Rafael V. C. Guido

Center for Research and Innovation in Biodiversity and Drugs

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

In vivo Assays

Inhibitor Candidates

Inhibitors Inhibitors

Lead candidates

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

Mg+2

Dr. Fernando Maluf

9

SuperpositionAPO / PEP+Mg2+

APO / PEP+Mg2+

Dr. Fernando Maluf

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

In vitroInhibitory activity and toxicity evaluation

Dr. Anna Caroline C. Aguiar

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