Andre S. Godoy

2015-16

January 2016

ConfirmedReported

Zika cases

http://www.healthmap.org/zika/

ConfirmedReported

http://www.healthmap.org/zika/

March 2016

Zika cases

C E NS1

NS2A NS3 NS4

BNS5

Mtase NS5-RdRp

NS4A

pr M

NS2BNH2- -OH

Structural proteins

Non-structural proteins

Science, Vol. 352, Issue 6284, pp. 467-470, 2016

• RNA-dependent RNA polymerase (RdRp) is an enzyme that catalyzes the replication of RNA

• Initiation is primer-independent (de novo)

• Essential for virus replication – exciting target !

Sofosbuvir in complex with HCV NS5 RdRP – Science (2015) v. 347, p 771- 775

Godoy, AS. et al. Nat. Commun. v8, 2017

Palm

Fing

ers

Thum

b

Prim

ing

loop

Zinc site

Zinc site

Fragment-based drug design (FBDD)

• Each heavy atom (HA) increases required Chemical Space (CS) by 8 fold

• 103 molecules of MW 190 Da explore similar CS of 1018 molecules of MW 440 Da

J. Med. Chem., 2016, 59 (18), pp 8189–8206

• 500 fragments tested• About 450 datasets collected and processed (From 1.3 – 3.0

Å)• 166 events (33%)• about 16 sites (a lot in interfaces)• 44 reliable and on interesting sites (8%)

• Several hits at the RNA binding site, previously unexplored

SDII

SDI

SDIII

NTP binding site

RNA cleft

ZIKV NS3-Hel structure Theorical predicted sites

• Several hits at the RNA binding site, previously unexplored

protein ligand complexes for all flaviviral NS3-Hel in the PDB Results from the X-Chem

experiment

• Three fragments close within binding distance at the RNA binding site

under development…

Cell lines expressing reporter containing viral replicons

Dra. Rafaela FernandesProfa. Laura Gil, Fiocruz, PE

TOGAVIRIDAEAnsP1 nsP2 nsP3 nsP4 C E3 E15’ Cap AAA

3’ E2UTR UTR

BnsP1 nsP2 nsP3 nsP4 Reporter Resistance5’ Cap AAA

3’

UTR UTRUbi

6K

Antiviral assay using BHK-21 cell line expressing YFV reporter replicon. Indirect immunofluorescence assay (IFA). NS1 protein expression was evidenced using a monoclonal primary antibody (mAbF2) antibody and FITC-conjugated secondary antibody.

Flaviviral RepliconsB

HK

-21

BH

K-2

1-R

epY

FV7D

-LU

C

Transmembrane viral complex

Transmembrane viral complex

Ms. Edwin QuesnayProfa. Andre Ambrosio

Transmembrane viral complex

15 nm

4 nm

6 nm

6 nm16 nm

4.4 nm

5.8 nm

NS3 PDB 6HM3NS4B Rosetta ModelGFP

Gabriela NoskeVictor OliveiraMs. Nathalya FurtadoDr. Myrna Bonaldo

NS2B

NS3 protease

NS2B NS3 protease NS3 helicase46 92 1 H53 D77 S138 176

46 92 G4SG4 5 H53 D77 S138 176

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Inhi

bitio

n

Concentration M)

CfFS 2

IC50= 0.152 uM 0.06

0.1 1 10 100

0.0

0.5

1.0

Inhi

bitio

n

Concentration (uM)

IC50= 4.1 uM 0.3

CfFS51

0.1 1 10 100

0.0

0.2

0.4

0.6

0.8

1.0

Inhi

bitio

n

Concentration (uM)

IC50= 8.0 uM

CfFS52

0.1 1 10 100

0.0

0.2

0.4

0.6

0.8

Inhi

bitio

n

Concentration (uM)

IC50= 8.7 uM 0.8

CfFS 28

NS2B-NS3protease YFV

Enzyme kinetics of YFV protease versus synthetic

peptide

compounds series examples

CfFS2

CfFS28

CfFS51

CfFS52

(about 1,000 tested so far)

- Recombinant proteins- Enzyme assays- Biochemical assays- Cell-based assays- X-ray XTAL- Cryo-EM

• Zika Virus• Yellow Fever Virus• Chikungunya Virus• Mayaro Virus

Congratulations and thank you !

Gustavo Lima Andre Ambrósio

Bruna Macedo