/Designed

RQIKIWFQNRRMKWKK-NH2 Penetratin

Tat48-60 GRKKRRQRRRPPQ

pVEC LLIILRRRIRKQAHAHSK

AGYLLGKaINLKALAALAKKIL-NH2

Transportan 10

Transportan GWTLNSAGYLLGKaINLKALAALAKKIL-NH2

RRRRRRRR Polyarginine

(RXR)4 (R(Ahx)R)4

CADY Ac-GLWRALWRLLRSLWRLLWRA-Cya

PepFects

Primary Sequence Name/Source

HIV-1 Tat-derived peptides

GRKKRRQRRRPPQ Tat48-60

QPPRRRQRRKKRG RI-Tat

Penetratins

RQIKIWFQNRRMKWKK AntHD43-58 (Penetratin)

RRRRRRRQIKIWFQNRRMKWKK R6-Penetratin

Other arginine-rich peptides

RRRRRRRRR R8 and R9 are the most common

(R(Ahx)R)4 (RXR)4

Predominantly amphipathic sequences

AGYLLGKINLKALAALAKKIL Transportan10

INLKKLAKL(Aib)KKIL Mitoparan

LLIILRRRIRKQAHAHSK pVec

Hydrophobic Sequences

CSIPPEVKFNKPFVYLI C105Y

Most CPP are polycationic sequences

Fixation artifacts (Live cell imaging) Direct membrane translocation (4oC)

Endocytosis (37oC, ATP) Clathrin-mediated Caveolin-mediated Macropinocytosis

Dependent upon sequence, cell type, concentration and cargo (biochemical properties and size)

Uptake mechanisms, a fixation!

Mishra A et al. Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions PNAS (2011) 108 16883

To Endocytose or not to Endocytose? That is the question

Stearyl-AGYLLGKINLKALAALAKKIL-NH2

Progress in CPP Technologies

PepFect Technologies • Oligonucleotide therapeutics-siRNA and

splice correcting oligonucleotides

• Ecsapes endosomal entrapment associated with the delivery of larger cargoes

CPP Technologies in Clinical Development • PsorBan (CellGate) – heptaarginine coupled to cyclsporin A (psoriasis)

• KAI-9803 (Kai Pharmaceuticals) - tat coupled to a peptide inhibitor of PKCd (reperfusion injury)

• XG-102 (Auris Medical)– tat coupled to a JNK inhibiting peptide (traumatic hearing loss)

AGYLLGKINLKALAALAKKIL-NH2 TP10

PepFect3 Stearyl-AGYLLGKINLKALAALAKKIL-NH2

PepFect6

PepFect14 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2

Ülo Langel Stockholm University

•Its molecular weight is less than 500. •The compound's lipophilicity, expressed as a quantity known as logP (the logarithm of the partition coefficient between water and 1-octanol), is less than 5. •The number of groups in the molecule that can donate hydrogen atoms to hydrogen bonds (usually the sum of hydroxyl and amine groups in a drug molecule) is less than 5

•The number of groups that can accept hydrogen atoms to form hydrogen bonds (estimated by the sum of oxygen and nitrogen atoms) is less than 10.

The Lipinski Rule of Five

What Cell Penetrating Peptides DO NOT DO!

Expansion of Druggable Targets Intracellular Drug Target Space

Bioactive Cell Penetrating Peptides (Bioportides) Enhance the Repertoire of Druggable Targets

It is estimated that 8-10% of the human genome encodes disease-modifying proteins

Only 10% of the druggable genome can be targeted by conventional approaches (SMDs)

New Chemical Entities which target Protein-Protein Interactions are gaining momentum as an attractive therapeutic modality

Proteomics and interactomics will identify many other PPIs that can be modulated by peptides

Mast cell secretion Activation of

p42/44

MAPK

Sychnologic CPP Rhegnylogic CPP

Sequence Target Activity Pathology

Tat-DI1 Raf dimerization

Inhibits proliferation of NSCLC cell lines

Cancer

Antp-MEK1 ERK Inhibition of ERK2 activation Cancer

Tat48-60-P10 PCNA Apoptogenic Cancer

STAT-6-IP STAT-6 Inhibitor of TH2 cytokine production

Allergic airways disease

Hph-1-ctCTLA-4

TcR signalling

Inhibition of TcR signalling. Reduction in Th2 cytokines, serum IgE

Allergic airways disease

Tat-acp-TrkA666-676

TrkA activation loop

TrkA antagonism Inflammatory pain

Nup153-Cyt c

NPC Apoptogenic Cancer

Sequence Target Activity

Nosangiotide ND Anti-angiogenic

Camptide G proteins cAMP modulation

Cyt c77-101 ER Apoptogenic

BIP BAX Anti-apoptogenic (neuroprotection)

Mouse PrP1-28 Prion Proteins Anti-prion infection

AT1AR304-318 G proteins Blood vessel contraction

Vasostatin-1 Heparan Sulphate Proteoglycans

eNOS activation (vasodilatory)

Arf (1-22) ND Apoptogenic

Mitoparan VDAC (mitochondria) Apoptogenic

Stapled p53-derived peptides

p53-hDM2 Reactivation of p53 apoptosis pathway

Bioactive Cell Penetrating Peptides - Bioportides

Prediction algorithm: Hällbrink et al. Int. J. Peptide Res. Ther. 11, 249 (2005)

CPP mimic of GPCRs: Ostlund et al. Int. J. Peptide Res. Ther. 11, 237 (2005)

QSAR Prediction Algorithm

No. of

Amino

Acids

Position Sequence CPP

Probability

16 85 IKKKEERADLIAYLKK 2

16 86 KKKEERADLIAYLKKA 3

17 84 GIKKKEERADLIAYLKK 3

17 85 IKKKEERADLIAYLKKA 3

17 86 KKKEERADLIAYLKKAT 2

Whole

Protein

Signal

Transduction

Domain

Later nuclear and

cytoplasmic

distribution

Cells were incubated for

80 mins with rho-

eNOS492-507 (5mM) in

endothelial cell medium

plus growth supplement

then transferred to DMEM

w/o phenol red for

confocal visualization

Early distribution

Cells were incubated for

45 mins with rho-

eNOS492-507 (5mM) in

endothelial cell medium

plus growth supplement

then transferred to

DMEM w/o phenol red

for confocal visualization

Nosangiotide is

located within the

a-helix domain

(shown in yellow)

that tightly binds

calmodulin. Diagram

adapted from Aoyagi et

al., EMBO J. 22, 766-775

(2003)

RKKTFKEVANAVKISASLMG

RKKTFKEVANA

RKKTFKEVANAVK

RKKTFKEVANAVKI

RKKTFKEVANAVKISA

Anti-angiogenic Properties of Nosangiotide (eNOS492-507)

A Bioportide Derived from Endothelial Nitric Oxide Synthase

Sequence CPP Index

1

3

3

3

3 Nosangiotide

Nosangiotide is a potent inhibitor of FGF-2 (25 ng/ml)-induced proliferation (a), migration (b) and tube

formation (c) of primary endothelial cells

Nosangiotide (eNOS492-507) Inhibits Biological Features of FGF-induced Angiogenesis

in vitro

500

750

1000

1250

1500

1750

-8 -7 -6 -5 -4

log { [eNOS492-507

] (M) }

nu

mb

er

of

mig

rate

d c

ell

s

50

100

150

200

250

-9 -8 -7 -6 -5 -4 -3

log { [eNOS492-507

] (M) }

mean

of

clo

sed

are

as p

er

field

60,000

70,000

80,000

90,000

-9 -8 -7 -6 -5

log { [eNOS492-507

] (M) }

cell

nu

mb

er

(a) (b) (c)

Proliferation

IC50 = 83.7 nM

Migration

IC50 = 38.2

nM

Tube Formation

IC50 = 509 nM

Nosangiotide inhibits FGF-induced angiogenesis

A) Carrier control

B) FGF-2 (200ng)

C) Nosangiotide (0.5 nmole)

D) FGF-2 plus nosangiotide (0.5

nmole)

E) FGF-2 with nosangiotide (0.05

nmole)

F) FGF-2 with nosangiotide

(0.005 nmole).

Howl et al. (2012) Cell. Mol. Life Sci.

69, 2951 .

Biotinylated CPP as

Molecular Fishing Rods

TRYPSIN

DIGEST

MALDI

TOF/TOF

Streptavidin-coated multiwell

plates

Biotinylated CPP

Swiss 3T3

Dr Ashley Martin

Proteomics Unit Cancer Studies

University of Birmingham, UK

Cross linking of peptide side chains

Metabolic stability

Enhance propensity for cellular

penetration

VGIKKK 83-88

KMIFVGIKKK 79-88

KMIFVGIKKKEERA 79-92

KKKEERADLIAYLKKA 86-101

GIKKKEERADLIAYLKK 84-100

IKKKEERADLIAYLKKA 85-101

VGIKKKEERADLIAYLKK 83-100

GIKKKEERADLIAYLKKA 84-101

FVGIKKKEERADLIAYLKK 82-100

VGIKKKEERADLIAYLKKA 83-101

IFVGIKKKEERADLIAYLKK 81-100

FVGIKKKEERADLIAYLKKA 82-101

KMIFVGIKKKEERADLIAYLK 79-99

MIFVGIKKKEERADLIAYLKK 80-100

IFVGIKKKEERADLIAYLKKA 81-101

KMIFVGIKKKEERADLIAYLKK 79-100

MIFVGIKKKEERADLIAYLKKA 80-101

TKMIFVGIKKKEERADLIAYLKK 78-100

KMIFVGIKKKEERADLIAYLKKA79-101

GTKMIFVGIKKKEERADLIAYLKKA 77-101

TKMIFVGIKKKEERADLIAYLKKA 78-101

KMIFVGIKKKEERADLIAYLKKAT 79-102

GTKMIFVGIKKKEERADLIAYLKKA77-101

TKMIFVGIKKKEERADLIAYLKKAT 78-102

KMIFVGIKKKEERADLIAYLKKATN 79-103

C-terminal helix

Primary Sequence Position

KGKKIF 5-10

KGKKIFI 5-11

GKKIFIMK 6-13

KGKKIFIM 5-12

KGKKIFIMK 5-13

EKGKKIFIMK 4-13

N-terminal helix

Primary Sequence Position

Future Work Stapled

Peptides

Identify Bioportide

Binding Partners

a-aminoisobutyric

acid

TLKKLIVRLNNVQEGKQI

RKILLSKGIHLN

KLVLAALNRFIG

RKALTISIGKGDSQIISLLLRR

NLRKQTNIASTLARMVIRYQMK

LLKRKRKILSSDDSLRSSKLQ

SPLRLKELKILNLSKN

HIGCKAKDIIRFLQQRLKKAVP

LQQRLKKAVPYNRMKLMIV

GSGKTTLLQQLMKTKK

ATVGIDVKDWPIQIRDKRKR

KQRKACMSKITKELLNKRGFPA

LAKLRKTIINESLNFKIRDQLV

PVIDRKRLLQLVRENQLQL

AVKIFNKHTSLRLLRQ

LISLLAAGIRPR

RLLQQDKASLTRTLQHRIA

LTRRILLPKNVI

KTVKLKGAAPLKILNIGN

RVKTLCLQKNTALWI

LLLDLSTRRLIRVI

IEVRKELAEKMRRT

KKQCFKNDIHKLVLAALNRFI

SPKLVELLLNSGSREQDVRKAL

QIISLLLRRLALDVANNSICLG

GNKISGICSPLRLKELKILNL

ATVGIDVKDWPIQIRDKRKRDL

LISLLAAGIRPRMLVMELASKG

KKTEKLCGLIDCVHFLREVMVK

15-32

433-444

544-555

772-793

828-849

945-965

1124-1139

1310-1331

1322-1340

1344-1359

1367-1386

1460-1481

1497-1518

1546-1564

1904-1919

1932-1943

1957-1975

2140-2151

2278-2295

2413-2427

2435-2448

2511-2524

534-554 (K544E)

754-775 (P755L)

785-806 (R793M)

1116-1136 (I1122V)

1367-1388 (I1371V)

1932-1953 (R1941H)

2378-2399 (G2385R)

Sequence Position Sequence Position (mutation)

Highly Probable CPPs collectively

spanning the entire LRRK2 protein

CPPs containing mutations known to cause

Parkinson’s disease

LRRK2-derived Bioportides

> 5000 Probable

CPP

NFAT Luciferase reporter assay (Armesilla, A.L. et al. (1999) Mol. Cell. Biol. 19, 2032-2043)

Bioportides facilitate relocation of NFAT from the cytoplasm to the nucleus?

Bioportides abrogate PMA and Ca2+ ionophore induced relocation of NFAT?

The role of the Immune System in PD

LRRK2 role in Inflammatory

Bowel Disease (Liu, Z. et al.,

(2011) Nature Immunology 12,1063-

1070)

LRRK2 sequesters NFAT in the

cytoplasm

LRRK2 deficiency enhances

susceptibility to experimental

colitis and enhances nuclear

localization of NFAT

LRRK2: Not just Parkinsons!

LRRK2

GNKISGICSPLRLKELKILNL GNKISGVCSPLRLKELKILNL

WT inhibits NFAT translocation

Mutant enhances NFAT translocation

Candidate Bioportides for Modulation of NFAT Translocation

LRRK21116-1136

LRRK21116-1136 (I1122V)

KLEQLILEGNKISGICSPLRLK

Van Craenenbroeck et al., (2012) Purification and preliminary biochemical and structural characterisation of the leucine rich repeat domain of LRRK2. Biochem. Biophys. Acta 1824, 450

LRRK21124-1139 SPLRLKELKILNLSKN Enhances NFAT translocation

LRRK21310-1331 (C-terminal helix) HIGCKAKDIIRFLQQRLKKAVP Inhibits NFAT translocation

ANK LRR ROC COR Kinase WD401 2527

G2385R

ARM

LRRK215-32 LRRK21367-1386

LRRK22378-2399

LRRK21116-1136

LRRK21124-1139

LRRK21310-1331

I1122V

T2397M

Leucine-Rich

Repeat domain

ANK LRR ROC COR Kinase WD40 1 2527

G2385R

ARM

LRRK215-32 LRRK21367-1386 LRRK22378-2399

LRRK21116-1136

LRRK21124-1139

LRRK21310-1331

I1122V

T2397M

LRRK22378-2399 includes the very common T2397M risk allele for Crohn’s Disease

LRRK2-derived bioportides

modulate NFAT translocation. HEK-

293 cells were transiently transfected

with a luciferase reporter vector

(pNFAT-Ta-Luc; Clontech). Luciferase

activity was measured as an indicator

of NFAT translocation to the nucleus.

Cells were stimulated with PMA (20

ng/ml) and the calcium ionophore

A23187 (1 mM) for 16 hours.

Luciferase activity was calculated as

fold induction over the value of the

reporter vector in un-stimulated cells.

• CPP can be readily incorporated into

multimeric complexes

• An inherent lack of specificity can be

surmounted by the inclusion of

tissue specific targeting peptides

• Phage Display Technology has

generated an abundance of tissue

homing peptides

Building Cell Selectivity into CPP Delivery

Homing Peptides CPP

Sequence

Building Cell Selectivity into CPP Delivery

Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2

Scale 100 μm.

Coronal section of a mouse brain with U87 tumor in the right striatum. The tumour area is circled with a

dotted line. (B) H&E stained hemisphere of brain and glioma. (C) The animals received an i.v. injection of

FAM-labeled gHoPe2 3 h before tissue collection

Intracranial Tumour Model

FAM-gHo FAM-gHoPe2

Intr

ac

ran

ial

tum

ou

r In

tac

t b

rain

K

idn

ey

Liv

er

Eriste, E., Kurrikoff, K., Suhorutsenko, J., et al. Bioconjugate Chemistry (2013). In press

Building Cell Selectivity into CPP Delivery

Activatable CPPs

++++++++++++++

- - - - - - - - - - - -

Cellular internalization

impaired

cargo ++++++++++++++ cargo

Cellular internalization

Matthias Hallbrink – NoPe (YTA4 + MMP-2 cleavage site + inactivationg region) -tumour imaging in vivo.

Exploitation of tissue specific endopeptidases

MMP2

http://www.flickr.com/photos/wellcomeimages/5814253423/

Internal Volume

• Is reduced. • Mature sperm lacks a variety of

organelles: endoplasmic reticulum, Golgi apparatus, cytosolic ribosomes.

Nucleus

• Once Spermatozoa are released into the seminiferous tubules, genomic transcription and translation have largely been silenced.

• Conventional molecular biology techniques are thus redundant. Modulation of sperm cell biology is restricted to cell permeable agents.

Plasma Membrane

• Lipid composition of plasma membrane is highly polarized and compartmentalized.

• It appears to be incapable of endocytotic events. • Static physical barrier • Detergents are detrimental to protein function

The Challenge: Penetrating the Impenetrable

Understanding Sperm Biology with CPP

Sperm are transcriptionally and translationary inactive

Studies of their intracellular biology are restricted to cell permeable agents

CPP are ideal vehicles for the study of sperm intracellular biology, from motility to capacitation and the acrosome reaction

Can be easily isolated from bulls semen with supplemented EBSS containing 0.3% BSA (“swim up method”)

semen

sEBSS (0.3% BSA)

Differential Intracellular Distribution of CPP

Tat Penetratin C105Y

Rho-tat (5mM) + DIC Rho-penetratin (5mM)

Rho-penetratin (5mM)

Mitotracker (500nM)

Rho-C105Y (5mM)

Rho-C105Y (5mM) Rho-tat (5mM) + DIC

Pen

etra

tin Tat

C10

5YM

itP

TP10

iMitP iM

P10

2-11

3

R1a

rV

0

1

2

3

4

5

6

7

8

Flu

ore

scen

ce m

inu

s b

ackg

rou

nd

(A

.U.)

Comparative analysis of CPP translocation

efficacies into bovine spermatozoa.

Spermatozoa were incubated with TAMRA-

labelled CPP (5 mM) for 1 h at 37 oC. Data are

mean + S.E.M. from 3 experiments performed

in triplicate.

The Mitochondrial-localising CPP Mitoparan Rho-MitP (5mM) Mitotracker (500 nM)

Mitotracker (500 nM) merge Rho-TP10 (5mM)

Rho-MitP (5mM)

Mitotracker (500 nM) merge

Mitochondrial Midpiecei

Rho-TP10 (3 mM) + avidin Alexa Fluor® 488 (1 mM)

Swiss 3T3

Spermatozoa Rho-TP10 (3 mM) + avidin Alexa Fluor® 488 (1 mM)

Rho-TP10 (1 mM) + avidin Alexa Fluor® 488 (0.33 mM)

Swiss 3T3

Avidin

TP10

Merge

Merge DIC

Merge Avidin

TP10 Merge DIC

Merge DIC Merge Avidin TP10

. Protein Delivery

0 10 20 30 40 50 600

2

4

6

8

10

12

C105Y

tat

rV1aR102-113

Time (min)

Flu

ore

scen

ce m

inu

s b

ackg

rou

nd

(A

.U.)

0 10 20 30 40 50 600

2

4

6

8

10

C105Y

tat

rV1aR102-113

Time (min)

Flu

ore

scen

ce m

inu

s b

ackg

rou

nd

(A

.U.)

Translocation Kinetics of C105Y

Internalisation occurred with first order saturable

kinetics (F = Fmax x t/t0.5 + t, GraphPad Prism 5).

Spermatozoa Swiss 3T3

Cells were incubated at 37oC with TAMRA-labelled peptides (5mM)

for the times indicated. Normalised data (compared to tat-assigned

a value of 1) are expressed as mean fluorescence (minus

background) + s.e.m. from 3 experiments performed in triplicate.

t0.5 = 0.70 min

t0.5 = 7.02 min

Swiss 3T3 Spermatozoa

Transferrin Alexa Fluor® 488 (50 mg/ml)

Transferrin Texas Red® (50 mg/ml)

LysoTracker® Red (75 nM))

Dextran Texas Red® (10 mM)

Endocytosis Incompetent Spermatozoa

LysoTracker® Red (75 nM))

Dextran Texas Red® (10 mM)

Direct membrane translocation is the sole mechanism of CPP import into sperm

Why are CPP unable to deliver large proteins into sperm?

H-KQLLVAKEGAEKIKKKRNTLFG-NH2 H-LKNKFKGVKLAEIEKQALKGTR-NH2 STIM1371-392 Scr- STIM1371-392

STIM1

Orai1

Endoplasmic reticulum

Plasma membrane

Store depletion

CC2 CC3

CC1

CC2 CC3

CC1 KIKKK

EEEL

E

Occluded SOAR Free SOAR

? STIM1

(485-

685)

? STIM1

(485-

685) +++

+ - - -

-

STIM1 ORAI1 Activating Region

CC2 (364-388)

CC3 (399-432)

CC1 (248-342)

334 442

KIKKK EEELE

Progesterone-the

best-characterised

agonist of human

sperm [Ca2+]i

signalling.

Motility and the

acrosome reaction

Biphasic [Ca2+]i

response

Bioportides as Modulators of Human Sperm [Ca2+]i Signalling

CPP Import is Compatible with Sperm Motility and Viability

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

MitP

nosangiotide

tat

Time (min)

% m

oti

le c

ells (

*rap

id)

rela

tive t

o c

on

tro

ls

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

120

140

camptide

Cyt c5-13

C105Y

Time (min)

% m

oti

le c

ells (

*rap

id)

rela

tive t

o c

on

tro

ls

0

10

20

30

40

50

60

70

80

90

100

110

-6.0 -5.5 -5.0 -4.5

C105Y

MitP

tat

iMitP

iMP

penetratin

nosangiotide

log { [peptide] (M) }

% c

ell v

iab

ilit

y

Non-capacitating Capacitating

A, B. Motility data were collected from human sperm cells treated with 5 mM CPP.

Each peptide was tested on samples from 3 individual donors. Data are shown as %

rapid cells from treated samples relative to that of controls and expressed as mean +

s.e.m. *rapid cells = velocity (average path) ≥25 µms-1 and straightness ≥ 80%.

C. Isolated bovine spermatozoa were treated with CPP for 1 h at the concentrations

indicated. Cell viability was measured by MTS conversion and expressed as a

percentage of those spermatozoa treated with vehicle alone (sEBSS).

A B

C

Conclusions

Pantechnia

CPPs for site-specific delivery of bioactive

cargoes into mammalian sperm

Cargo size is critical

CPP technologies as valuable tools for the

investigation and modulation of

fundamental processes of sperm

physiology such as maturation,

capacitation, motility, hyperactivation and

fertilisation.

Jones, S., Lukanowska, L., Suhorutsenko, J., Oxenham, S., Barratt, C., Publicover, S., Copolovici, D.M., Langel, Ü. and Howl, J. (2013) Intracellular translocation of cell penetrating peptides into spermatozoa. Human Reproduction DOI: 10.1093/humrep/def064.

The Future for CPP Therapeutics Modifications to enhance stability are now surmountable

Routes of administration Mechanisms for moving forward Marcus Evans Discovery and Evolution Summits

Mass Screening, Formulation and Analogues

Acknowledgements Scientific contributors

University of Wolverhampton, UK John Howl Monika Lukanowska

University of Manchester, UK Shant Kumar

University of Birmingham, UK Michelle Farquhar Ashley Martin Steve Publicover

University of Dundee, UK Chris Barratt Senga Oxenham

Department of Neurochemistry, Stockholm University, Sweden University of Tartu, Estonia Ulo Langel

MRC Protein Phosphorylation Unit, University of Dundee, UK Dario Alessi