apresentação do powerpoint - eventos.fct.unl.pt · natural products research group project design...

Drug Discovery

Romeu A. Videira, Fátima Fernandes, Patrícia Valentão, Andreia P. Oliveira, M. Figueiredo González,

João Bernardo, Daniela Correia, Rui Gonçalves, David M. Pereira, Paula B. Andrade

NATURAL PRODUCTS RESEARCH GROUP

1st Workshop - Novel Compounds from

Renewable Sources

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPNatural Products And Drug Discovery

Alzheimer's disease

Parkinson's disease

Diabetes type 2

Chronic kidney disease

Cardiovascular diseases

What is common at all age-related degenerative diseases?

AGE-RELATED DEGENERATIVE DISEASES - THE GREAT CHALLENGE OF OUR TIME

Chronic low-grade inflammationMitochondrial dysfunctionsOxidative stress

How we can address this challenge?

How these pathological markers are connected in biological

systems and how they can trigger “apparently” different

diseases?

1st Workshop -

Novel Compounds

from Renewable

Sources


Mitochondrial dysfunctions -- decay of complex I activity

Organs/ tissues / cells affected by age-related degenerative process

PL withn-6 PUFA

iPLA2PL with

n-3 PUFA

Free n-6 PUFA Free n-3 PUFA

Cyclooxygenases

Lipoxygenases

CyP450

Pro-inflammatory lipid mediators

Anti-inflammatory lipid mediators

Cross talk among mitochondrial function, oxidative stress and Chronic low grade inflammation

ROS

NLRP3inflammasome

CASPASE 1

Pro-IL-1βPro-IL-18

IL-1βIL-18

Pyroptosis

Car

dio

lipin

s

Ca2

+

mt

DN

A

iPLA2 affinity: PL (PUFAoxidized) > PL(PUFA) >> PL(MFU) >>PL (SFA)

Membrane LysoPL content increasing – changes in the functional membrane organization and dynamics

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPProject Proposal: Natural bioactive compounds to

control age-related chronic low-grade inflammation using

an integrative multi-target strategy

Our long-term vision is to develop a multi-target therapy and/orspecific diets to reduce chronic low-grade inflammation process,associated with several age-related diseases in order to promotehealthy aging and increasing Human longevity.

Natural Products And Drug Discovery

Strategy: Natural bioactive compounds:

i) To increase cell capacity to quenching ROS and other reactive species

ii) To increase n-3/n-6 PUFA in membrane phospholipids, decreasing the production

pro-inflammatory lipid mediators;

iii) To improve the fitness of IMM to allow the functional assembling of protein

complexes involved in the oxidative phosphorylation, decreasing ROS release and

avoiding the NLRP3 inflammasome activation

1st Workshop -

Novel Compounds

from Renewable

Sources


Know how to characterize the antioxidant and antiradical activities of theextracts as well as to evaluate theirs effects on the activity of key enzymesinvolved in the inflammation cascade, by using cell-free assays;

Know how to obtain, from natural sources, extracts with a well-definedchemical composition and to use them to prepare mixtures enriched in(pre)selected physiologically-active compounds, using methodologies withscalable potential;

Skills and Expertise

Know how to evaluate the extracts ability to modulate the biochemicalpathways connected with the age-related chronic low-grade inflammationprocess and with the redox state of cells (oxidative stress), using in vitro(different types of cells) and in vivo/ex vivo (animal models for differentdiseases such as Alzheimer's disease) assays;

Know how to evaluate the extracts ability to modulate the bioenergeticand lipidomic profiles of different types of cells, tissues and organs and itsrelationship with both chronic low-grade inflammation process and redoxstate of cells

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPPólen de Echium

plantagineumGrãos de pólen

(1000×)

Flor de Echium

plantagineum L.

Echium plantagineum L. bee pollen flavonoids-rich extract


HPLC-DAD chromatogram

Moita E, Gil-Izquierdo A, Sousa C, Ferreres F, Silva LR, Valentão P, Domínguez-Perles R, Baenas N, Andrade PB..

PLoS ONE (2013) 8(3): e59131.

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPPólen de Echium

plantagineumGrãos de pólen

(1000×)

Flor de Echium

plantagineum L.


PLoS ONE (2013) 8(3): e59131.

Echium plantagineum L. bee pollen flavonoids-rich extract exhibit antioxidant/antiradical activity


NADH/PMS Sodium nitroprusside

IC25 = 1.9 mg/mL0.8 mg/mL

Standardized Cell-Free Assaysto evaluate the antioxidant/antiradical activity as well as the effects on key enzymes (e.g. phospholipases A2, Lipoxygenases, Cyclooxygenases)

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPPólen de

Echium

plantagineumGrãos de

pólen (1000×)

Flor de

Echium

plantagineum

L.


O2 -

L-Citrulline

L-Arginine

NO

ONOO -

NOS

O2

NADPH

NADP+

iNOS inhibition


PLoS ONE (2013) 8(3): e59131.

Echium plantagineum L. bee pollen flavonoids-rich extract RAW 264.7macrophages

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUPPólen de

Echium

plantagineumGrãos de

pólen (1000×)

Flor de

Echium

plantagineum

L.

Moita E, Gil-Izquierdo A, Sousa C, Ferreres F, Silva LR, Valentão P, Domínguez-Perles R, Baenas N, Andrade PB.. PLoS ONE (2013) 8(3): e59131.


Echium plantagineum L. bee pollen flavonoids-rich extract has ability tomodulate the prostaglandins release by RAW macrophages stimulated by LPS

PGs metabolites UPLC-MS

RAW 264.7macrophages

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise Brassica oleracea L. vs Pieris brassicae L.Ecological duo as source of bioactive compounds

Xanthine oxidase inhibition (XO)

0 250 500 750 1000-25

0

25

50

75

100 X/XO system

XO inhibition

NADH/PMS system

Concentration (g/mL)

Inhibition (%)

X/XO System

XO Inhibition

NADH/PMS System

0 500 1000 1500 20000

25

50

75

100 X/XO system

XO inhibition

NADH/PMS system


Inhibition (%)

P.

brassicae

Concentration (µg/mL)

Inh

ibit

ion

(%)

0 250 500 750 1000-25

0

25

50

75

100 X/XO system

XO inhibition

NADH/PMS system


Inhibition (%)

B. oleracea var. costata

Concentration (µg/mL)

Inh

ibit

ion

(%)

Cont c/ MMS4 µg

extract/mL

4 µg extract/mL +

MMS100 µg extract/mL +

MMSCont s/ MMS

B

Antigenotoxic activity detected by comet assay

C

1st PRIZE – CIENTIFIC

PHOTOGRAPHY CONTEST

(Fátima Fernandes)

Patent: Andrade, P. B.; Valentão, P.; Sousa,

C.; Pereira, D. M.; Ferreres, F. Aqueous

extract of Pieris brassicae larvae and its use

as antioxidant. Portuguese Patent nr. 103931.

Standardized aqueous extracts of Pieris brassicae larvae exhibit antioxidant

and antigenetotoxic activities

Sousa C, et al. (2009). J Agric Food Chem, 57:2288-2294

1st Workshop -

Novel Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise

Naphthoquinones from Diospyros chamaethamnus Dinter ex Mildbr.

Pinho BR, Sousa C, Valentão P, Andrade PB. PLoS ONE (2011) 6(8): e24098.

(A) p-Hydroquinone

(B) p-Benzoquinone

(C) 1,4-Naphthoquinone

(D) Menadione

(E) Plumbagin

(F) Juglone

(G) Naphthazarin

(H) Diosquinone

1st Workshop -

Novel Compounds

from Renewable

Sources


Biological behaviour of Naphthoquinones from Diospyros chamaethamnusDinter ex Mildbr. - molecular mechanism of action


Nucleophilic attack to thiol groups involves C2

and C3

Diosquinone

Introduction of electron-donating hydroxyl groups

increases the pro-oxidant potential

Menadione Plumbagin Juglone

Naphthazarin


1st Workshop -

Novel Compounds

from Renewable

Sources




Diosquinone dampens the inflammation cascade induced by LPS, as detected by the reduction of proinflammatory cytokines: tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6.

Biological behaviour of Naphthoquinones from Diospyros chamaethamnusDinter ex Mildbr. - molecular mechanism of action

iNOS NO O2- ONOO ¯

Superoxide radical quantification.

TNF-: 51% IL-6: 39%

Semi-quantitative analysis of 3-nitrotyrosine. Image of slot-blot film,

where 3-nitrotyrosine was detected by immunoblotting and respective

surface plot analysis (Y axis represents intensity of bands).

1st Workshop -

Novel Compounds

from Renewable

Sources


3xTg-AD mitochondrial dysfunction connected with a decay of complex I activityand with an aberrant mitochondrial in CL profile occurs mainly in synapticmitochondria preceding the traditional pathological hallmarks of disease (senileplaques and neurofibrillary tangles)

Age-dependent low catalase activity and high levels of ROS detected in 3xTg-ADmice suggests that oxidative stress should be connected with mitochondrialdysfunction

The increased LPC levels simultaneously with a decreased levels of PC containingarachidonic acid in both brain and skeletal muscle of 3xTg-AD mice indicate anenhancement of cPLA2 activity and is an evidence of systemic low gradeinflammation.

Studies with Animal model for Alzheimer`s disease

3 96 12 18

Brain amyloid-β pathology Brain tau pathology

Youth Adulthood Elderly

NonTg

3xTg-AD

Human APPSW

TauP301L

PS1M146V

1st Workshop -

Novel

Compounds

from Renewable

Sources


Monteiro-Cardoso et al. (2015). J Alzheimers Dis. 43: 1375-1392. Monteiro-Cardoso et al. (20015). Current Alzheimer Research 12: 100-115.

Studies with Animal model for Alzheimer`s disease

Membrane Lipid Changes and Chronic Low Grade Inflammation

Re

lati

ve

Ab

un

da

nc

e (

mo

l%)

73

2.8

(3

2:1

)

73

4.5

(3

2:0

)

75

4.6

(3

4:2

)

76

0.6

(3

4:1

)

78

0.7

(3

6:5

)

78

2.7

(3

6:4

)

78

4.7

(3

6:3

)

78

6.7

(3

6:2

)

80

6.5

(3

8:6

)

80

8.5

(3

8:5

)

81

0.6

(3

8:4

)

81

2.7

(3

8:3

)

82

8.5

(4

0:9

)

83

2.5

(4

0:7

)

83

4.7

(4

0:6

)

83

6.6

(4

0:5

)

85

4.5

(4

2:1

0)

85

6.6

(4

2:9

)

86

8.5

(4

2:3

)

87

0.5

(4

2:5

)

87

2.8

(4

2:1

)

87

4.6

(4

2:0

)

87

8.5

(4

4:1

2)

89

6.7

(4

4:3

)

89

8.6

(4

4:2

)

82

2.6

(o

40

:5)

83

0.7

(o

40

:1)

0

2

4

6

8

1 0

*

*

* *

**

*

* *

P C m o le c u la r s p e c ie s [M + H ]+

m /z ( c a rb o n :d o u b le b o u n d )

* **

PLA2+

PC (16:0/20:4)

LPC

(16:0/0:0)AA

C20:4

Re

lati

ve

Ab

un

da

nc

e (

mo

l%)

49

6.5

(1

6:0

)

51

8.5

(1

8:3

)

52

0.5

(1

8:2

)

52

2.5

(1

8:1

)

52

4.5

(1

8:0

)

54

2.5

(2

0:5

)

54

4.5

(2

0:4

)

54

6.6

(2

0:3

)

54

8.7

(2

0:2

)

56

8.4

(2

2:6

)

57

0.4

(2

2:5

)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0

*

*

*

**

*

*

*

*

L P C m o le c u la r s p e c ie s [M + H ]+

m /z ( c a rb o n :d o u b le b o u n d )

1st Workshop -

Novel

Compounds

from Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUP Project Design

Extraction and purification of Polyphenols, Flavonoids,

Naphthoquinones(mainly of water-soluble ) from

dietary sources

Extraction and purification of a selected pool of phospholipids (PC, PE, PG) with high-content of n-3 PUFA (n-6 /n-3 ≤ 1) from Red algae (Palmaria palmate) and

brawn algae of genus Sargassum

Work package 1 – Preparation and analysis of standardized bioactive formulationsfor clinical applications and food industry

Water solublePolyphenols,

Naphthoquinones Flavonoids

Incorporation in food matrices (e.g. dairy products) for the production of specialized foods for the elderly and to be used in pilot and interventions studies

Nanomedical platform for oral and intravenous administration to control the chronic low grade inflammation in peripheral organs

MitochondriotropicSM+

Unilamellar liposomes with size close to 100 nm and Zeta potential positive or close to zero

Nanomedical platform for nasaladministration to control the chroniclow grade inflammation in brain

1st Workshop

- Novel

Compounds

from

Renewable

Sources

NATURAL PRODUCTS RESEARCH GROUP Project DesignWork package 2 – In vitro studies: molecular mechanisms of action andtoxicological screening

The therapeutic success of nanomedical platform is dependent of five main steps to which the

experimental approaches will be addressed using immortalized cell lines and primary cell cultures:

i) cell internalization pathway in order to escape from the endosome and subsequent fusion with

mitochondrial membranes;

ii) ability to modulate da lipid composition of cell membranes, increasing the phospholipid content

with n-3 PUFA (increasing n-3/n-6 PUFA rate);

iii) ability to restore both mitochondrial functionality and redox state of cells induced by different

types of mitochondria-active drugs that also promote oxidative stress;

iV) ability to modulate the inflammation cascade induced by different types of pro-inflammatory

agents

V) Toxicological screening to find the dose range with potential therapeutic effects without cytotoxicity

1st Workshop -

Novel

Compounds

from Renewable

Sources


Water phasePolyphenols,

NaphthoquinonesFlavonoids

Work package 3 – Validation the novel therapeutic concept (liposomal formulations)to control the chronic low-grade inflammation associated with age-related diseasesby using animal models: e.g. Alzheimer’s disease, diabetes type 2, chronic kidneydisease

The therapeutic approach should be tested considering, at least, three different diseases: oneassociated with brain degeneration (e.g. AD) and two with chronic low grade inflammation inperipheral organs, such us diabetes type 2 and chronic kidney disease. However, the choice ofdiseases and corresponding animal models for the investigation are dependent on theexpertise of the research partners that we join to form the project consortium

Require the use of the integrative “omics” approaches to investigate molecularmechanisms underlying the biological effects of the liposomal formulations indifferent organs, tissues and cell types and may have also the goal to find newdisease biomarkers.

1st Workshop -

Novel

Compounds

from Renewable

Sources


Water phasePolyphenols,

NaphthoquinonesFlavonoids

Work package 4 – Evaluation the effectiveness of a bifunctional diet enriched inomega-3 fatty acids and specific pool of polyphenols/flavonoids/naphthoquinonesto control the chronic low-grade inflammation and prevent/delay the degenerationassociated to several age-related diseases, by using animal models and, eventually,performing pilot studies with humans.

Work package 5 – Management, Coordination, Dissemination and Exploitation

Objectives

To ensure the project meets its objectives within the time and the budget limits

To manage the evolution of the project and coordinate activities

To facilitate communication between the partners, and with the EU commission

To disseminate the project’s findings and promote the results on a national and European

level to stakeholders, media and society.

To establish an exploitation plan for the milestones achieved in the project

Drug Discovery

Romeu A. Videira, Fátima Fernandes, Patrícia Valentão, Andreia P. Oliveira, M. Figueiredo González,

João Bernardo, Daniela Correia, Rui Gonçalves, David M. Pereira, Paula B. Andrade


1st Workshop - Novel Compounds from Renewable Sources

apresentação do powerpoint - eventos.fct.unl.pt · natural products research group project design...

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