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TRANSCRIPT
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