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Professor Doutor, MS-3.1 2010-2014
Professor Doutor, MS-3.2 2014-2019
Professor Associado, MS-5.1 2019-atualINBEQMeDIInstituto Nacional de Biotecnologia e Química Medicinal em Doenças Infecciosas
Pesquisador AssociadoCoordenadores: Prof. Dr. Glaucius Oliva Prof. Richard C. Garratt
Pesquisador AssociadoCoordenador:Prof. Dr. Glaucius Oliva
Integração da Biologia Estrutural com a Química Medicinal:• elucidação e compreensão das bases moleculares responsáveis
pela estrutura, função e reconhecimento molecular de alvos biológicos;
• descoberta de candidatos a novos agroquímicos e fármacos
6
Distribution91 countries on the tropical e subtropical regions of the planet
Causative AgentProtozoan parasites of the genus Plasmodium
Global Impact (2017)Cases: 203–262 millions/year (219 millions)Mortality: 235.000–639.000/year (435.000/year)435.000 deaths / 518.400 min ≃ 1 death/min!60% of deaths occurring in children under the age of 5 yearsPeople at risk: > 3.4 billions
TreatmentFirst-line treatment for P. falciparum infections (in regions where chloroquine resistant parasites are present): Combination therapy of artemisinin derivatives with partner drugs (ACT) • artemether–lumefantrine (Coartem; Novartis) • amodiaquine–artesunate (Coarsucam; Sanofi-Aventis)
World Health Organization. World Malaria Report 2018
Malaria is a devastating infectious disease that is characterized by intermittent high fevers and, in the case of cerebral malaria, neurological complications, such as brain injury and coma.
• Glycolytic enzymes play important roles in Plasmodium biology• Intra-erythrocytic stages of P. falciparum lack the functional tricarboxylic acid cycle • Enzymes of the glycolytic pathways are attractive targets for antimalarial drug discovery and development• Enolase (E.C. 4.2.1.11), the ninth enzyme of the glycolytic pathways, is a dimeric enzyme of 100 KDa molecular mass and magnesium dependent
7phosphoenolpyruvate2-phosphoglycerate
F
NH
N
N N
NH
NH
HNOOH2N
O
IC50 = 0.32 ± 0.04 µM
10-2 10-1 1 101
0
20
40
60
80
100
Parameter Value Std. Error
IC 50 0,3212 0,0490Slope factor -0,7640 0,0852
[enoblk] µM10-2 10-1 1 101
%Inibição
0
20
40
60
80
100
% In
hibi
tion
11
F
NH
N
N N
NH
NH
HNOOH2N
O
1/[PGA] (µM)-1
-0,02 0 0,02 0,04
1/V (µmol/s) -1
0
20
4060
80
100120
140
160180 Controle
0,16 µM
0,31 µM
0,60 µM
1,0 µM
1,80 µM
Ki = 0.20 ± 0.01 µM K’i = 0.82 ± 0.05 µM
Control
Non-competitive Inhibitor
Ki K’i
ENOblockDifratômetro Micromax-
007R-AXIS IV ++
Resolution (Å) 89,61 – 1,80
Space Grup P21
Cell Parameters (Å) a=59,9 b=77,4 c=90,19
Cell Parameters (°) α=90,00 β=96,51 γ=90,00
Total number
reflections
61.761
Unique number
reflections
9.392
Multiplicity 2,1 (2,0)
Completeness (%) 89,9 (89,8)
Rmerge (%) 6,7 (48,0)
<(I)/σ(I)> 10,9 (2,1)
Rwork (%) 20,1
ENOBLOCKPlasmodium falciparum
IC50 (µM)Cytotoxicity
IC50 (µM)Ring Schizont BGM HepG2 SI
3D7 2.2 ± 0.1 2.5 ± 0.3 > 750 > 750 > 375K1 1.9 ± 0.2 3 ± 1 > 750 > 750 > 375
1E-3 0.01 0.1 1 100
10
20
30
40
50
60
70
80
90
100
IC90 = 4,3
Enoblock 3d7
Via
bilid
ade
Concentração (uM)
IC50 = 2,4
1E-3 0.01 0.1 1 100
10
20
30
40
50
60
70
80
90
100
IC90 = 4,7
Enoblock 3d7Esquizonte
Via
bilid
ade
Concentração (uM)
IC50 = 2,2
1E-3 0.01 0.1 1 100
10
20
30
40
50
60
70
80
90
100
IC90 = 5,5
Enoblock K1
Via
bilid
ade
Concentração (uM)
IC50 = 2,3
1E-3 0.01 0.1 1 100
10
20
30
40
50
60
70
80
90
100
IC90 = 6,7
Enoblock K1Esquizonte
Via
bilid
ade
Concentração (uM)
IC50 = 4,3
ENOBLOCKPlasmodium falciparum
IC90 (µM)Ring Schizont
3D7 4.3 ± 0.2 4.7 ± 0.3K1 5.5 ± 0.2 6.7 ± 0.8
Dr. Anna Caroline C. Aguiar
(Peters, 1965) Parasitemia evaluation
Infection: 5x105 iRBC P. berghei (strain NK65)
VO tratment for 3 days(100 mg/Kg)
Blood smear
Dr. Anna Caroline C. Aguiar
Para
site
mia
(%)
Not tre
ated
Enoblo
ck 10
0 mg/K
g
Chloroqu
ine 20
mg/Kg
Not tre
ated
Enoblo
ck 10
0mg/Kg
Chloroqu
ine 20
mg/Kg 0
5
10
15
20
*
*
*
*Day 5
Day 7
16
82%
70%
*p<0.05
Plasmodium berghei parasitemia reduction after oral treatment with Enoblock (100 mg/Kg, during 3 consecutives days)
Not treated
Enoblock
Day 5 Day 7
Day 5 Day 7
Dr. Anna Caroline C. Aguiar
17
Prof. Luiz Carlos Dias (Unicamp) and Prof. Rafael Guido (USP)
Defeating Malaria Together
MMV 12/0103 Long duration heterocycles_(Aza)Benzimidazole
17
• Inhibitory activity evaluation (SYBR Green assay)
against sensitive and resistant strains of P. falciparum
• 140 compounds tested in 2018
• Generation of resistant clones to unravel the mode of action
• Cytotoxicity testing (HepG2 cells)
• Schizont maturation test in P. vivax and P. falciparum Brazilian isolates
Center of Excellence for MMV projects in Porto Velho at Rondonia Tropical Medicine Center in cooperation with Dr. Dhelio B. Pereira
40 compounds/year from MMV projects
USP-CEPEM-MMV Team
Prof. Rafael Guido Prof. Glaucius Oliva Dr. Anna Caroline Aguiar Me. Guilherme SouzaDr. Dhelio B. Pereira
FCF-USP• Prof. Celia R. S. Garcia
Collaborators
UNIFESP• Prof. Fabio C. Cruz Dr. Paul Willis
Dr. Javier Gamo
UNICAMP• Prof. Luiz Carlos Dias• Prof. Carlos R. D. Correia• Prof. Jose Luiz Costa
Ao Prof. Dr. Glaucius Oliva pela confiança, suporte, orientação, exemplo de liderança e amizade.
Guido RVC, 2008
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