Identification of Inhibitors for the DEDDh Family of ... - ACS Publications

Aug 16, 2016 - inhibition mechanism by inducing the general base His179 to shift out of the active site. ... designing general inhibitors targeted to ...
1 downloads 11 Views 2MB Size
Download PDF
Article pubs.acs.org/jmc

Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN‑4 Bound with 2‑Morpholin-4-ylethanesulfonate (MES) Kuan-Wei Huang,†,‡ Kai-Cheng Hsu,§ Lee-Ya Chu,‡,∥,⊥ Jinn-Moon Yang,†,#,∇ Hanna S. Yuan,*,‡ and Yu-Yuan Hsiao*,†,#,○ †

Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan, ROC Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, ROC § Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan ∥ Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Nankang, Taipei 11529, Taiwan ⊥ Institute of Bioinformatics and Structural Biology, National Tsing Hua University, 101 Kuang-Fu Road Section 2, Hsinchu 30013, Taiwan # Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu, 30050, Taiwan ∇ Center for Bioinformatics Research, National Chiao Tung University, Hsinchu 30068, Taiwan ○ Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan ‡

S Supporting Information *

ABSTRACT: The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.

■

INTRODUCTION The DEDDh family of exonucleases, also named DnaQ-like or RNase T family, constitutes more than 70 000 members widely distributed in all species from prokaryotes to eukaryotes (see RNase T family in the Pfam database). These DEDDh exonucleases bear 3′-to-5′ exonuclease activity, capable of digesting RNA and/or DNA by removing one nucleotide at a time from the 3′ end of a nucleic acid chain. They all have a DEDDh domain with a similar mixed αβ fold and four conserved acidic aspartate and glutamate residues (DEDD) for © XXXX American Chemical Society

binding two magnesium ions in the active site with a nearby general base residue, histidine or tyrosine.1,2 Although they all have a conserved fold and active site, DEDDh exonucleases have distinctive preferences in substrate binding and digestion. As examples, CRN-4 and NP exonuclease prefer to digest double-stranded DNA or RNA.3,4 Exo I and ISG20 prefer to digest single-stranded DNA or RNA,5,6 while Snp, Received: May 26, 2016

A

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3′hExo, and RNase T digest 3′ overhang of duplex nucleic acids.7,8 DEDDh exonucleases play essential roles in various cellular processes, including RNA maturation, RNA turnover, DNA replication, and DNA repair.1,2 Dysfunction of human DEDDh exonucleases is deleterious and associated with diseases. Mutations in the exonuclease domain of WRN are linked to Werner syndrome,9,10 whereas mutations in TREX1 are associated with the autoimmune diseases Aicardi-Goutieres syndrome and systemic lupus erythematosus.11,12 Moreover, several viral and human DEDDh exonucleases are essential for virus infection and gene replication, including Nsp14 of SARS coronavirus,13,14 NP exonuclease of Lassa fever virus,3,15 and human TREX1 that is hijacked by HIV to digest viral nonproductive transcripts.16,17 NP exonuclease in Lassa fever virus specifically digests double-stranded RNA to prevent viral RNA detected by the host cytosolic RNA sensors, causing suppression of innate immune signaling in the infected cells.3,15 Lassa fever virus causes severe hemorrhagic fever and infects 300 000 to 500 000 people per year in Western Africa, but there is no vaccine and limited therapeutic treatment.18 These viral or human exonucleases, including NP exonuclease, can serve as antiviral drug targets; however, no inhibitor for DEDDh exonucleases has ever been identified. All DEDDh exonucleases contain a conserved fold and active site but have divergent substrate-binding sites due to their different substrate preferences. These features are beneficial for designing general inhibitors targeted to the conserved active site and specific inhibitors targeted to different substrate-binding sites. In the first step toward a search for specific inhibitors, here we use RNase T and CRN-4 as model systems to screen for general inhibitors of DEDDh exonucleases. RNase T is a well-studied exonuclease digesting both RNA and DNA during RNA maturation and DNA repair in E. coli.19−21 CRN-4 is an apoptotic nuclease participating in chromosomal DNA fragmentation during apoptosis in C. elegans.4,22 The crystal structures of both RNase T and CRN-4 are already reported, and their biochemical properties and catalytic mechanisms are well studied. We searched in the literature and found the following potential nuclease inhibitors: citric acid (citrate), 2-(N-morpholino)ethanesulfonic acid (MES), 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid (DR396), pontacyl violet 6R (PV6R), fmoc-D -Cha-OH (FDCO), p-chloromercuriphenyl sulfonate (PCMPS), 5,5′-dithiobis(2nitrobenzoic acid) (DTNB), 7-nitroindole-2-carboxylic acid (NCA), and aurintricarboxylic acid (ATA) (see the full name and chemical structure of each inhibitor in Table 1 and Table S1 in Supporting Information). Citrate is a metabolite of the Krebs cycle that binds polynucleotide phosphorylase (PNPase) and inhibits its exoribonuclease activity in Escherichia coli.23 In addition, the nuclease activity of thermophilic bacteriophage GBSV1 (GBSV1-NSN) was reduced by 40% upon application of 10 mM citrate.24 The sulfuric group in MES (2-(N-morpholino)ethanesulfonic acid) likely mimics phosphate groups in nucleic acids, as contaminant oligomers of vinylsulfonic acid (OVS) of a MES buffer were potent inhibitors of RNase A.25−27 DR396, PV6R, and FDCO inhibit DNase γ in a dose-dependent manner,27,28 whereas the two phosphatase inhibitors (PCMPS and DTNB) have been used during nuclei isolation to prevent chromatin digestion by DNase I, DNase II, and micrococcal nuclease.29 DTNB has been used as a sulfhydryl modifying agent to react with cysteine residues in RNase T to inactivate its nuclease activity.30 NCA is a small molecule inhibitor of the

Table 1. Summary of Inhibitor Activities for the Inhibition of Exonuclease and Nucleic Acid Binding Activities for CRN-4 and RNase T inhibition of exonuclease activityb

inhibition of nucleic acidbinding activityc

inhibitorsa

CRN-4

RNase T

CRN-4

RNase T

citrate MES PV6R PCMPS NCA DR396 DTNB ATA FDCO

− + +++ +++ − + ++ ++ +

+ + ++ +++ + + ++ +++ +

− − + + − − + + +

− − − + − − + + −

a

Full names are the following: citrate, citrate acid; MES, 2-morpholin4-ylethanesulfonate; PV6R, pontacyl violet 6R; PCMPS, p-chloromercuriphenyl sulfonate; NCA, 7-nitroindole-2-carboxylic acid; DR396, 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]-2-(3-hydroxy-6-oxoxanthen9-yl)benzoic acid; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ATA, aurintricarboxylic acid; FDCO, fmoc-D-Cha-OH. bInhibitors in inhibitorcoupled nuclease activity assays are grouped into strong (+++), medium (++), weak (+), and noninhibitor (−). cInhibitors in inhibitor-coupled nucleic acid-binding assays are grouped into effective (+) and ineffective (−) compounds.

apurinic/apyrimidinic (AP) endonuclease,31 and preclinical and clinical data indicate that inhibitors of APE1 can block base excision repair, resulting in potentiation of radiotherapy for cancer cells.32,33 ATA is a general inhibitor of nucleases widely inhibiting of DNase I, RNase A, Sl nuclease, exonuclease III, and the restriction endonucleases Sal I, Bam HI, Pst I, and Sma I.34 Besides nuclease inhibitor activity, ATA is also an inhibitor of nucleic acid processing enzymes,35 kinase,36 and the JAK-STAT pathway.37 Recently, ATA has been reported as a potent inhibitor of various viruses, such as influenza A and B,38,39 human immunodeficiency virus,35,40 hepatitis C virus,41 SARS-CoV,42 and vaccinia virus.43 It is intriguing why ATA is such a broadspectrum inhibitor. It is likely that ATA has many hydroxyl and keto groups that can form hydrogen bonds with the active-site residues39,44 and that ATA may polymerize to block both nucleic acid and ATP binding on target proteins.45 Here, using inhibitor-coupled nuclease activity and nucleic acid-binding assays, we found that PV6R, PCMPS, DTNB, and ATA can efficiently inhibit the nuclease activities of RNase T and CRN-4. As proof-of-concept, we further treated NP exonuclease from Lassa fever virus with the potential inhibitors and show that indeed some of these compounds can efficiently inhibit the viral protein in vitro. The crystal structure of CRN-4 in complex with the inhibitor MES revealed a unique inhibition mechanism that involved inducing the general base His179 to shift out of the active site upon inhibitor binding. Molecular docking analysis identified the key residues for inhibitor binding that will be helpful for the design of more effective and selective inhibitors in the future. Our biochemical, structural, and molecular docking studies suggest the general lead inhibitors for the DEDDh family of exonucleases and pave the way for designing nuclease inhibitors for biochemical and biomedical applications.

■

RESULTS Screening Inhibitors for CRN-4 and RNase T. We searched the literature and found nine potential inhibitors that have been used to inhibit the activities of various nucleases B

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(Table 1 and Table S1). We first set out to identify a good substrate for both CRN-4 and RNase T that can be used for inhibitor screening experiments. RNase T digests single-stranded 11-nucleotide DNA with a higher efficiency than RNA.21 We tested the nuclease activity of CRN-4 in digesting these singlestranded 11-nt DNA and RNA. We found that CRN-4 digested 5′-end 32P-labeled single-stranded RNA with a high efficiency at concentrations greater than 0.25 μM (Figure 1A). In contrast,

On the basis of the cleavage results, the inhibitors were classified into four groups: strong, medium, weak, and noninhibiting. Strong inhibitors can limit nuclease activity at inhibitor concentrations of 0.01−0.1 mM, whereas medium inhibitors are effective at 0.1−1 mM. Weak inhibitors can only moderately inhibit nuclease activity at 1 mM, whereas noninhibitors cannot interfere with nuclease activity at concentrations up to 1 mM. For CRN-4, both PV6R and PCMPS were strong inhibitors, DTNB and ATA were medium inhibitors, MES, DR396, and FDCO were weak inhibitors, and citrate and NCA were noninhibitors (Figure 2A). For RNase T, both PCMPS and ATA were strong inhibitors, PV6R and DTNB were medium inhibitors, whereas citrate, MES, NCA, DR396, and FDCO were all weak inhibitors (Figure 2B). The inhibitory effects of these agents on CRN-4 and RNase T were consistent in that PV6R, PCMPS, DTNB, and ATA were all strong-to-medium inhibitors, whereas citrate, MES, NCA, DR396, and FDCO were weak to noninhibiting (Table 1). This result suggests that these inhibitors may share similar inhibition mechanisms or binding sites on CRN-4 and RNase T. To understand the inhibition mechanism for these inhibitors, we further examined the RNA-binding activity of CRN-4 in the presence of the inhibitors by gel shift assays. In the absence of an inhibitor, CRN-4 bound the single-stranded 11-nt RNA at a concentration of 100 μM in a buffer with or without 10% dimethyl sulfoxide (DMSO) (Figure 3A). Except for FDCO (1 mM), all the other weak-to-noninhibitors (citrate (1 mM), MES (1 and 10 mM), NCA (1 mM), and DR396 (1 mM)) did not interfere with the interactions between CRN-4 and RNA. Among the medium-to-strong inhibitors, PV6R (1 mM) reduced the interactions between CRN-4 and RNA, whereas PCMPS (1 mM), DTNB (1 mM), and ATA (1 mM) altered the binding between CRN-4 and RNA, as the location of the bands of the CRN-4-RNA complex differed from that of the control group. On the basis of the results of these RNA-binding assays, we could classify these inhibitors into two groups: the effective inhibitors that interfere with RNA-binding activity, including PV6R, PCMPS, DTNB, ATA, and FDCO, and the ineffective compounds that do not interfere with the RNA-binding activity of CRN-4, including citrate, MES, NCA, and DR396 (see summary in Table 1). For a comparison, we tested the DNA-binding activity of RNase T with or without each inhibitor using gel shift assays. We found that similar to the effect on CRN-4, PCMPS, DTNB, and ATA interfered with the DNA-binding activity of RNase T (Figure 3B). The remaining inhibitors (citrate, MES, PC6R, NCA, DR396, and FDCO) did not interfere with the DNA-binding activity of RNase T (see summary in Table 1). Taken together, these results suggest that PCMPS, DTNB, and ATA are general inhibitors for the DEDDh family of exonucleases, since they inhibit the exonuclease activities and block the nucleic acid-binding activities for both CRN-4 and RNase T. Inhibitors for Viral Protein NP Exonuclease. To demonstrate that these compounds could inhibit the exonuclease activity of viral proteins in the DEDDh family of exonucleases, we further cloned and purified the NP exonuclease from Lassa fever virus (Figure S1A). We found that NP exonuclease digests both single-stranded and stem-loop RNA (Figure S1B), and therefore a 20-nucleotide ssRNA and stem-loop RNA were used as substrates for inhibitor-coupled nuclease activity assays. We found that ATA was a strong inhibitor for NP exonuclease as the inhibitor concentration of 0.01 mM was sufficient for enzyme inhibition, whereas PV6R was a medium inhibitor as the concentration of at least 0.1 mM was required for effective

Figure 1. CRN-4 prefers to digest and bind single-stranded RNA than DNA. (A) CRN-4 cleaved a single-stranded 11-nt RNA more efficiently than DNA. (B) CRN-4 prefers to bind single-stranded RNA than DNA.

CRN-4 exhibited a low activity in digesting the 5′-end 32P-labeled single-stranded DNA at concentrations up to 4 μM (Figure 1A). The difference between 0.25 μM and 4 μM is 16-fold, so these results suggest that CRN-4 is a more efficient RNase than DNase, as RNA digestion efficiency is more than 16 times that of DNA. Moreover, CRN-4 bound RNA at a low concentration of 12.5 μM, but it bound DNA weakly at a high concentration of 200 μM as revealed by the gel retardation assays (Figure 1B). Taken together, these results show that CRN-4 binds RNA with a higher affinity and that it prefers to digest single-stranded RNA over DNA. To screen for effective inhibitors, we incubated CRN-4 (2 μM) and RNase T (0.1 μM) with each inhibitor for 10 min and then added the single-stranded 11-nt RNA or DNA for the digestion assays, respectively. After the RNA digestion reaction (see Experimental Section), the exonuclease/RNA/inhibitor mixture was resolved by 20% denaturing polyacrylamide gels to determine if the RNA substrate had been digested (see Figure 2). C

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. The exonuclease activities of CRN-4 and RNase T are inhibited by several compounds. (A) The exonuclease activity of CRN-4 was assayed by incubating with citrate, PV6R, PCMPS, NCA, DR396, DTNB, ATA, and FDCO in concentrations ranging from 0.01 to 1 mM. The concentration for MES was from 0.1 to 10 mM. (B) The exonuclease activity of RNase T was analyzed by incubating with the same compounds as in (A). The 5′-end 32Plabeled single-stranded 11-nt DNA or RNA was used in the inhibitor-coupled nuclease assays.

related to each other by a noncrystallographic 2-fold axis, similar to the structures of the apo-form of CRN-44 (see Figure 5A). CRN-4 consists of an N-terminal DEDDh exonuclease domain and a C-terminal Zn domain (see Figure 5A). The electrostatic surface potential of the Zn domain is basic, suggesting that it is involved in nucleic acid binding (Figure 5B). The nucleic acid substrate is likely bound at the Zn domain, and its 3′ end is extended and inserted into the active site in the DEDDh exonuclease domain.4 MES in the CRN-4-MES complex was bound to Thr161 and Asp184 by hydrogen bonds in the active site of the DEDDh exonuclease domain (Figure 5C). The electron density for the MES molecule in the active site of CRN-4 clearly revealed the shape of MES as a ring-like domain linked to a sulfuric group (Figure 5C). Next to MES, only one Mg2+ was bound in the active site in an octahedral geometry with six coordinated ligands: Asp15, Asp184, and four water molecules (Figure 5D). Due to the presence of MES in the active site, the general base His179 in the CRN-4-MES complex had shifted out of the active site. The superimposition of the CRN-4-MES complex, Mn2+-bound apo-form of CRN-4 (PDB entry 3CM5), and DNA/Mg2+-bound RNase T (PDB entry 3V9W) shows that their active sites are highly conserved, with the four acidic DEDD residues positioned at the same locations and orientations (Figure 6A). The MES molecule in the CRN-4-MES complex is superimposed on the general base His179 in the Mn2+-bound apo-CRN-4 (Figure 6B). As a result, the general base His179 in the CRN-4-MES complex is shifted more than 6 Å away from the active site. In summary, the MES-bound CRN-4 adopts an inactive conformation that is different from the active conformation observed in several DEDDh exonucleases with two Mg2+ ions and a nearby general base in the active site.

enzyme inhibition (Figure 4A and Figure 4B). On the other hand, citrate, MES, PCMPS, NCA, DTNB, FDCO, and DR396 were weak inhibitors, as a concentration of at least 1 mM was required for effective enzyme inhibition. The inhibition efficiencies of these inhibitors are similar to those of CRN-4 and RNase T (see Table 1), confirming that the strong inhibitors, PV6R and ATA, are likely general inhibitors for the DEDDh family of exonucleases. Moreover, we also found that PV6R and ATA inhibited NP in a dose-dependent manner when we incubated NP with a stem-loop RNA with a 3′-end overhang (Figure 4C, Figure 4D, and Figure S1C). The IC50 values for ATA, PV6R, and MES were estimated to be 6.8, 66.2, and 6693 μM at the NP concentration of 2 μM and the DNA concentration of 1 μM (Figure 4E). Taken together, these results confirm that the strong-to-medium inhibitors for CRN-4 and RNase T can also inhibit the exonuclease activity of the Lassa fever NP exonuclease in vitro. ATA and PV6R are therefore potential lead compounds for inhibition of viral exonucleases of the DEDDh subfamily. Crystal Structure of the CRN-4-MES Complex. To reveal the inhibition mechanisms of these inhibitors, we screened the conditions for cocrystallization of CRN-4 with inhibitors. We obtained co-crystals of the CRN-4-MES complex by mixing CRN-4 with DNA in 50 mM MES by the hanging-drop vapor diffusion method. The crystal structure of the CRN-4-MES complex was solved by molecular replacement using the apoform CRN-4 structure (PDB accession code 3CG7) as the searching model. The structure was refined to a resolution of 2.1 Å, and all of the diffraction and refinement statistics are listed in Table S3. No DNA molecule was observed in the structure, but only a CRN-4 dimer bound with two MES molecules was crystallized in one asymmetric unit of the orthorhombic unit cell. CRN-4 folds into a dimeric structure, with the two protomers D

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. Inhibitor-coupled RNA/DNA-binding assays of CRN-4 and RNase T. (A) The RNA-binding activity of CRN-4 was analyzed by electrophoresis mobility shift assays with or without the presence of different inhibitors. The concentrations of MES were 1 and 10 mM, and for all other inhibitors it was 1 mM. The 5′-end 32P-labeled single-stranded 11-nt RNA was used in the assays. (B) The DNA-binding activity of RNase T was analyzed by electrophoresis mobility shift assays with or without the presence of different inhibitors. The 5′-end 32P-labeled single-stranded 11-nt DNA was used in the assays.

Key Inhibitor-Binding Residues in CRN-4. To further decipher the possible inhibitor-binding sites, we used the docking program iGEMDOCK46 to fit six of the weak-to-strong exonuclease activity inhibitors (listed in Table 1), except for MES, into CRN-4. Two noninhibitors, citrate and NCA, were also used in docking analysis to serve as the negative controls. The six inhibitors and two noninhibitors were all bound in the active site pocket in the docking models, probably because the active site is located in the most concave surface of CRN-4. On the basis of the interacting residues in the docking models, the six inhibitors were classified into three groups (groups 2−4), while the noninhibitors were classified as group 5 (Figure 7A). The six inhibitors all overlapped

MES does not interfere with the RNA-binding activity of CRN-4 as shown in Figure 3A. To understand the inhibition mechanism of MES, we generated a model of the CRN-4-RNA complex using the structure of the 3′hExo-RNA complex (PDB entry 4L8R) as the template. The stem-loop RNA substrate is bound on the Zn domain and fitted well onto the CRN-4 surface. The 3′ end of the stem-loop RNA is inserted into the active site of CRN-4 (Figure 6C). The MES molecule is located in the active site, but it does not interfere with the binding of the substrate, as the scissile phosphate is bound next to MES (see Figure 6D). This result is consistent with the RNA-binding assays, suggesting that MES is a noncompetitive inhibitor of CRN-4. E

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. NP exonuclease of Lassa fever virus is inhibited by several potential inhibitors. (A, B) The exonuclease activity of NP exonuclease was analyzed by incubating NP exonuclease with singled-stranded 20-nt RNA and stem loop RNA labeled with FAM at 5′ end in the presence of each inhibitor. DR396 absorbed UV light, so the cleavage pattern was different from those of other inhibitors. (C) IC50 of ATA in inhibiting NP exonuclease was estimated by incubating NP exonuclease (2 μM) and stem-loop RNA (1 μM) with different concentrations of ATA, and the digested DNA was analyzed by 20% denaturing polyacrylamide gels. (D) Quantification results of IC50 of three independent experiments. (E) Summary of inhibition activity and IC50 for the potential inhibitors of NP exonuclease.

The inhibitors in groups 2 and 3 had similar binding modes and interacted with similar residues, such as Asp15, Glu17, Thr18, and Phe71. The main difference between the inhibitors in groups 2 and 3 was that group 2 inhibitors presented additional interactions with Arg118 and Gln25 (Figure 7A−C). These additional interactions may account for the enhanced inhibition activity of the group 2 inhibitors (ATA, DTNB, and PV6R) compared to the group 3 inhibitors (DR396 and FDCO). The docking study also revealed that PCMPS (group 4) could be anchored through a covalent linkage to the sulfur atom of Cys68, and so PCMPS inhibited CRN-4 with a higher potency than other compounds (Figure 7D). Taken together, these docking studies suggest that inhibitors that could interact with Glu17, Gln25, Phe71, Asp111, Asp115, Arg118 and the metal ion would be potent inhibitors of the exonuclease activity of CRN-4. To verify if these key residues are conserved in viral DEDDh exonucleases, we superposed two structures, apo-CRN-4 and NP exonuclease. Even though there is no significant sequence similarity between these two proteins, their overall structure and orientation of the

with RNA substrates in the CRN-4-RNA model, suggesting that they were bound at the RNA-binding sites in the active site of CRN-4 (see Figure 7B−D and Figure S2). In comparison to the crystal structure of the CRN-4-MES complex, MES was bound at the active site but did not overlap with RNA (see Figure 6D). The interaction profile shows that Glu17 and Phe71 were the two key residues that most frequently formed interactions with inhibitors in CRN-4 (Figure 7A). The inhibitors interacted with the side chains of these two residues through their ring functional groups, such as the morpholine group in MES in the crystal structure and the naphthalene group in PV6R in the docking model (Figure S2). Furthermore, all inhibitors consistently formed electrostatic interactions with the catalytic Mg2+. In contrast, citrate and NCA did not interact with these two residues (Glu17 and Phe71) or Mg2+, suggesting that these two compounds could not make stable interactions with CRN-4. This docking study thereby may explain why the two compounds, citrate and NCA, are noninhibitors for CRN-4 (Figure 7E and Figure S2). F

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 5. Crystal structure of the CRN-4-MES complex. (A) Overall crystal structure of the dimeric CRN-4 in complex with MES. The DEDDh domain is displayed in light blue, whereas the Zn domain is displayed in pink. Two MES molecules are bound in the active sites of the CRN-4 dimer. (B) Electrostatic surface potential of one CRN-4 monomer. The MES molecule is bound deeply in the active site of CRN-4. (C) OMIT electron density map (2Fo − Fc, 1σ) and the interacting residues of the MES molecule. Hydrogen bonds are displayed as red dotted lines. (D) Active site of MES-bound CRN-4. One Mg2+ ion is bound in the active site. The general base His179 shifts out of the active site upon MES binding there.

Figure 6. MES inhibits the exonuclease activity of CRN-4 by inducing a conformational change in the active site but not by blocking substrate binding. (A) Superimposition of apo-form CRN-4/Mn2+, the CRN-4-MES complex, and the RNase T-DNA complex reveals a similar geometry of the active site with four acidic residues (DEDD) and one general base (H) residue. His179 in the CRN-4-MES complex is shifted away from the active site. (B) Superimposition of apo-CRN-4/Mn2+ and the CRN-4-MES complex shows that MES occupies the position of His179 in apo-CRN-4. The general base His179 in the CRN-4-MES complex is consequently shifted away from the active site. (C) Structural model of CRN-4 bound with RNA. One monomer is displayed as a molecular surface, whereas the other is displayed as a ribbon model. The 3′ end of the stem-looped RNA is inserted into the active site. (D) Close look at the active site of the MES-bound CRN-4. MES does not overlap with the RNA in the CRN-4-RNA model. G

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

exonuclease activity of NP exonuclease in vitro with an IC50 value in the single or double-digit μM range. Our study thus identifies, for the first time, a group of effective inhibitors for the DEDDh family of exonucleases that have potential to be developed into antiviral agents. We also reveal a unique inhibition mechanism for DEDDh exonucleases through crystal structure determination of the CRN-4-MES complex. In our biochemical assays, we showed that MES is a weak inhibitor and does not interfere with the RNA-binding activity of CRN-4. The crystal structure of this association reveals that MES indeed does not interfere with substrate binding, but it does occupy the position of the general base residue. As a result, the general base, His179, which is located on a flexible loop, is shifted away from the active site upon MES binding, thereby inhibiting the activity of CRN-4. Interestingly, previous structural studies of RNase T7,47 and TREX148 also revealed a similar mechanism whereby the general base located on a flexible loop is shifted from the active site when unfavored substrates are bound to the enzyme, which consequently inhibits exonuclease activity (see Figure S3). We note that the general base His residue is always located on a loop region in the DEDDh family of exonucleases (see Table S2). Therefore, it seems feasible to trap the exonuclease in an inactive conformation by means of a small molecule, such as MES, to inhibit the nuclease activity of the DEDDh family of exonucleases. Nevertheless, the interactions between MES and CRN-4 are only maintained by a few hydrogen bonds and van der Waals interactions (Figure 5C), suggesting that MES binds weakly to CRN-4. As MES does not bind at the same location as the other four potent inhibitors, MES could be modified by adding small functional groups to increase its interactions and improve its specificity toward a specific DEDDh exonuclease. Many enzyme inhibitors have been used in antiviral therapy, including inhibitors for neuraminidases, proteases, polymerases, integrases, and reverse transcriptases.49,50 Neuraminidase inhibitors are usually the first choice for influenza prevention and treatment, although many cases of viral resistance to neuraminidase inhibitors have been reported.51,52 Similarly, a significant degree of drug resistance through mutations in reverse transcriptases, integrases, and proteases has been reported for HIV.53−55 Thus, designing new types of antiviral drugs or combining new drugs with existing ones in combination therapies is an emerging trend of drug development.50,56 The design of inhibitors for exonucleases and endonucleases is a new area for exploration. In the identification of lead inhibitors for the DEDDh family of exonucleases, our study is the first step in discovering new targets and new inhibitors for biochemical and biomedical applications.

Figure 7. Important amino acids for inhibitor binding in CRN-4 are revealed by molecular docking. (A) Eight compounds, except MES, are docked into CRN-4 using iGEMDOCK,46 and they are divided into four groups (groups 2−5) based on the interactions with CRN-4 in the docking models. The interactions of MES in CRN-4 are derived from the crystal structure of CRN-4-MES complex, and it is classified into a single group (group 1). Interactions listed on the top row marked by E indicate electrostatic interactions, by H indicate hydrogen bonding, by C indicate covalent bond, and by V indicate van der Waals interactions. A cell is colored green for a positive interaction; otherwise the cell is colored black. The symbols +++, ++, + , and − represent strong, medium, weak, and no inhibition, respectively, based on the inhibitorcoupled nuclease activity assay from Figure 3A. (B−E) Docking models for ATA, DR396, PCMPS, and citrate in the active site of CRN-4. Scissile phosphates are displayed as orange balls marked by P. Hydrogen and covalent bonds are displayed as red and green dotted lines, respectively.

five active-site residues are comparable (Figures S4A and S4B). The metal ions in these two structures, Mn2+ and Mg2+, are located at a similar position (Figure S4B). Key residues for inhibitor binding in CRN-4, including Glu17, Asp111, Asp115, and Arg118, are also superimposed well with those in NP exonuclease, Glu388, Gln459, Asp463, and Lys466 (Figure S4C). These structural features suggest that these inhibitor binding residues are conserved in viral DEDDh exonucleases.

■

■

EXPERIMENTAL SECTION

Protein Expression and Purification of CRN-4, RNase T, and NP exonuclease. The plasmid pGEX4T-2-CRN-422 was transformed into the E. coli BL21(DE3)pLysS strain and cultured in LB medium supplemented with 50 μg/mL ampicillin to an OD600 of 0.5−0.6. Expression of GST-tagged CRN-4 was induced by 1 mM IPTG at 18 °C for 20 h. The harvested cells were disrupted by a microfluidizer in lysis buffer (1 mM KH2PO4, 10 mM Na2HPO4, 20 mM NaCl, 2.7 mM KCl, pH 7.4). The crude cell extract was passed through a glutathioneSepharose column (GE Healthcare) in a standard protocol, and the collected protein fractions were digested by thrombin at room temperature for 16 h to remove the GST tag. The protein sample was further purified by a HiTrap heparin column (GE Healthcare) and a gel filtration column (Superdex 75, GE Healthcare). Purified CRN-4 samples were concentrated to 10−15 mg/mL in 50 mM Tris-HCl,

DISCUSSION AND CONCLUSIONS In this study, we identified seven compounds that can inhibit the activity of CRN-4 and RNase T from the DEDDh family of exonucleases, with weak (∼mM) to strong effects (∼10 μM). The four inhibitors PCMPS, PV6R, ATA, and DTNB are the most potent in inhibiting the exonuclease activity of both CRN-4 and RNase T (Figure 3). As proof-of-concept for showing these inhibitors may serve as leading compounds to inhibit the viral proteins in the DEDDh family of exonucleases, we further tested if these compounds could inhibit NP exonuclease from Lassa fever virus. Indeed, ATA and PV6R efficiently inhibit the H

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

pH 7.5, 300 mM NaCl. His-tagged RNase T was purified as described21 and concentrated to 20−30 mg/mL in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl. The exonuclease domain of nucleoprotein protein (NP exonuclease) from Lassa virus was cloned into expression vectors pET-28c (Novagen, USA) to generate the N-terminal His-tagged fused protein (342−569 aa). The plasmid was transformed into E. coli Tuner (DE3) strain cultured in LB medium supplemented with 50 μg/mL kanamycin. Cells were grown to an OD600 of 0.5−0.6 and induced by 1 mM IPTG at 18 °C for 18−22 h. The harvested cells were disrupted by a microfluidizer and passed through Ni-NTA resin affinity column (Qiagen Inc., USA) using a standard protocol. The protein sample was further purified by HiTrap heparin column (GE Healthcare, USA) and gel filtration chromatography column (Superdex 200, GE Healthcare, USA). Purified NP exonuclease samples were concentrated to 24 mg/mL in 300 mM NaCl and 50 mM Tris-HCl (pH 7.0). The purity of CRN-4, RNase T, and NP exonuclease was higher than 95% by SDS−PAGE analysis (Figures S1 and S5). Nuclease Activity Assays. The single-stranded 11-nt DNA (5′-AACCTTACAAA-3′) or RNA (5′-AACCUUACAAA-3′) were labeled respectively at the 5′ end with [γ-32P]ATP by T4 polynucleotide kinase and then purified by a Microspin G-25 column (GE Healthcare) to remove the nonincorporated nucleotides. Inhibitors were mixed with CRN-4 or RNase T at room temperature for 10 min. The full names and chemical structures of each inhibitor are given in Table 1 and Table S1. Citrate, MES, PV6R, and PCMPS were dissolved in water. NCA, DR396, DTNB, ATA, and FDCO were dissolved in 100% DMSO. The 5′-end labeled 11-nt ssDNA/ssRNA (20 nM) was then incubated with the exonuclease/inhibitor mixture in 120 mM NaCl, 2 mM MgCl2, and 20 mM Tris-HCl pH 7.0, with or without the presence of 10% DMSO, at room temperature for 30−60 min. The reaction times for CRN-4, RNase T, and NP exonuclease are 30, 40, and 60 min, respectively. The reaction was stopped by the addition of DNA loading dye at 95 °C for 5 min. The DNA/RNA digestion patterns were resolved on 20% denaturing polyacrylamide gels and visualized by autoradiography (Fujifilm, FLA-5000). The 5′-end FAM labeled 20-nt ssRNA (5′-ACUGGACAAAUACUCCGAGG-3′) and a stem-loop RNA with a 4-nt 3′ overhang (5′-GGCCCTCTTTAGGGCCTTGG-3′) were used as substrates for NP exonuclease activity assays. These substrates (1 μM) were incubated with the NP or NP/inhibitor in 120 mM NaCl, 2 mM MgCl2, and 20 mM Tris-HCl pH 7.0, with or without the presence of 10% DMSO, at room temperature for 60 min. The samples were resolved on 20% denaturing polyacrylamide gels and visualized by ultraviolet light. Software programs, ImageJ and GraphPad Prism 6, were used in quantification of cleavage patterns and calculation of the half maximal inhibitory concentration (IC50). DNA/RNA-Binding Assays. The single-stranded 11-nt DNA or RNA substrates were labeled as described in the previous section. Inhibitors in different concentrations as shown in Figures 2 and 3 were mixed with CRN-4 or RNase T at room temperature for 10 min. The 5′-end labeled 11-nt DNA/RNA substrates (20 nM) were then incubated respectively with different exonuclease/inhibitor mixtures in a buffer containing 50 mM Tris-HCl, pH 7.0, 120 mM NaCl, 10 mM EGTA, and 40 mM EDTA, with or without 10% DMSO, at room temperature for 20 min. After incubation, the reaction mixture was applied to 20% TBE gels, which were exposed to a phosphorimaging plate (Fujifilm) for autoradiographic visualization. Crystallization, Data Collection, and Structure Determination of the CRN-4-MES Complex. CRN-4-MES complex crystals were grown by the hanging-drop vapor diffusion method at 4 °C. Purified CRN-4 samples were mixed with single-stranded DNA (5′-GCTTAC-3′) in the molar ratio of 1:1.2. The crystallization drop was made by mixing 1 μL of protein−DNA mixed solution and 1 μL of reservoir solution (50 mM MES monohydrate, pH 6.0, 20 mM magnesium chloride hexahydrate, 15% v/v 2-propanol). Crystals appeared after 3−5 months. The crystal was cryoprotected by a soaking buffer (50 mM MES monohydrate, pH 6.0, 20 mM magnesium chloride hexahydrate, 15% v/v 2-propanol, 30% glycerol) for data collection at 100 K. The singlewavelength diffraction data of the CRN-4-MES crystal were collected via beamline 13B1 at NSRRC, Taiwan.

The diffraction data were processed by HKL2000. The structure was solved by a molecular replacement method using the crystal structure of the apo-form CRN-4 (PDB accession code 3CG7) as the search model by MOLREP in CCP4 suite.57 Interestingly, no DNA was found in the crystal structure, but only two CRN-4-MES complexes were identified in one asymmetric unit. The models were built by Coot58 and refined by Phenix.59 Structural coordinates and diffraction structure factors were deposited in the RCSB Protein Data Bank with the PDB code of 5DK5 for CRN-4-MES complex. Molecular Docking. The Mn-bound (PDB accession code 3CM5) and MES-bound structures of CRN-4 were used for the molecular docking. To determine the substrate-binding site, a structure of a homologous exonuclease (3′hExo, PDB code 4L8R) in complex with a stemloop RNA was aligned with CRN-4 using a structural alignment tool.60 The inhibitor-binding site was defined by residues ≤10 Å from the inhibitor. The structures of each inhibitor were generated using the CORINA software.61 These inhibitor structures were docked into CRN-4 using the in-house docking tool, iGEMDOCK.46 iGEMDOCK is an extension of GEMDOCK62 and provides a graphical interface to visualize protein−inhibitor interactions. Since the chlorine atom could be dissociated from PCMPS, which formed a covalent linkage with the cysteine,63−65 PCMPS was in the dissociated form during docking and the scoring function of GEMDOCK was modified to include the possible covalent linkage. Finally, iGEMDOCK was used to generate interactions between inhibitors and amino acid residues, including electrostatic, hydrogen-bonding, and van der Waals interactions.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00794. Tables S1−S3 listing candidate inhibitors, general base residues, and crystal data; Figures S1−S5 showing IC50, docking models, general base residues, structural comparisons, and SDS−PAGE analysis (PDF) Accession Codes

PDB entry code for CRN-4-MES complex is 5DK5. Authors will release the atomic coordinates and experimental data upon article publication.

■

AUTHOR INFORMATION

Corresponding Authors

*H.S.Y.: e-mail, [email protected]; phone, +886-2-27884151. *Y.-Y.H.: e-mail, [email protected]; phone, +886-3-571-2121, extension 56999. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Portions of this research were carried out at the National Synchrotron Radiation Research Center. The Synchrotron Radiation Protein Crystallography Facility is supported by the National Core Facility Program for Biotechnology. Additional assistance was provided by the Institute of Molecular Biology English Editing Core. Funding is from Academia Sinica and Ministry of Science and Technology, Taiwan, ROC, to Y.-Y.H. (Grant MOST103-2311-B-009-001-MY3) and H.S.Y. Funding for open access charge is from Academia Sinica, Taiwan.

■

ABBREVIATIONS USED MES, 2-morpholin-4-ylethanesulfonate; citrate, citrate acid; PV6R, pontacyl violet 6R; PCMPS, p-chloromercuriphenyl sulfonate; NCA, 7-nitroindole-2-carboxylic acid; DR396, 4-[(4,6-dichloro1,3,5-triazin-2-yl)amino]-2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic I

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes. Nat. Immunol. 2013, 14, 61−71. (17) Yan, N.; Regalado-Magdos, A. D.; Stiggelbout, B.; Lee-Kirsch, M. A.; Lieberman, J. The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nat. Immunol. 2010, 11, 1005−1013. (18) Ogbu, O.; Ajuluchukwu, E.; Uneke, C. J. Lassa fever in West African sub-region: an overview. J. Vector Borne Dis. 2007, 44, 1−11. (19) Li, Z.; Pandit, S.; Deutscher, M. P. 3′ exoribonucleolytic trimming is a common feature of the maturation of small, stable RNAs in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 2856−2861. (20) Li, Z.; Pandit, S.; Deutscher, M. P. Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T. RNA 1999, 5, 139−146. (21) Hsiao, Y. Y.; Fang, W. H.; Lee, C. C.; Chen, Y. P.; Yuan, H. S. Structural insights into DNA repair by RNase T–an exonuclease processing 3′ end of structured DNA in repair pathways. PLoS Biol. 2014, 12, e1001803. (22) Parrish, J. Z.; Xue, D. Functional genomic analysis of apoptotic DNA degradation in C. elegans. Mol. Cell 2003, 11, 987−996. (23) Nurmohamed, S.; Vincent, H. A.; Titman, C. M.; Chandran, V.; Pears, M. R.; Du, D. J.; Griffin, J. L.; Callaghan, A. J.; Luisi, B. F. Polynucleotide phosphorylase activity may be modulated by metabolites in escherichia coli. J. Biol. Chem. 2011, 286, 14315−14323. (24) Song, Q.; Zhang, X. Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1. BMC Biotechnol. 2008, 8, 43. (25) Zhang, M.; Stauffacher, C. V.; Lin, D.; Van Etten, R. L. Crystal structure of a human low molecular weight phosphotyrosyl phosphatase. Implications for substrate specificity. J. Biol. Chem. 1998, 273, 21714− 21720. (26) Smith, B. D.; Soellner, M. B.; Raines, R. T. Potent inhibition of ribonuclease A by oligo(vinylsulfonic acid). J. Biol. Chem. 2003, 278, 20934−20938. (27) Park, C.; Raines, R. T. Origin of the “inactivation” of ribonuclease A at low salt concentration. FEBS Lett. 2000, 468, 199−202. (28) Sunaga, S.; Kobayashi, T.; Yoshimori, A.; Shiokawa, D.; Tanuma, S. A novel inhibitor that protects apoptotic DNA fragmentation catalyzed by DNase gamma. Biochem. Biophys. Res. Commun. 2004, 325, 1292−1297. (29) Prentice, D. A.; Kitos, P. A.; Gurley, L. R. Effects of phosphatase inhibitors on nuclease activity. Cell Biol. Int. Rep. 1985, 9, 1027−1034. (30) Li, Z. W.; Zhan, L. J.; Deutscher, M. P. The role of individual cysteine residues in the activity of Escherichia coli RNase T. J. Biol. Chem. 1996, 271, 1127−1132. (31) Madhusudan, S.; Smart, F.; Shrimpton, P.; Parsons, J. L.; Gardiner, L.; Houlbrook, S.; Talbot, D. C.; Hammonds, T.; Freemont, P. A.; Sternberg, M. J.; Dianov, G. L.; Hickson, I. D. Isolation of a small molecule inhibitor of DNA base excision repair. Nucleic Acids Res. 2005, 33, 4711−4724. (32) Raffoul, J. J.; Heydari, A. R.; Hillman, G. G. DNA repair and cancer therapy: targeting APE1/Ref-1 using dietary agents. J. Oncol. 2012, 2012, 370481. (33) Fishel, M. L.; Kelley, M. R. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target. Mol. Aspects Med. 2007, 28, 375−395. (34) Hallick, R. B.; Chelm, B. K.; Gray, P. W.; Orozco, E. M. Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic-acid isolation. Nucleic Acids Res. 1977, 4, 3055−3064. (35) Cushman, M.; Wang, P. L.; Chang, S. H.; Wild, C.; De Clercq, E.; Schols, D.; Goldman, M. E.; Bowen, J. A. Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight. J. Med. Chem. 1991, 34, 329−337. (36) Tsi, C. J.; Chao, Y.; Chen, C. W.; Lin, W. W. Aurintricarboxylic acid protects against cell death caused by lipopolysaccharide in macrophages by decreasing inducible nitric-oxide synthase induction via IkappaB kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase inhibition. Mol. Pharmacol. 2002, 62, 90−101.

acid; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ATA, aurintricarboxylic acid; FDCO, fmoc-D-Cha-OH; PNPase, polynucleotide phosphorylase; GBSV1-NSN, thermophilic bacteriophage GBSV1; OVS, vinylsulfonic acid; AP, apurinic/apyrimidinic; IC50, half-maximum inhibitory concentration

■

REFERENCES

(1) Zuo, Y.; Deutscher, M. P. Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res. 2001, 29, 1017−1026. (2) Yang, W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 2011, 44, 1−93. (3) Hastie, K. M.; King, L. B.; Zandonatti, M. A.; Saphire, E. O. Structural basis for the dsRNA specificity of the Lassa virus NP exonuclease. PLoS One 2012, 7, e44211. (4) Hsiao, Y. Y.; Nakagawa, A.; Shi, Z.; Mitani, S.; Xue, D.; Yuan, H. S. Crystal structure of CRN-4: implications for domain function in apoptotic DNA degradation. Mol. Cell. Biol. 2009, 29, 448−457. (5) Korada, S. K.; Johns, T. D.; Smith, C. E.; Jones, N. D.; McCabe, K. A.; Bell, C. E. Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion. Nucleic Acids Res. 2013, 41, 5887−5897. (6) Horio, T.; Murai, M.; Inoue, T.; Hamasaki, T.; Tanaka, T.; Ohgi, T. Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease. FEBS Lett. 2004, 577, 111−116. (7) Hsiao, Y. Y.; Duh, Y.; Chen, Y. P.; Wang, Y. T.; Yuan, H. S. How an exonuclease decides where to stop in trimming of nucleic acids: crystal structures of RNase T-product complexes. Nucleic Acids Res. 2012, 40, 8144−8154. (8) Kupsco, J. M.; Wu, M. J.; Marzluff, W. F.; Thapar, R.; Duronio, R. J. Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3′hExo/ERI-1 family of 3′ to 5′ exonucleases. RNA 2006, 12, 2103−2117. (9) Zhao, N.; Hao, F.; Qu, T.; Zuo, Y. G.; Wang, B. X. A novel mutation of the WRN gene in a Chinese patient with Werner syndrome. Clin. Exp. Dermatol. 2008, 33, 278−281. (10) Perry, J. J.; Yannone, S. M.; Holden, L. G.; Hitomi, C.; Asaithamby, A.; Han, S.; Cooper, P. K.; Chen, D. J.; Tainer, J. A. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 2006, 13, 414−422. (11) Crow, Y. J.; Hayward, B. E.; Parmar, R.; Robins, P.; Leitch, A.; Ali, M.; Black, D. N.; van Bokhoven, H.; Brunner, H. G.; Hamel, B. C.; Corry, P. C.; Cowan, F. M.; Frints, S. G.; Klepper, J.; Livingston, J. H.; Lynch, S. A.; Massey, R. F.; Meritet, J. F.; Michaud, J. L.; Ponsot, G.; Voit, T.; Lebon, P.; Bonthron, D. T.; Jackson, A. P.; Barnes, D. E.; Lindahl, T. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 cause Aicardi-Goutieres syndrome at the AGS1 locus. Nat. Genet. 2006, 38, 917−920. (12) Lee-Kirsch, M. A.; Gong, M.; Chowdhury, D.; Senenko, L.; Engel, K.; Lee, Y. A.; de Silva, U.; Bailey, S. L.; Witte, T.; Vyse, T. J.; Kere, J.; Pfeiffer, C.; Harvey, S.; Wong, A.; Koskenmies, S.; Hummel, O.; Rohde, K.; Schmidt, R. E.; Dominiczak, A. F.; Gahr, M.; Hollis, T.; Perrino, F. W.; Lieberman, J.; Hubner, N. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 are associated with systemic lupus erythematosus. Nat. Genet. 2007, 39, 1065−1067. (13) Graham, R. L.; Becker, M. M.; Eckerle, L. D.; Bolles, M.; Denison, M. R.; Baric, R. S. A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease. Nat. Med. 2012, 18, 1820−1826. (14) Minskaia, E.; Hertzig, T.; Gorbalenya, A. E.; Campanacci, V.; Cambillau, C.; Canard, B.; Ziebuhr, J. Discovery of an RNA virus 3′- > 5′ exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5108−5113. (15) Qi, X.; Lan, S.; Wang, W.; Schelde, L. M.; Dong, H.; Wallat, G. D.; Ly, H.; Liang, Y.; Dong, C. Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 2010, 468, 779−783. (16) Hasan, M.; Koch, J.; Rakheja, D.; Pattnaik, A. K.; Brugarolas, J.; Dozmorov, I.; Levine, B.; Wakeland, E. K.; Lee-Kirsch, M. A.; Yan, N. J

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(58) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126− 2132. (59) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; GrosseKunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213−221. (60) Shindyalov, I. N.; Bourne, P. E. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng., Des. Sel. 1998, 11, 739−747. (61) Sadowski, J.; Gasteiger, J.; Klebe, G. Comparison of Automatic Three-Dimensional Model Builders Using 639 X-ray Structures. J. Chem. Inf. Model. 1994, 34, 1000−1008. (62) Yang, J. M.; Chen, C. C. GEMDOCK: a generic evolutionary method for molecular docking. Proteins: Struct., Funct., Genet. 2004, 55, 288−304. (63) Pike, A. C.; Brzozowski, A. M.; Walton, J.; Hubbard, R. E.; Thorsell, A. G.; Li, Y. L.; Gustafsson, J. A.; Carlquist, M. Structural insights into the mode of action of a pure antiestrogen. Structure 2001, 9, 145−153. (64) Birck, C.; Poch, O.; Romier, C.; Ruff, M.; Mengus, G.; Lavigne, A. C.; Davidson, I.; Moras, D. Human TAF(II)28 and TAF(II)18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family. Cell 1998, 94, 239−249. (65) Ackerman, S. J.; Liu, L.; Kwatia, M. A.; Savage, M. P.; Leonidas, D. D.; Swaminathan, G. J.; Acharya, K. R. Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion. J. Biol. Chem. 2002, 277, 14859−14868.

(37) Chen, C. W.; Chao, Y.; Chang, Y. H.; Hsu, M. J.; Lin, W. W. Inhibition of cytokine-induced JAK-STAT signalling pathways by an endonuclease inhibitor aurintricarboxylic acid. Br. J. Pharmacol. 2002, 137, 1011−1020. (38) Hashem, A. M.; Flaman, A. S.; Farnsworth, A.; Brown, E. G.; Van Domselaar, G.; He, R.; Li, X. Aurintricarboxylic acid is a potent inhibitor of influenza A and B virus neuraminidases. PLoS One 2009, 4, e8350. (39) Hung, H. C.; Tseng, C. P.; Yang, J. M.; Ju, Y. W.; Tseng, S. N.; Chen, Y. F.; Chao, Y. S.; Hsieh, H. P.; Shih, S. R.; Hsu, J. T. Aurintricarboxylic acid inhibits influenza virus neuraminidase. Antiviral Res. 2009, 81, 123−131. (40) Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 3322−3326. (41) Chen, Y.; Bopda-Waffo, A.; Basu, A.; Krishnan, R.; Silberstein, E.; Taylor, D. R.; Talele, T. T.; Arora, P.; Kaushik-Basu, N. Characterization of aurintricarboxylic acid as a potent hepatitis C virus replicase inhibitor. Antivir. Chem. Chemother. 2009, 20, 19−36. (42) He, R.; Adonov, A.; Traykova-Adonova, M.; Cao, J.; Cutts, T.; Grudesky, E.; Deschambaul, Y.; Berry, J.; Drebot, M.; Li, X. Potent and selective inhibition of SARS coronavirus replication by aurintricarboxylic acid. Biochem. Biophys. Res. Commun. 2004, 320, 1199−1203. (43) Myskiw, C.; Deschambault, Y.; Jefferies, K.; He, R.; Cao, J. Aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors. J. Virol. 2007, 81, 3027−3032. (44) Yap, Y.; Zhang, X.; Andonov, A.; He, R. Structural analysis of inhibition mechanisms of aurintricarboxylic acid on SARS-CoV polymerase and other proteins. Comput. Biol. Chem. 2005, 29, 212−219. (45) Shadrick, W. R.; Mukherjee, S.; Hanson, A. M.; Sweeney, N. L.; Frick, D. N. Aurintricarboxylic acid modulates the affinity of hepatitis C virus NS3 helicase for both nucleic acid and ATP. Biochemistry 2013, 52, 6151−6159. (46) Hsu, K. C.; Chen, Y. F.; Lin, S. R.; Yang, J. M. iGEMDOCK: a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis. BMC Bioinf. 2011, 12 (Suppl. 1), S33. (47) Hsiao, Y. Y.; Yang, C. C.; Lin, C. L.; Lin, J. L.; Duh, Y.; Yuan, H. S. Structural basis for RNA trimming by RNase T in stable RNA 3′-end maturation. Nat. Chem. Biol. 2011, 7, 236−243. (48) de Silva, U.; Choudhury, S.; Bailey, S. L.; Harvey, S.; Perrino, F. W.; Hollis, T. The crystal structure of TREX1 explains the 3′ nucleotide specificity and reveals a polyproline II helix for protein partnering. J. Biol. Chem. 2007, 282, 10537−10543. (49) De Clercq, E. Strategies in the design of antiviral drugs. Nat. Rev. Drug Discovery 2002, 1, 13−25. (50) 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. (51) Ison, M. G. Antivirals and resistance: influenza virus. Curr. Opin. Virol. 2011, 1, 563−573. (52) Samson, M.; Pizzorno, A.; Abed, Y.; Boivin, G. Influenza virus resistance to neuraminidase inhibitors. Antiviral Res. 2013, 98, 174−185. (53) Hosseinipour, M. C.; Gupta, R. K.; Van Zyl, G.; Eron, J. J.; Nachega, J. B. Emergence of HIV drug resistance during first- and second-line antiretroviral therapy in resource-limited settings. J. Infect. Dis. 2013, 207 (Suppl. 2), S49−S56. (54) Berkhout, B.; Eggink, D.; Sanders, R. W. Is there a future for antiviral fusion inhibitors? Curr. Opin. Virol. 2012, 2, 50−59. (55) Das, K.; Arnold, E. HIV-1 reverse transcriptase and antiviral drug resistance. Part 1. Curr. Opin. Virol. 2013, 3, 111−118. (56) Bean, P. New drug targets for HIV. Clin. Infect. Dis. 2005, 41 (Suppl. 1), S96−S100. (57) Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1131−1137. K

DOI: 10.1021/acs.jmedchem.6b00794 J. Med. Chem. XXXX, XXX, XXX−XXX