Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening
Abstract
:1. Introduction
2. Results
2.1. FBS-X Reveals Two Novel Drug Binding Sites on ZIKV NS3-Hel
2.2. Site A Is at a Critical Interface of the NS3-NS5 Flaviviral Replication Complex (RC)
2.3. Binding of Fragments 1 and 2 to Site A Offers Multiple Drug Design Prospects
2.3.1. Fragment 1
2.3.2. Fragment 2
2.4. Site B Is at a Flexible, Interdomain Hinge That is Highly Conserved across Flaviviruses
2.5. Fragments 3 and 4 Collectively Reveal Features Enabling Efficient Binding at Site B
2.5.1. Fragment 3
2.5.2. Fragment 4
3. Discussion
4. Materials and Methods
4.1. Fragment Libraries
4.2. Protein Purification and Crystallization
4.3. Fragment Soaking and X-ray Data Collection
4.4. NMR STD Experiments
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Virus | ZIKV | DENV | Kunjin | JEV | YFV | MVEV | TBEV |
---|---|---|---|---|---|---|---|
PDB Code | This Work | 2JLQ | 2QEQ | 2Z83 | 1YKS | 2V8O | - |
Site A | R439 | R | R | R | K | R | R |
I441 | I | I | I | A | I | E | |
N568 | N | T | T | E | S | A | |
N569 | N | N | N | H | N | N | |
T570 | Q | T | A | E | I | A | |
I571 | I | I | I | I | I | V | |
M572 | L | L | L | L | L | D | |
M595 | L | I | L | C | L | K | |
A597 | A | A | A | E | A | A | |
C600 | Y | Y | Y | S | Y | F | |
S601 | A | S | A | S | S | K | |
Site B | V191 | V | V | V | V | V | V |
A287 | A | A | A | A | A | A | |
H288 | H | H | H | H | H | H | |
F314 | F | F | F | A | F | L | |
T316 | T | T | T | T | T | T | |
P320 | P | P | P | P | P | P | |
E413 | E | I | S | I | E | E | |
T449 | T | T | T | S | T | T | |
S452 | S | S | S | S | S | S | |
Y508 | Y | Y | Y | Y | Y | Y | |
P510 | P | P | P | V | P | P |
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Parameters | Results |
---|---|
Total number of compounds selected for screening | 200 |
Number of compounds successfully soaked into individual crystals for data collection 1 | 154 |
Number of datasets that diffracted to better than 3 Å | 88 |
Median resolution of collected datasets | 2.2 Å |
Number of hits 2 | 4 |
Binding Site | A (NS3-NS5 interface) | B (Interdomain hinge region) | ||
---|---|---|---|---|
Fragment Hits | 1 | 2 | 3 | 4 |
Chemical name | 2,6-di-aminopurine | 5-methylisatin | 3-amino-1-[2-(4-chlorophenyl)ethyl] thiourea | 2,4-dicholorobenzamide |
Structural formula | ||||
Molecular weight (Da) | 150 | 160 | 230 | 190 |
Data collection 1 | ||||
Cell parameters (space group P21): | ||||
a, b, c (Å) | 49.92, 73.22, 58.45 | 50.12, 73.35, 59.06 | 49.89, 73.46, 60.72 | 49.95, 74.17, 58.58 |
β (°) | 90.85 | 91.66 | 99.51 | 90.7 |
Resolution range (Å) | 46–1.9 | 46.6–2.2 | 49.21–2.35 | 49.8–1.80 |
Unique reflections | 33305 (2063) | 21559 (1783) | 17920 (1742) | 39559 (2239) |
Rmerge | 0.10 (1.21) | 0.187 (1.63) | 0.178 (1.62) | 0.12 (1.74) |
Rmeas | 0.12(1.44) | 0.22 (1.93) | 0.21 (1.95) | 0.14 (2.05) |
I/σ(I) | 10.3 (1.3) | 6.8 (1.1) | 7.4 (1.1) | 11.1 (1.2) |
CC1/2 | 0.99 (0.99) | 0.97 (0.99) | 0.99 (0.36) | 0.99 (0.51) |
Completeness (%) | 99.6 (94.4) | 98.7 (95.1) | 98.6 (96.8) | 99.6 (95.2) |
Redundancy | 6.8 (6.5) | 6.9 (6.9) | 7.0(6.5) | 6.8 (6.6) |
Refinement | ||||
Rwork | 0.194 | 0.219 | 0.202 | 0.185 |
Rfree | 0.224 | 0.242 | 0.264 | 0.217 |
No. of atoms: | ||||
Protein | 3544 | 3544 | 3544 | 3434 |
Ligand/ions | 81 | 69 | 107 | 43 |
Water molecules | 354 | 276 | 149 | 491 |
Mean B factor (Å2) | 36.6 | 44.5 | 52.0 | 28.1 |
R.m.s. deviations: | ||||
Bond lengths (Å) | 0.008 | 0.008 | 0.011 | 0.008 |
Bond angles (°) | 0.96 | 0.96 | 0.91 | 0.94 |
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Munawar, A.; Beelen, S.; Munawar, A.; Lescrinier, E.; Strelkov, S.V. Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening. Int. J. Mol. Sci. 2018, 19, 3664. https://doi.org/10.3390/ijms19113664
Munawar A, Beelen S, Munawar A, Lescrinier E, Strelkov SV. Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening. International Journal of Molecular Sciences. 2018; 19(11):3664. https://doi.org/10.3390/ijms19113664
Chicago/Turabian StyleMunawar, Ali, Steven Beelen, Ahmad Munawar, Eveline Lescrinier, and Sergei V. Strelkov. 2018. "Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening" International Journal of Molecular Sciences 19, no. 11: 3664. https://doi.org/10.3390/ijms19113664