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. 2018 Aug 28;9(10):1025-1029.
doi: 10.1021/acsmedchemlett.8b00316. eCollection 2018 Oct 11.

One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease

Natalia Gruba  1 , Jose Ignacio Rodriguez Martinez  2 , Renata Grzywa  3 , Magdalena Wysocka  1 , Marcin Skoreński  3 , Agnieszka Dabrowska  2   4 , Maria Łęcka  3 , Piotr Suder  5 , Marcin Sieńczyk  3 , Krzysztof Pyrc  2   4 , Adam Lesner  1
Affiliations

One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease

Natalia Gruba et al. ACS Med Chem Lett. .

Abstract

Although the mosquito-borne Zika virus was discovered in the late 1940s of the 20th century, for years it was neglected, as the disease in humans was rare and relatively mild. Viral NS2B-NS3 protease is essential for virus replication, and except for maturation of viral proteins, it also modulates the infection microenvironment to facilitate virus invasion. Here, we report the combinatorial chemistry approach for the synthesis of internally quenched substrates of the Zika virus NS2B-NS3 protease that were optimized in prime positions of the peptide chain. Final substrate ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 displays an excellent kinetic parameter (k cat/K M reaching nearly 1.26 × 108 M-1 × s-1), which is over 10 times greater than previously reported (7.7 × 106 M-1 × s-1) substrate. Moreover, it was found to be selective over West Nile virus protease.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Deconvolution of the ABZ-Val-Lys-Lys-Arg-X1′-X2′-X′-Tyr(3-NO2)-NH2 library against ZIKV NS2BLNNS3pro. Enzymatic hydrolysis of the library was performed in PBS (pH 7.4) at 37 °C for 30 min. The fluorescence increase of the ABZ-peptide (excited at 320 nm) was monitored at 450 nm.
Figure 2
Figure 2
HPLC analysis of prime site libraries. (A,C) Chromatography of peptide library and single peptide (E) in assay buffer, while chromatograms (B), (D), and (F) show library analysis after incubation with enzyme. Fluorescence detection mode was used to measure the fluorescence of ABZ-peptide (Ex. 320 nm, Em. 450 nm).
Figure 3
Figure 3
Kinetic parameters of selected ZIKV NS2BLNNS3pro substrate ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 determined in PBS (pH 7.4) (A) and Tris-CHAPS-glycerol (pH 7.4) (B) buffers.
Figure 4
Figure 4
pH dependence of ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 substrate processing by NS2BLNNS3pro at different pH values ranging from 3 to 10.
Figure 5
Figure 5
Titration of ZIKV NS2BLNNS3pro with ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 substrate. Enzymatic hydrolysis was performed in PBS (pH 7.4) at 37 °C for 30 min.
Figure 6
Figure 6
Cleavage rates of peptide ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 incubated with ZIKV protease (1) and West Nile Virus protease (2). Enzymatic hydrolysis was performed in PBS (pH 7.4) at 37 °C for 30 min.
Figure 7
Figure 7
Binding model of ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 in the active center of ZIKV NS2B-NS3 protease (PDB code 5LC0). The oxygen atoms are indicated in red, nitrogen in blue, and sulfur in yellow.
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