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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
NATURAL PRODUCTS RESEARCH GROUP
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
NATURAL PRODUCTS RESEARCH GROUP
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
Skills and Expertise
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.
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 exhibit antioxidant/antiradical activity
Skills and Expertise
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.
Skills and Expertise
O2 -
L-Citrulline
L-Arginine
NO
ONOO -
NOS
O2
NADPH
NADP+
iNOS inhibition
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 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.
Skills and Expertise
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
Concentration (g/mL)
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
Concentration (g/mL)
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
NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise
Biological behaviour of Naphthoquinones from Diospyros chamaethamnusDinter ex Mildbr. - molecular mechanism of action
Pinho BR, Sousa C, Valentão P, Andrade PB. PLoS ONE (2011) 6(8): e24098.
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
RAW 264.7macrophages
1st Workshop -
Novel Compounds
from Renewable
Sources
NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise
Pinho BR, Sousa C, Valentão P, Andrade PB. PLoS ONE (2011) 6(8): e24098.
RAW 264.7macrophages
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
NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise
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
NATURAL PRODUCTS RESEARCH GROUP Skills and Expertise
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
NATURAL PRODUCTS RESEARCH GROUP Project Design
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
NATURAL PRODUCTS RESEARCH GROUP Project Design
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
NATURAL PRODUCTS RESEARCH GROUP
1st Workshop - Novel Compounds from Renewable Sources
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