NS3 Protease by Amino Terminal

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Attenuation of West Nile Virus NS2B/NS3 Protease by Amino Terminal Copper and Nickel Bind-ing (ATCUN) Peptides Andrew M. Pinkham, Zhen Yu, and James A. Cowan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01409 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Journal of Medicinal Chemistry

Attenuation of West Nile Virus NS2B/NS3 Protease by Amino Terminal Copper and Nickel Binding (ATCUN) Peptides

Andrew M. Pinkham,† Zhen Yu,† and James A. Cowan†,*

†

Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Co-

lumbus, OH, 43210, USA.

* Correspondence to: Dr. J. A. Cowan, Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210.

tel: 614-292-2703, e-mail: cow-

[email protected]

1 ACS Paragon Plus Environment

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ABSTRACT West Nile Virus NS2B/NS3 protease (WNVP) is a viable target for the development of antiviral compounds. To that end, catalytic metallopeptides that incorporate the copper-binding ATCUN motif into either the N- or C-terminus of known WNVP targeting peptides have been developed as new families of peptide-based inhibitors. Each metallopeptide was evaluated based on its inhibitory constant (KI), timedependent inactivation of the protein, Michaelis-Menten parameters, and the ability to oxidatively modify WNVP. Following catalytic inactivation of WNVP, sequencing by LC-MS/MS demonstrated active site residues Ser135, Thr134, and Thr132, as well as residues in the S2 binding pocket, to be modified by oxidative chemistry. Results from a DNPH-based assay to detect oxidative damage showed the formation of carbonyls in WNVP treated with metallopeptides. These results suggest that the metallopeptides are attenuating WNVP activity by irreversible oxidation of amino acids essential to substrate binding and catalysis.

Key words: West Nile Virus; protease; ATCUN motif; mass spectrometry, copper,


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INTRODUCTION The genus Flavivirus contains many human pathogens of current concern, including dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), Zika virus (ZKV), and West Nile virus (WNV).1 WNV has rapidly spread across five continents in the last decade, causing disease and fatalities in humans and other mammals.1 It is an arthropod-borne virus (arbovirus), transmitted from an avian reservoir to its dead-end hosts of humans and horses via Culex mosquitoes.2 Until recently, infection with WNV was found to be asymptomatic, or to cause a mild febrile disease, West Nile fever.3 However, current reports show the development of flu-like symptoms and fatal WNV neuroinvasive disease.4 Global warming, climate change, urbanization, and insufficient vector controls have caused the continuous growth of the Culex mosquitoes habitat, resulting in the rapid spread of infection beyond their original geographical boundaries.5-6 The development of antiviral chemotherapeutic or vaccines against WNV remains a critical unmet medical need. WNV has an ~11.0 kb positive single-stranded RNA genome typical of the genus Flavivirus.3 The genome is translated into a precursor polyprotein that is then cleaved and co- and posttranslationally modified into various structural and nonstructural (NS) proteins.7-8 When noncovalently complexed with a NS2B cofactor, the NS3/NS2B complex comprises an active serine protease that contains a trypsin-like catalytic triad at its active site.9 The NS2B/NS3 protease is responsible for the proteolytic processing of the viral polyprotein.8 As such, the activity of the NS2B/NS3 protease is vital for viral viability, making it an attractive target for antiviral strategies.10 Substrate-mimetic inhibitor therapies have provided a successful strategy for treatment of HIV and HCV infections.11-12 WNVP has the added challenge of a solvent exposed and topologically-shallow active site with selectivity for substrates containing basic amino acids (arginine and lysine) at the P1 and P2 positions.10 Inclusion of these highly polar amino acids obstructs membrane permeability, resulting in poor pharmacokinetic profiles for substrate mimetic inhibitors. 3 ACS Paragon Plus Environment


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Peptidic inhibitors for WNVP were synthesized by attaching an amino terminal copper- and nickel-binding (ATCUN) motif to sequences based on natural substrates of WNVP and other antivirals currently in development. The attachment of the ATCUN motif allows for the creation of catalyticallyactive protease inhibitors when used under oxidative conditions. Our laboratory has previously implemented this strategy to develop antiviral, anticancer, and antimicrobial agents,13-15 and has also demonstrated catalytic inactivation of proteins through the generation of metal-associated reactive oxygen species (ROS) that can modify neighboring amino acid sidechains.13-17 The ATCUN motif was transposed to the C-terminal of a WNVP binding domain by replacing the N-terminal glycine of the tripeptide GlyGly-His with D-2,3-diaminopropionic acid (DDap), resulting in DDap-Gly-His. Targeting sequences were coupled through the 2’ amine of diaminopropionic acid allowing the 3’ amine to coordinate copper. The DDap-Gly-His motif was attached to targeting domains with poly-glycine linkers of various lengths. Inactivation studies of WNVP were performed by use of a well characterized chromophore substrate (2napthoyl-Lys-Lys-Arg-pNA),18 while characterization of oxidatively modified WNVP was achieved by use of LC-MS/MS analysis of tryptic fragments, kinetic assays to determine the catalytic parameters of attenuated protease, and detection of carbonyl formation with 2,4-dinitrophenylhydrazine (DNPH) labeling.

RESULTS

Metallopeptide Design. Catalytic metallodrug design principles have been described before and typically consist of a targeting sequence, an optional linker sequence, and an N-terminal Gly-Gly-His ATCUN motif.16, 19 The protease-targeting sequence Np-Lys-Lys-Arg (Np: 2-naphthoyl) has been shown to not only be a high affinity substrate,18 but also a potent aldehyde inhibitor20 and was co-crystallized with a truncated WNVP mu4 ACS Paragon Plus Environment

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tant.21 Given the plethora of kinetic and crystallographic information available for this sequence, NpLys-Lys-Arg was chosen to be a targeting domain. The metallopeptide Gly-Gly-His-DNap-Lys-LysArg-NH2 (5) (DNap: DN-2-Napthyl-glycine) was synthesized to evaluate reactivity when transposing the ATCUN motif to the N-terminus of the targeting domain. However, use of the standard metallodrug template would result in a peptide with suboptimal placement of the catalytic ATCUN motif at the Nterminus, away from the protease active site. In this case the N-terminal glycine of the ATCUN motif was replaced with a diaminopropionic acid residue to allow peptides to be made with a free amine (3’ amine of diaminopropionic acid) for appropriate Cu coordination while also providing a linker to continue to synthesize the targeting domain. Another targeting sequence, Bz-Arg-Lys-DPhg-NH2 (Βz: Benzoyl; DPhg: D-α-Phenylglycine), was also selected due to its high affinity for both DENV and WNV proteases (IC50 3.79 and 3.78 µM respectively) and detailed studies into binding modes.22-23 To evaluate alternative linkers, Lys-Gly-His was coupled to the targeting domain Bz-Arg-Lys-DPhg-NH2 through the lysine side chain. Additionally, Bz-Arg-Lys-DPhg-NH2 has been shown to have very little off-target binding to other biomolecules.22 Finally, ATCUN motifs were also attached to some acetylated (Ac: Acetyl) DENV and WNV protease substrate sequences (Table 1). To ensure metal binding and determine the concentrations of metallopeptides in solution, copper titration to the high affinity ATCUN sequences was performed from a copper stock of known concentration.24 Copper binding was determined by measuring the increase of absorbance at 525 nm using UV/vis spectroscopy. The resulting data was plotted as copper concentration vs absorbance and fit to a one site binding equation. All metallopeptides tested were able to bind copper with KD’s similar to the native ATCUN motif. The inclusion of a C-terminal ATCUN motif allows the placement of the catalytic domain directly towards the enzyme active site and mimics the positioning of natural substrate peptides of proteases.



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Table 1. Metallopeptide sequences. Sequences of metallopeptides with unnatural amino acids D-N-2Napthyl-glycine (DNap), D-2,3-diaminopropionic (DDap), and D-Phenylglycine (DPhg). Metallopeptide Sequence Cu2+ Cu C

NH2-Gly-Gly-His-OH

1

Np-Lys-Lys-Arg-(DDap)-Gly-His-NH2

2

Np-Lys-Lys-Arg-Gly-(DDap)-Gly-His-NH2

3

Np-Lys-Lys-Arg-Gly-(DDap)-Gly-Gly-His-NH2

4

Np-Arg-Lys-Lys-Arg- (DDap)-Gly-His-NH2

5

Gly-Gly-His-DNap-Lys-Lys-Arg-NH2

6

Bz-Arg-Lys- DPhg-(DDap)-Gly-His-NH2

7

Bz-Arg-Lys-DPhg-Gly-(DDap)-Gly-His-NH2

8

Bz-Arg-Lys-DPhg-Gly-Gly-(DDap)-Gly-His-NH2

9

Bz-Arg-Lys-DPhg-(Lys)-Gly-His-NH2

10

Ac-Gly-Phe-Ala-Ala-Gln-Arg-Arg-Ser-(DDap)-Gly-His-NH2

11

Ac-Arg-Gly-Phe-Lys-Arg-Arg-Ser-(DDap)-Gly-His-NH2

12

Ac-Asp-Pro-Asn-Arg-Lys-Arg-(DDap)-Gly-His-NH2

13

Ac-Lys-Pro-Gly-Leu-Lys-Arg-(DDap)-Gly-His-NH2

Binding Constant Determination. KI values of metallopeptides were obtained by use of a Dixon plot with substrate concentrations of 12.5, 25, and 50 µM while metallopeptide concentration was varied from 1.0 to 50 µM and reported in Table 2 and Figure S1 to S16. Metallopeptide KI values range from 8.0 µM to 63.5 µM. Exceptions include Cu-12 and Cu-C that have KI’s in excess of 200 µM. The addition of a poly-glycine linker to naphthoylated peptides resulted in an overall decrease of KI. An ~3-fold decrease of KI for Cu-1, over Cu-3 was observed. Due to the proximity of the surface-exposed Trp50 residue, Förster resonance energy transfer (FRET) from Trp50 emission to benzoylated groups of metallopeptides is a good indication of proximity to the active site. Unfortunately, due to the similar excitation wavelengths for 2-naphthoyl and Trp50, the naphthoylated inhibitors were not amenable to this approach. Also, other substrate-based 6 ACS Paragon Plus Environment

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metallopeptides lacking a benzoyl group have no mechanism to quench Trp50. However, all of the benzoylated inhibitors were able to promote a significant decrease of active site tryptophan emission (Figure S17). Binding plots were fit to a one-site quadratic binding equation25 to obtain the KD values ~1.02.0 µM (Table 3).

Table 2. Metallopeptide inhibition constants (KI). KI metallopeptides determined of (Cu denotes copper bound form) in 300 µL of activity buffer (50 mM Tris-HCl, pH 9.5, 30% Glycerol, 1 mM CHAPS) with 0.5 µM WNVP. Metallopeptide

KI (µM)

Cu Cu-C Cu-1 Cu-2 Cu-3 3 Cu-4 Cu-5 Cu-6 Cu-7 Cu-8 8 Cu-9 Cu-10 Cu-11 Cu-12 Cu-13

>200 >200 30.5 ± 3.0 20.0 ± 1.0 10.5 ± 0.5 12.3 ± 1.8 23.6 ± 1.2 8.0 ± 2.0 18.0 ± 1.0 18.0 ± 2.0 13.0 ± 1.7 12.0 ± 0.7 12.0 ± 3.0 63.5 ± 4.0 30.0 ± 5.0 >200 63.0 ± 8.0

Table 3. KD values of benzoylated peptides from Trp50 fluorescence assays. Metallopeptides tested in 300 µL aliquots at varying concentrations with 3.0 µM WNVP in activity buffer. Metallopeptide

KD (µM)

Cu-C

>1000

Cu-6

2.0 ± 1.0

Cu-7

1.6 ± 0.7

Cu-8

1.0 ± 0.1

Cu-9

1.0 ± 0.5 7

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Effects of Metallopeptides on WNVP. For Michaelis-Menten kinetic studies of Cu bound metallopeptides, the concentration of each metallopeptide was kept constant at 1 µM, with reactions containing 5 to 60 µM of WNVP and coreactants (1 mM H2O2, 1 mM ascorbate, or both). For each time point of the inactivation profile, an aliquot was withdrawn and WNVP activity was measured at that time point (Figure 1). Overall, no change was observed when WNVP was treated solely with any combination of 1 mM ascorbate and/or hydrogen peroxide or with metallopeptide only. It was only when WNVP was treated with metallopeptide and both coreagents was any noticeable effect on enzyme activity observed. Additionally, Cu-C was able to promote a slight decrease in activity when both co-reagents were present (Figure 1). The Michaelis-Menten parameters for all Cu-peptide complexes are summarized in Table 4 and displayed in Figure S18. Overall, Cu-C displayed little activity, demonstrating that the targeting domain is necessary to promote effective attenuation of the protease. The naphthoylated series of metallopeptides displayed decreasing KM with the inclusion of a polyglycine linker, with two glycines showing optimal KM. This trend was also observed in the kcat for naphthoylated series of metallopeptides, with the inclusion of a single glycine showing optimal turnover. The results of the decrease in KM and increase in kcat, lead to a synergistic effect with Cu-3 showing the best kcat/KM ratio. The metallopeptide Cu-1 showed the smallest KM, but also the second smallest kcat for naphthoylated metallopeptides. This is also seen when the ATCUN motif was transposed to the N-terminus of the targeting domain, resulting in a metallopeptide derivative Cu-5, that exhibits the least efficient kcat/KM. Overall, the benzoylated metallopeptides displayed slightly lower KM’s, but an approximate 10fold higher kcat than other series of metallopeptides. The benzoylated metallopeptides displayed kcat/KM ratios 2- to 3-fold higher as a result of increased kcat. The inclusion of the glycine linker follows the same trend as in the naphthoylated series, albeit at a smaller magnitude. The change of the orientation


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from DDap-Gly-His to Lys-Gly-His, displayed a drastic decrease in kcat, possibly reflecting the orientation of the copper center away from the active site by switching the orientation of the stereocenter.

Figure 1. (A) Representative time-dependent inactivation profiles for 10 µM WNVP (), 1 µM Cu-1 with 1 mM ascorbate and H2O2 (), 1 mM ascorbate and H2O2 (), 1 mM H2O2 (▲), 1 mM ascorbate (▼), and Cu-C with 1 mM ascorbate and H2O2 (). (B) Representative Michaelis-Menten plot for the inactivation of WNVP by Cu-1 with 1 mM ascorbate and H2O2. 9 ACS Paragon Plus Environment


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Table 4. Michaelis-Menten parameters for Cu complexes. Metallopeptides where kept at 1.0 µM in the presence of 1 mM ascorbate and H2O2, while WNVP concentration was varied from 1 to 60 µM. Metallopeptide

KM (µM)

kcat (h-1)

Cu

-

-

kcat/KM (µM-1h-1) -4

Cu-C

46.0 ± 6.6

0.05 ± 0.01

8.9 × 10 ± 5.0 × 10-4

Cu-1

16.0 ± 3.0

2.0 ± 0.5

0.1 ± 0.08

Cu-2

20.0 ± 9.0

4.0 ± 0.8

0.3 ± 0.1

Cu-3

10.0 ± 3.0

4.0 ± 0.3

0.4 ± 0.1

Cu-4

6.6 ± 1.5

1.7 ± 0.1

0.30 ± 0.06

Cu-5

40.0 ± 20.0

0.9 ± 0.4

0.02 ± 0.01

Cu-6

13.0 ± 4.0

21.1 ± 1.5

1.6 ± 0.5

Cu-7

8.4 ± 0.8

19.0 ± 1.6

2.3 ± 0.3

Cu-8

8.5 ± 0.3

21.0 ± 1.0

3.5 ± 0.1

Cu-9

12.0 ± 0.3

0.25 ± 0.1

0.02 ± 6.0 × 10-3

Cu-10

3.0 ± 1.0

1.0 ± 0.1

0.3 ± 0.1

Cu-11

2.0 ± 1.5

1.0 ± 0.1

0.5 ± 0.4

Cu-12

10.0 ± 3.0

0.1 ± 0.03

0.01 ± 4.0 × 10-3

Cu-13

8.5 ± 0.6

0.11 ± 0.10

0.013 ± 0.010

Metallopeptides based on Dengue and WNVP substrates, Cu-10 and Cu-11, displayed overall the smallest KM’s. However, these metallopeptides also showed some of the lowest kcat’s, which results in poor kcat/KM for this family of metallopeptides. At least one member of each family of metallopeptides was selected to test the attenuation of WNVP activity over time to determine the resulting effects on altered enzyme KM and kcat parameters after incubation with metallopeptides for 3 h (Table 5). Overall, the benzoylated and naphthoylated metallopeptides resulted in the largest decrease in kcat/KM, largely due to an ~200-fold decrease in kcat. Naphthoylated metallopeptides seem to function solely through modification of the protease kcat, with


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KM being only modestly affected. The large decrease in kcat suggests oxidation of those amino acid side chains in the active site of protease that are important for catalysis. The benzoylated metallopeptides display a similar trend in attenuating protease activity. However, the increase in KM is more pronounced in this family of metallopeptides, suggesting modification of amino acids that play a role in substrate binding, or possibly active site structure, rather than residues at the catalytic site.

Table 5. WNVP kinetic parameters after 3 h incubation with select metallopeptides. Sequence

KM (µM)

kcat (s-1)

kcat/KM (s-1M-1)

No metallopeptide & no coreagents

30 ± 2

1.1 ±0.1

37,000 ± 4000

1 mM H2O2 & Ascorbate

60 ± 3

1.3 ± 0.1

22,000 ± 2000

Cu-1

95 ± 5

7.5 ± 1.0 x10-3

80 ± 10

Cu-2

60 ± 4

2.2 ± 0.5 x10-3

37 ± 9

Cu-3 Cu-6 Cu-7

150 ± 20 230 ± 75 240 ± 50

9.0 ± 1.x10

-3

60 ± 10

1.1 ± 0.2 x10

-2

40 ± 10

1.0 ± 0.2 x10

-2

40 ± 12

-3

60 ± 20

Cu-8

150 ± 20

9.5 ± 1.0 x10

Cu-11

380 ± 100

0.5 ± 0.1

1300 ± 200

Mass Spectrometry. Oxidatively-modified WNVP was studied by use of LC-MS/MS following digestion with trypsin or chymotrypsin. A number of minor oxidation events were observed around the active site. These low intensity oxidation events most likely reflect either the flexibility imparted by the polyglycine linker, or non-selective activity of metallopeptides. Following treatment with any naphthoylated metallopeptide, the major oxidation event is associated with a protein fragment containing the active site Ser135 with sequence TPEGEIGAVTLDFPTGTSGSPIVDK. While disappearance of the intact Ser135-containing 11 ACS Paragon Plus Environment


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fragment (m/z = 830.0, [M+3H]3+) (Figure S19) was observed, the oxidized Ser135-containing fragment displayed a shift of +64 amu (m/z = 851.2), a mass change consistent with the uptake of four oxygen atoms. The appearance of the +64 amu peak is only observed in samples treated with metallopeptide, ruling out the alternative possibility of an ACN+Na adduct. Following fragmentation of the 851.2 peak by CID (collision-induced dissociation), it became clear that oxidized amino acids were located solely around the active site of the protein. Specifically, Ser135 displayed a +16 amu shift, Thr134 displayed a +32 amu shift, and Thr132 displayed a +16 amu shift (Figure S4). The appearance of +16 peaks is consistent with the uptake of oxygen atoms into the amino acids side chains. The mass list with assigned sequences for each naphthoylated metallopeptides is summarized in Tables S1, S2, S3 and raw LC-MS/MS sequence data in Figure S20. The normalized fraction of each oxidized amino acid is displayed in Figure 2A. It appears Cu-1 is able to mediate modest oxidation of Ser135 compared to the other two metallopeptides in the Np series. However, Cu-3 promotes significant oxidation of Thr132. All benzoylated metallopeptides exhibited similar chemistry, but target the S2 pocket of the protease. An oxidized fragment mass of m/z 428.8 (-47 amu) was observed for all benzoyl peptides (Figure S4) and is attributed to oxidation of an Asp75-containing chymotryptic fragment, with sequence GSVKEDRL (m/z = 452.5, [M+2H]2+) (Figure S21). The MS/MS degradation profiles were examined to further assess oxidative damage to amino acid sidechains (Tables S4-S6 and raw LC-MS/MS sequence data in Figure S22). In all samples, Ser71 and Lys73 were oxidized to the diol and corresponding aldehyde, displaying +16 and -1 peaks, respectively. Additionally, it appears that both Glu74 and Asp75 are both decarboxylated via the loss of CO2 and oxidized to aspartic semialdehyde and 3oxoalanine, respectively.


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Figure 2. LC-MS/MS analysis of, (A) oxidized Ser135-containing peptides (m/z = 851.2) promoted by naphthoylated metallopeptides, and (B) oxidized Asp75-containing peptide (m/z = 428.6) promoted by benzoylated metallopeptides.

DNPH Assay. While oxidative chemistry is clearly evident as the mechanism for addition of oxygens, or the loss of amino acids side chains, the products of oxidative chemistry are not readily apparent. For this reason, DNPH was used to qualitatively detect the appearance of carbonyl functionality in WNVP. Following incubation for 3 h with various metallopeptides, the reaction solutions were mixed with DNPH 13 ACS Paragon Plus Environment


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and the absorbance at 365 nm was measured. No DNPH content was seen in either the WNVP control, 1 mM ascorbate and H2O2, or Cu-C samples, as illustrated in Table 6. Copper-naphthoylated metallopeptides catalyzed oxidative modifications to WNVP that react with approximately two moles of DNPH per WNVP, indicating the formation of two ketone or aldehyde sites. Oxidation of WNVP by the benzoylated series of metallopeptides on average shows addition of approximately four moles of DNPH to one mole of WNVP, while the sample treated by the substrate-based metallopeptide Cu-11 only showed the addition of one DNPH per WNVP.

Table 6. Ratio of moles of DNPH to WNVP. Determination of DNPH content with 10 µM WNVP exposed to 1 µM Cu bound metallopeptides with 1 mM Ascorbate and 1 mM H2O2 in 50 mM Tris, pH 7.5. Sequence

DNPH:WNVP

WNVP

< 0.1

1 mM H2O2 and Ascorbate

< 0.1

Cu 1 mM H2O2 and 1 mM coreagents

8.0 ± 1.0

Cu-C

< 0.1

Cu-11

1.1 ± 1.0

Cu-1

2.5 ± 0.6

Cu-3

2.0 ± 1.0

Cu-6

3.0 ± 0.2

Cu-7

5.5 ± 0.4

Cu-8

3.0 ± 0.5

Docking of Metallopeptides. Docking studies were performed with Autodock (AD) using the crystal structure of WNV NS2B-NS3 (2FP7).20,24 Metallopeptides Cu-1 and Cu-8 were selected due to their distinct oxidation patterns characterized by LC-MS/MS and to show the effects of the polyglycine linker on placement of the 14 ACS Paragon Plus Environment

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ATCUN motif. Longer linkers positioned the Cu-C moiety directly against the catalytic triad. Overall, the targeting domain of the naphthoylated metallopeptide assumed an orientation similar to the crystal structure of WNVP complexes with Np-Lys-Lys-Arg,19 displayed in Figure 3A. The arginine was seated in the S1 pocket, while lysine was displaced from the S2 into the S1 pocket by the ATCUN motif. The last lysine of the targeting domain was located in the S3 pocket, while the naphthalene is positioned against the hydrophobic S4 pocket. The benzoylated metallopeptide assumed a cyclic-like orientation as seen in Figure 3B. The Dphenylglycine is in the S1 site, the lysine in the S3 site and the arginine in the S3 site. The benzoic acid cap is placed into the S4 site while the ATCUN motif is displaced deep into the S2 pocket.

Figure 3. Docking of (A) Cu-1 and (B) Cu-8 into WNVP crystal structure (2FP7).

DISCUSSION

Inactivation of WNVP. Inhibition is a consequence of the low KI values demonstrated by the Cu-peptides, relative to the higher values observed for Cu-C and free Cu2+(aq), both of which lack a binding motif. This drastic 15 ACS Paragon Plus Environment


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change in KI supports targeting of WNVP by the binding motifs of the metallopeptides. Incorporation of the Gly-Gly-His motif to Bz-Arg-Lys-DPhg resulted in no change of affinity for WNVP, within error. Moreover, Cu-peptides that supported longer targeting domains and/or glycine linker segments generally saw a slight enhancement in binding affinity via a decrease in KI. This trend is also repeated in the observed KD values for the benzoylated metallopeptides. The slight differences observed for KI and KD could be the result of using two different methods to determine the constants, but overall the values agree favorably. No significant in KI change between Cu-8, 8, Cu-3, and 3 was observed. This is due to the square planar coordination of copper by the ATCUN motif leaving only the axial positions open for coordination with amino acid side chains. Due to Jahn-Teller distortions, the axial positions have weakened binding, suggesting a majority of the binding interaction is dictated by the targeting sequence. No cleavage of metallopeptides by WNVP was seen (data not shown), possibly due to the added steric bulk of the copper ATCUN motif preventing favorable interaction with S1’, which has a requirement for small, hydrophobic amino acids. Additionally, the docking model in Figure 3 also seems to suggest that the ATCUN motif would be placed either in the S2 pocket, or over the active site. Inactivation studies were carried out under Michaelis-Menten conditions with metallopeptides kept at a constant 1 µM concentration and WNVP concentration varied from 5 to 100 µM. Irreversible inactivation of WNVP was only observed when both ascorbate and peroxide were present under aerobic conditions (Figure 1). The need for both ascorbate and hydrogen peroxide to be present is similar to previously reported metallodrug candidates.13 It is assumed that modification of WNVP proceeds through the addition of an oxygen-based radical that arises via the generation of a reactive copperassociated oxygen species on the metallopeptide. The copper-ATCUN motif redox cycles through the Cu3+/2+ couple17 with examples of Cu-GGH catalyzing oxidative chemistry on DNA, RNA, and various proteins. Most likely a copper-associated reactive oxygen species (ROS) promotes hydrogen abstraction from protein sidechains, where formation of ROS is most likely mediated by transient formation of 16 ACS Paragon Plus Environment

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Cu3+. An important distinction from regular Fenton chemistry is the production of metal-associated rather than diffusible ROS.16 Consequently, a specific orientation of the Cu-NH2-Gly-Gly-His-OH moiety would be needed in order to successfully deliver the ROS to its intended target, resulting in different rates for different orientations of the Gly-Gly-His. This is exemplified in the comparison of Cu-8, Cu-9, Cu-5, and Cu-1. The addition of a Lys-Gly-His moiety most likely directs the copper bound-ROS away from important active site residues. Placement of the ATCUN motif on the N-, rather than C-terminus, appears to orient the copper-bound ROS away from vital binding and catalytic residues resulting in low activity for the Cu-5 metallopeptide. The higher flexibility of the polyglycine linker may also allow the Cu-Gly-Gly-His moiety to sample more catalytically-active conformations, thus imparting a higher rate of inactivation. The physiological relevance of the coreagents has been shown to range from micromolar to millimolar for ascorbate, and picomolar to micromolar for hydrogen peroxide.26-27 While it is possible that other in vivo cellular reducing agents and coreagents may have similar effects within the cell, for the studies described herein reactive oxygen species are the most likely key intermediates. The use of hydrogen peroxide as a mechanistic “shunt” to accelerate the formation of key reaction steps is widely used in the study of peroxidase, p450 models, and our previous reports,24 including cellular studies of HCV metallodrugs that demonstrated the necessity for copper to promote cellular efficacy under redox conditions.28-29 The observation that the benzoylated metallopeptides resulted in overall higher levels of activity most likely results from several multiple factors. The actual targeting domain Bz-Arg-Lys-NH2 is reported to have an IC50 of 3.8 µM for WNV. Peptide Np-Lys-Lys-Arg-NH2 has not previously been characterized, with only aldehyde inhibitors being synthesized. The observed tighter binding by the BzArg-Lys-NH2 could result in a longer “dwell time” and with a greater chance to promote chemistry and more efficient in catalysis. This is supported by the observed kcat/KM, and reflected to a smaller extent in the KM’s of the metallopeptide families. Small movements in how the actual targeting domain sits in the 17 ACS Paragon Plus Environment


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active site of the protease could also determine which residues the ATCUN motif encounters. In the case of the benzoylated metallopeptides, the S2 pocket is lined by residues that are known to undergo oxidative chemistry (Lys73 and Glu74), while the naphthoylated metallopeptides are in contact with less oxidatively labile residues in the S1 site (Thr132, Thr134). Taken together these functions result in the benzoyl series demonstrating overall more efficient catalysis. The use of Dengue virus substrate sequences for targeting domains generally results in poorer performing catalysts than their non-native counterparts. The KI’s for these metallopeptides vary significantly from 30 to 200 µM, while the KM’s also vary from 1 to 10 µM. This could be reflective of suboptimal alignment of the dengue virus sequences in the WNVP active site, and positioning the ATCUN motif that results in little to no chemistry.

Chemistry Promoted by Metallopeptides and Characterization of Attenuated WNVP. The observed decrease of kcat/KM for the attenuated WNVP seems largely to depend on the ~ 200-fold decrease in kcat over time, reflecting the oxidation of residues that are known to be important in enzyme catalysis, and in particular Ser135 and Asp75. Residues that are consistently oxidized by the benzoylated and naphthoylated metallopeptides are outlined in Figure 4. Naphthoylated metallopeptides were observed to oxidize the active site nucleophile, Ser135, to an aldehyde and the corresponding diol would disrupt the chemistry needed to make a transient covalent bond with the substrate, resulting in an enzyme with significantly lowered activity. Additionally, a slight increase in KM for the naphthoylated series of metallopeptides is also observed, reflecting the oxidation of amino acids known to play a role in substrate binding. The oxidation of Thr134 and Thr132 could influence WNVP cleavage preference by forming a hydrogen bond with residues in the P1’ and P2’ positions, thereby limiting their mobility.36 Oxidation of Thr132 could further influence these binding interactions, resulting in a further restriction of the P1’ and P2,’ sites. The difference in oxidation events at Thr134 (+32) vs Thr132 (+16) could result from positioning of the ATCUN motif relative to each amino acid. The finding that the naphthoylated 18 ACS Paragon Plus Environment

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metallopeptide with the di-Gly linker is able to oxidize Thr132 more effectively suggests that greater flexibility and length is required to reach this amino acid.

Figure 4. Map showing oxidative damage to WNVP. Residues that are consistently modified by the naphthoylated (red) and benzoylated (cyan) metallopeptides are shown in space-filling mode.

Differences in oxidative chemistry toward WNVP was expected for the naphthoylated and benzoylated metallopeptides. While the docking for benzoylated agrees with the oxidative modifications observed, the ATCUN motif of the naphthoylated peptides appears to be displaced into the S2 subsite, while modified amino acids appear to lie around the S1 site. The addition of the flexibility imparted by DDap

and poly-glycine linker should allow the ATCUN domain to sample other conformations then

those shown in Figure 3, with only more reactive conformations showing chemistry. The KI’s between Cu-3 and 3 do not differ, within error, suggesting that the open axial coordination sites on the copper are not interacting with WNVP, and so the catalytic metal is free to sample alternative conformations around the targeting domain and the catalytically active conformation of the ATCUN domain of the naphthoylated metallopeptides may differ from the thermodynamically preferred binding conformation. 19 ACS Paragon Plus Environment


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This distinction of kinetic versus thermodynamic control of the site of oxidative damage on a target biomolecule has previously been observed in metallopeptide-promoted cleavage of HCV IRES RNA.25 The benzoylated series of metallopeptides appears to function by oxidation of residues in the S2 site, but does not appear to directly modify the active site serine. However, it appears that all benzoylated metallopeptides modify Asp75, the acid in the catalytic triad. The change in mass observed is consistent with decarboxylation of both the Asp75 side chain (-30 amu) and Glu74 sidechain (-30 amu) to 3-oxoalaline and aspartic semialdehyde

30

The differences in hydration between the N-terminal Ser71

diol and the Asp75 3-oxoalaline is possibly due to the steric constraints of the surrounding environment, with Ser71 being less sterically congested due to its location on the S2 pocket loop than the buried Asp75.31 Loss of the acidic side chain of Asp75 would also result in a protease with highly attenuated activity, thereby explaining the decrease in kcat observed after treatment with the benzoylated family of metallopeptides. Deamination of lysine to allysine is a classic sign of oxidative stress and well documented metal catalyzed oxidation reaction.32 Direct oxidation of alcohol side-chains by metal-generated ROS often proceeds through a carbonyl intermediate. DNPH, a chromophore specific for carbonyls, was used to detect these intermediates. Metallopeptide Cu-11 results in an approximately 1:1 ratio of DNPH:WNVP, with the most likely target being Thr134. Naphthoylated metallopeptides display a ratio of DNPH to WNVP of 2:1. The ratio of 2:1 DNPH: WNVP is in agreement with observations by MS sequencing that the Ser135 is converted to the corresponding vicinal diol and is in equilibrium with the aldehyde, thus accounting for one equivalent of DNPH. Thr132 has a mass shift of +16 amu, suggesting the formation of a ketone and accounting for the second equivalent of DNPH. Residue Thr134 displays a +32 shift, consistent with the uptake of two oxygen atoms. The addition of the second hydroxyl to Thr134 should proceed through a similar mechanism as C4’ hydrogen abstraction, a pathway previously observed in ATCUN chelates.16, 33 The resulting triple hydroxylated Thr residue is not reactive with DNPH and thus would not yield a signal. 20 ACS Paragon Plus Environment

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Oxidation of WNVP by benzoylated metallopeptides is exclusively seen in the S2 pocket of the protease. The oxidation of residues located on the loop of the S2 pocket results in the formation of a serine diol from Ser71 and conversion of Lys73 to allylysine. The formation of these two aldehydes results in the consumption of two moles DNPH. The remaining mole of DNPH results from decarboxylation of Glu74 and Asp75. Additionally, several minor areas of oxidation appear in samples treated with metallopeptides that include the polyglycine linker. These oxidized peptides are of low abundance and surround both the active site and the S2 site. Previous studies of small molecule metallodrugs have reported similar events with the inclusion of a flexible linker.34

CONCLUSIONS Three series of metallopeptides with naphthoylated, acetylated, and benzoylated N-terminal capping were designed by use of the catalytic metallodrug strategy. All three sets of metallopeptides are able to successfully target and inactivate WNVP. Benzoylated metallopeptides appear to function through modification of amino acid side chain residues in the S2 binding pocket via the formation of aldehydes in Ser72, Lys73, Glu74, and Asp75. The oxidation of amino acid side chains results in a decrease of enzyme kcat. Naphthoylated metallopeptides also demonstrated modification of active site residues important to substrate binding, including Thr132, Thr134, and the active site nucleophile Ser135. The oxidation of amino acid side chains by naphthoylated metallopeptides also resulted in a large decrease in kcat. Future studies will focus on the cellular permeability of such metallopeptides and their antiviral activity in cellular replicon assays



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EXPERIMENTAL SECTION General materials. The pET28(b+)-WNCf44(G4SG3) NS3pro184 construct was made by GenScript Inc. All Fmocprotected amino acids and other solid phase peptide synthesis materials were purchased from either Chem-Impex Int’l Inc., or Sigma. Trypsin and Chymotrypsin were purchased from Thermo Fischer Scientific and DNPH was purchased from Sigma. Peptides C, 4, 10, 11, 12, and 13 were purchased from Anaspec as 95% pure salts and further purified by HPLC.

pNA substrate synthesis. pNA substrates were synthesized according to the general method of Abbenante,35 on a Protein Technologies, Inc. PS3 peptide synthesizer. Substrates were characterized by analytical HPLC and mass spectrometry.

Enzyme preparation and activity assay. The previously described WNV protease plasmid pET28(b+)-WNCf44(G4SG3) NS3pro18436 was synthesized by GenScript and transformed into E. coli BL21(DE3) using standard molecular biology practices. Cells with BL21(DE3)-pET28(b+)-WNCf44(G4SG3)NS3pro184 were cultured overnight in 20 mL 2xYT medium at 37 °C with 50 µg/mL kanamycin in 50 mL sterile falcon tubes at 210 rpm. The overnight culture was transferred to 2.0 L of fresh 2xYT medium with 50 µg/mL of kanamycin and incubated at 37 °C until A600nm reached ~0.6. Expression of the recombinant protease was induced by addition of isopropyl-β-D-thiogalactopyranose (IPTG) to a final concentration of 1 mM and incubated for an additional 3 h at 22 °C. The cells were then harvested by centrifugation at 4500 g and stored at 80 °C.


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Cell pellets were thawed on ice and resuspended in 10 mL of lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM imidazole, 5% glycerol). Protease inhibitors were added to the lysis buffer to give final concentrations of 1 µg/mL benzamide and 1 mM PMSF. The resuspended cells were lysed by sonication and the insoluble fractions separated by pelleting via centrifugation at 15,000 rpm for 30 min at 4 °C. The cleared lysate was added to a 5 mL Ni2+-NTA column pre-equilbrated with 25 mL of standard buffer (50 mM HEPES , pH 7.5, 300 mM NaCl, 10 mM imidazole, 5% glycerol). The column was washed with 50 mL of standard buffer containing 40 mM imidazole and eluted with 50 mL of standard buffer with 500 mM imidazole and collected in 1 mL fractions, which were then analyzed by 12% SDS-PAGE. Protein-containing fractions were pooled and concentrated on a 10 kDa amicon membrane. The protease was then aliquoted, snap frozen in liquid N2, and stored at -80 °C until needed. The purified recombinant protease was assayed against the optimized substrate sequence of NpLys-Lys-Arg-pNA.18, 36 Assays were conducted in a 96-well plate with a final reaction volume of 300 µL that contained 0.5 µM of recombinant protease using the previously described conditions; 50 mM Tris-HCl (pH 9.5), 30% glycerol and 1 mM CHAPS. Substrate cleavage was monitored by the increase in absorbance at 405 nm and repeated in triplicate.

Preparation of metallopeptides. Peptide synthesis was performed on a Protein Technologies, Inc. PS3 peptide synthesizer by standard automated methods. Peptides were cleaved in 95% TFA, 1.25% thioanisole, 1.25% ethane dithiole, and 2.5 % diH2O at room temperature, with shaking, for four hours. The cleavage mixture was triturated three times with ice cold diethyl ether and dried overnight in a vacuum desiccator. The crude peptide was resuspended in diH2O with 0.1% TFA and purified on a prepscale Gemini C18 column from 100 % A (100% H2O, 0.1% TFA) to 100 % B (90% acetonitrile, 10% H2O, 0.1% TFA) over the course of 100 min with the UV/Vis detector set to 220 nm. Fractions were taken every 1.0 min and ana23 ACS Paragon Plus Environment


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lyzed by high resolution ESI, with fractions being >95% pure pooled together and lyophilized. All peptides were dissolved in deionized water and a stock solution of each peptide was made. Peptide purity was determined by analytical HPLC and ESI. Stocks of each peptide were run on a SunfireTM C18 5µm column with a UV detector set to 220 nm. The gradient for the HPLC assessment was developed from 100% buffer A (100% H2O, 0.1% TFA) to 100% buffer B (90% Acetonitrile, 10% H2O, 0.1 % TFA) over 30 min (Figure S23). The single peak was collected and assessed by ESI for purity. The sum of the mass intensities was determined and used to determine purity (Figures S24). The concentration of each peptide was determined by UV/vis titration (ε525 = 110 ± 6 M-1 cm-1) with a solution of known concentration of copper(II) chloride. Concentrations of Cu-peptide were prepared with a 1.1:1.0 ratio of peptide to Cu2+ in 50 mM Tris-HCl (pH 7.4) and incubated at room temperature for 30 min prior to further use.

Titration of Trp50 fluorescence quenching. A protein solution of 3 µM recombinant protease, with and without a range of inhibitors (0 to 200 µM), was made in a 96-well plate in WNVP activity buffer. After 1 h incubation at room temperature, the fluorescence was measured at 25 °C on a Spectra Max GEMINI XS with ex= 280 nm and em = 340 nm and repeated in triplicate.37 Slit widths were set to 10 and 20 nm for excitation and emission. Trp-50 fluorescence was fit to the corresponding quadratic one site binding equation Eq (1) that has been used in our previous work to determine KD‘s for the metallopeptides.21 = +

+ + − + + − 4 × × − + × 1 2

This equation is based on the intersection of two lines that represent binding limits, where is the y intercept of the first line and is the y-intercept of the second line, KD is the equilibrium dissociation constant, and is the corresponding inflection point. To account for any additional phases (i.e. a 24 ACS Paragon Plus Environment

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non-zero slope of the second line) an m term, or slope, was incorporated, and C is the independent variable in concentration. This equation was used to analyze binding plots and was used in Origin 7 to yield fitted parameters. All measurements were repeated in triplicate and reported with ± S.D.

Determination of metallopeptide kinetic parameters. Dixon plots were performed to determine KI values for all Cu-peptides (Figure S1 to S16). A 0.5 µM solution of the recombinant protease was assayed with 1, 2, 5, 10, 20, 30, 40, and 50 µM of metallopeptides and allowed to equilibrate at 37 °C for 10 min in activity buffer. Reactions were initiated by the addition of 12.5, 25, or 500 µM of Np-Lys-Lys-Arg-pNA. Measurements were repeated in triplicate and reported with standard error. Initial rates where expressed as 1/V and plots of 1/V vs Cu-peptide concentration were fit to a linear equation and KI determined. For Michaelis-Menten studies of Cu-peptides, reactions containing 1 µM of Cu-peptide were incubated with various concentrations of WNVP and coreagents (1 mM ascorbate and 1 mM H2O2) in 50 mM Tris-HCl (pH 7.4) at room temperature. At each time point, and aliquot containing 0.5 µM WNVP was transferred to the previously described activity assay and allowed to equilibrate at 37 °C for 10 min. The initial rate was collected by addition of 250 µM Np-Lys-Lys-Arg-pNA. Plots of initial velocity for the metallopeptide promoted reactions vs WNVP concentration were fit to a standard Michaelis-Menten equation using Origin 7 software to obtain Cu-peptide catalytic parameters. All measurements were repeated in triplicate and reported with standard error

Time-dependent attenuation of WNVP with metallopeptides. The following experiment was performed to determine the changes in KM and kcat that result from protease treatment with metallopeptides. Recombinant protease (25 µM) was incubated with 1 µM of metallopeptide in 50 mM Tris-HCl (pH 7.5), 1 mM ascorbate, and 1 mM H2O2 at 37.0 °C for 3 h. An aliquot of reaction mixture was taken to give a final recombinant protease concentration of 0.5 µM and 25 ACS Paragon Plus Environment


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assayed using 25, 50, 75, 100, 250, 500, 1000, and 2000 µM Np-Lys-Lys-Arg-pNA. Michaelis-Menten plots for enzyme solution taken from the solutions at each time point where used to determine the change in KM and kcat as the inactivation reaction proceeded. Each point was repeated in triplicate.

2,4-Dinitrophenylhydrazine (DNPH) assay. A 10 µM solution of WNVP was allowed to incubate with 1 µM metallopeptide under oxidative conditions in 50 mM Tris-HCl (pH 7.4) for 3 h. The reaction was stopped by addition of 500 µL 20% TCA on ice and centrifuged at maximum rpm for 10 min at 4 °C. The resulting pellet was resuspended in 6 M GuHCl pH 2.5, heated at 37 °C with gentle vortexing to dissolve. To this a 500 µL aliquot of fresh 10 mM DNPH in 2.5 M HCl was added and allowed to sit in the dark at room temperature for 1 h, with vortexing every 15 min. A control reaction was also made by the addition of 500 µL 2 M HCl to 10 µM WNVP with no coreagents or metallopeptides. After incubating at room temperature, 500 µL of 20% TCA was added and incubated on ice for 10 min. The mixture was centrifuged at max for 5 min at 4.0 °C and the supernatant was discarded. The pellets were washed 3-times with ice cold ethanol-ethyl acetate (1:1). The final precipitates are dissolved in 500 µL of 6 M GuHCl at 37 °C with gentle vortexing. The carbonyl content was calculated by obtaining the spectra at 365 nm of DNPH-treated samples (ε355 22,000 M-1 cm-1). All measurements were performed in triplicate.38

Enzyme digestion and LC-MS/MS studies. A 10 µM WNVP sample in 50 mM Tris-HCl (pH 7.4) was incubated with 50 µM metallopeptides in the presence or absence of 1 mM coreagents for 3 h, followed by three rounds of dialysis with 50 mM Tris-HCl (pH 8.0) for trypsin digestion or 100mM Tris-HCl (pH 8.0) and 10 mM CaCl2 for chymotrypsin digestion. This was performed to remove any metallopeptides or co-reagents. Samples were heated at 95 °C for 15 min to denature, and then cooled to room temperature and incubated overnight at 37 °C with trypsin or chymotrypsin in a ratio of 1:20. The resulting solution was subsequently 26 ACS Paragon Plus Environment

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injected to an LC-MS/MS instrument. Liquid chromatography was performed by use of an Agilent 1260 Series LC system equipped with a Zorbax SB-C18 column (1.8 µm, 2.1 × 50 mm). The mobile phase was composed of a mixture of acetonitrile/water with 0.1% formic acid, and a linear gradient was applied. The flow rate was 0.5 mL/min, and the temperature of the column oven was 30 °C. The LC system was directly connected to an Agilent 6460 Triple Quad mass spectrometer. The mass spectrometer was operated under the product ion mode with drying gas (N2, 350 °C) at a gas flow rate of 6 L/min and a nebulizing pressure of 30 psi. A collision energy of 30-70 V and fragmentor of 135 V were applied to promote CID (collision-induced dissociation) of the indicated protein fragments from trypsin/chymotrypsin digestion.13

Docking Studies. AutoDock 4.2.6 (AD)39 was used to perform the docking studies and investigate the binding mode of Cu-1, and Cu-8. All calculations were performed on an Intel ® Core™ i7-2600 CPU at 3.40GHz. The ligands were initially prepared using Chem3D Pro (PerkinElmer) with Ni in place of Cu to maintain the square planar geometry of the Cu-GGH motif. Autodock does not have parameters for either Cu or Ni ions, and so these were substituted by Fe ion, which should have similar atomic radii. A value of +0.8 was used to simulate the +2 charge in order to compensate for the tendency of AD to overestimate electrostatic interactions.25, 40 The crystal structure (PDB: 2FP7) of the WNV NS2B-NS3 protease in complex with the tetrapeptidyl aldehyde was used with water atoms present in the crystal structure and the bound ligand removed. Docking preparation was performed in Autodock Tools using the default setting. Polar hydrogens atoms were added, nonpolar hydrogen atoms were merged, Kollman charges were assigned to the protein, and Gasteiger charges to the ligand. AD was used with the docking parameters set at default values. The grid spacing was 1.0, and the size of the docking grid box was 25 Å, centered over the WNVP active site. Visualization was performed in Chimera.



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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website and includes details of metallopeptide synthesis, copper binding, KI, Michaelis-Menten parameters for metallopeptides, KD determinations for benzoylated metallopeptides, and raw LC-MS/MS data.

AUTHOR INFORMATION Corresponding Author *Correspondence to J. A. C., tel: 614-292-2703; fax: 614-292-1685; e-mail: [email protected]

ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (HL093446). Z.Y. was supported by the Pelotonia Fellowship Program.

ABBREVIATIONS Ac, Acetyl; AD, Auto Dock; Bz, Benzoyl;

DDap,

2,3-diaminopropionic acid; DNPH, 2,4-

Dinitrophenylhydrazine; DPhg D-α-Phenylglycine; Np, 2-Naphthoyl; DNap, D-α-2-Naphthoyl-glycine, WNV, West Nile virus; WNVP, WNCf44(G4SG3)NS3pro184.


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REFERENCES

1.

Hayes, C. G. West Nile Virus: Uganda, 1937, to New York City, 1999. Ann. N. Y. Acad. Sci. 2001, 951, 25-37.

2.

Chancey, C.; Grinev, A.; Volkova, E.; Rios, M. The Global Ecology and Epidemiology of West Nile Virus. Biomed. Res. Int. 2015, 2015, 1-20.

3.

Lanciotti, R. S. R., J. T. ; Deubel, V.; Smith, J.; Parker, M.', Steele, K.; Crise, B.; Volpe, K.E.; Crabtree, M.B.; Scherret, J.H.; Hall, R.A.; MacKenzie, J.S.; Cropp, C.B.; Panigrahy, B.; Ostlund, E.; Schmitt, B.; Malkinson, M.; Banet, C.; Weissman, J.; Komar, N; Savage, H.M.; Stone, W.; McNamara, T.; Gubler, D.J. Origin of the West Nile Virus Responsible for an Outbreak of Encephalitis in the Northeastern United States. Science 1999, 286, 2333-2337.

4.

Penn, R. G.; Guarner, J.; Sejvar, J. J.; Hartman, H.; McComb, R. D.; Nevins, D. L.; Bhatnagar, J.; Zaki, S. R. Persistent Neuroinvasive West Nile Virus Infection in an Immunocompromised Patient. Clin. Infect. Dis. 2006, 42, 680-683.

5.

Kilpatrick, A. M. Globalization, Land Use, and the Invasion of West Nile Virus. Science 2011, 334, 323-327.

6.

Hart, J.; Tillman, G.; Kraut, M. A.; Chiang, H.-S.; Strain, J. F.; Li, Y.; Agrawal, A.; Jester, P.; Gnann, J. W.; Whitley, R. J. West Nile Virus Neuroinvasive Disease: Neurological Manifestations and Prospective Longitudinal Outcomes BMC Infect. Dis. 2014, 14, 248-258

7.

Brinton, M. A. The molecular biology of West Nile virus: A New Invader of the Western Hemisphere. Annu. Rev. Microbiol. 2002, 56, 371-402.

8.

Lindenbach, B. D.; Rice, C.M. Molecular biology of flaviviruses. Adv. Virus Res. 2003, 59, 23-61.

9.

Bazan, J. F.; Fletterick, R.J. Detection of a Trypsin-like Serine Protease Domain in Flaviviruses and Pestiviruses. Virology 1989, 171, 637-639.



Page 30 of 34

10. Bastos Lima, A.; Behnam, M. A.; El Sherif, Y.; Nitsche, C.; Vechi, S. M.; Klein; C. D. Dual Inhibitors of the Dengue and West Nile Virus NS2B-NS3 Proteases: Synthesis, Biological Evaluation and Docking Studies of Novel Peptide-hybrids. Bioorg. Med. Chem. 2015, 23, 57485755. 11. De Clercq, E. Anti-HIV Drugs: 25 Compounds Approved within 25 Years After the Discovery of HIV. Int. J. Antimicrob. Agents 2009, 33, 307-320. 12.

Au, J. S.; Pockros, P. J. Novel therapeutic approaches for hepatitis C. Clin. Pharmacol. Ther. 2014, 95, 78-88.

13. Joyner, J. C.; Cowan, J. A. Targeted Cleavage of HIV RRE RNA by Rev-coupled Transition Metal Chelates. J. Am. Chem. Soc. 2011, 133, 9912-9922. 14.

Joyner, J. C.; Hodnick, W. F.; Cowan, A. S.; Tamuly, D., Boyd, R., Cowan, J. A. Antimicrobial Metallopeptides with Broad Nuclease and Ribonuclease Activity. Chem. Commun. (Camb) 2013, 49, 2118-2120.

15. Joyner, J. C.; Hocharoen, L.; Cowan, J. A. Targeted Catalytic Inactivation of Angiotensin Converting Enzyme by Lisinopril-coupled Transition-metal Chelates. J. Am. Chem. Soc. 2012, 134, 3396-3410. 16.

Joyner, J. C.; Reichfield, J.; Cowan, J. A. Factors Influencing the DNA Nuclease Activity of Iron, Cobalt, Nickel, and Copper Chelates. J. Am. Chem. Soc. 2011, 133, 15613-15626.

17.

Gokhale, N. H.; Cowan, J. A. Inactivation of Human Angiotensin Converting Enzyme by Copper Peptide Complexes Containing ATCUN Motifs. Chem. Commun. (Camb) 2005, 47, 5916-5918.

18.

Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young, P. R. Insights to Substrate Binding and Processing by West Nile Virus NS3 Protease Through Combined Modeling, Protease Mutagenesis, and Kinetic Studies. J. Biol. Chem. 2006, 281, 38448-38458.


Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19. Hocharoen, L.; Cowan, J. A. Metallotherapeutics: Novel Strategies in Drug Design. Chemistry, 2009, 15, 8670-8676. 20. Schuller, A.; Yin, Z.; Brian Chia, C. S.; Doan, D. N.; Kim, H. K.; Shang, L.; Loh, T. P.; Hill, J.; Vasudevan, S. G. Tripeptide Inhibitors of Dengue and West Nile Virus NS2B-NS3 Protease. Antiviral Res. 2011, 92, 96-101. 21.

Robin, G.; Chappell, K.; Stoermer, M. J.; Hu, S. H.; Young, P. R.; Fairlie, D. P.; Martin, J. L. Structure of West Nile Virus NS3 Protease: Ligand Stabilization of the Catalytic Conformation. JMB 2009, 385, 1568-1577.

22.

Behnam, M. A.; Graf, D.; Bartenschlager, R.; Zlotos, D. P.; Klein, C. D. Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue. J. Med. Chem. 2015, 58, 9354-9370.

23.

Behnam, M. A.; Nitsche, C.; Vechi, S. M.; Klein, C. D. C-terminal Residue Optimization and Fragment Merging: Discovery of a Potent Peptide-hybrid Inhibitor of Dengue Protease. ACS Med. Chem. Lett. 2014, 5, 1037-1042.

24.

Fidai, I.; Hocharoen, L.; Bradford, S.; Wachnowsky, C.; Cowan, J. A. Inactivation of Sortase A Mediated by Metal ATCUN Complexes. J. Biol. Inorg. Chem. 2014, 19, 1327-1339.

25. Bradford, S. S.; Ross, M. J.; Fidai, I.; Cowan, J. A. Insights into the Recognition, Binding and Reactivity of Catalytic Metallodrugs Targeting Stem Loop IIb of Hepatitis C IRES RNA. ChemMedChem 2014, 9, 1275–1285. 26. Margolis, S. A.; Duewer, D. L. Measurment of Ascorbic Acid in Human Pasma and Serum: Stability. Intralaboratory Repeatability, and Interlaboratory Reproducibility. Clin. Chem. 1996, 42, 1257-1262 27. Forman, H. J.; Bernardo, A.; Davies, K. J. What is the Concentration of Hydrogen Peroxide in Blood and Plasma? Arch. Biochem. Biophys. 2016, 603, 48-53. 31 ACS Paragon Plus Environment


Page 32 of 34

28. Bradford, S.; Cowan, J. A. Catalytic Metallodrugs Targeting HCV IRES RNA. Chem. Comm. 2012, 3118–3120. 29. Ross, M. J.; Bradford, S. S.; Cowan, J. A. Catalytic Metallodrugs Based on the LaR2C Peptide Target HCV SLIV IRES RNA. Dalton Trans. 2015, 44, 20972-20982. 30. Davies, M. J.; Fu, S.; Dean, R. T. Protein Hydroperoxides can Give Rise to Reactive Free Radicals. Biochem. J. 1995, 305, 643-649. 31.

Bell, R. P. The Reversible Hydration of Carbonyl Compounds Adv. Phys. Org. Chem. 1966, 4, 129

32.

Møller, I. M.; Rogowska-Wrzesinska, A.; Rao, R. S. Protein Carbonylation and Metal-catalyzed Protein Oxidation in a Cellular Perspective, . J. Proteomics 2011, 74, 2228-2242.

33. Joyner, J. C.; Keuper, K. D.; Cowan, J. A., Analysis of RNA Cleavage by MALDI-TOF ,ass Spectrometry. Nucleic Acids Res. 2013, 41, 1-11 . 34. Yu, Z.; Han, M.; Cowan, J. A. Toward the Design of a Catalytic Metallodrug: Selective Cleavage of G-Quadruplex Telomeric DNA by an Anticancer Copper–Acridine–ATCUN Complex. Angew. Chem. Int. Ed. Engl. 2015, 54, 1901-1905. 35.

Abbenante, G.; Leung, D.; Bond, T.; Fairlie, D. P. An Efficient Fmoc Strategy for the Rapid Synthesis of Peptide para-nitroanilides. Lett Peptide Sci. 2001, 7, 347-351

36.

Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young, P. R. Generation and Characterization of Proteolytically Active and Highly Stable Truncated and full-length Recombinant West Nile Virus NS3. Protein Expression Purif. 2007, 53, 87-96.

37.

Bodenreider, C.; Beer, D.; Keller, T. H.; Sonntag, S.; Wen, D.; Yap, L.; Yau, Y. H.; Shochat, S. G.; Huang, D.; Zhou, T.; Caflisch, A.; Su, X. C.; Ozawa, K.; Otting, G.; Vasudevan, S .G.; Lescar, J.; Lim, S. P. A Fluorescence Quenching Assay to Discriminate Between Specific and Nonspecific Inhibitors of Dengue Virus Protease. Anal. Biochem. 2009, 395, 195-204. 32 ACS Paragon Plus Environment

Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38. Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl Assays for Determination of Oxidatively Modified Proteins Methods Enzymol. 1994, 233, 346-357. 39. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M.; Belew, R. K.; Goodsell, D. S.; Olsom, A. J. Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 16, 2785-2791. 40.

Park, H.; Kim, S.; Kim, Y. E.; Lim, S.J. A Structure-Based Virtual Screening Approach toward the Discovery of Histone Deacetylase Inhibitors: Identification of Promising Zinc-Chelating Groups. ChemMedChem 2010, 5, 591–597.



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Table of Contents Graphic


NS3 Protease by Amino Terminal

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