Mining a KrÃ¶hnke Pyridine Library for Anti-Arenavirus Activity - ACS

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Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity Pedro O Miranda, Beatrice Cubitt, Nicholas T. Jacob, Kim D Janda, and Juan Carlos de la Torre ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00236 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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ACS Infectious Diseases

Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity Pedro O. Miranda#†, Beatrice Cubitt§†, Nicholas T. Jacob#, Kim D. Janda# and Juan C. De la Torre§* #

Departments of Chemistry, Immunology and Microbiology, The Skaggs Institute for Chemical Biology, The WIRM Institute, § Department of Immunology and Microbiology The Scripps Research Institute, La Jolla, CA 92037 10550 N. Torrey Pines Road, IMM-6, La Jolla, CA 92037 Corresponding author Email: [email protected] Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines and current anti-arenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus-host interactions suitable for targeting, as well as lead compounds to develop future anti-arenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3dihydrobenzo[b][1,4] dioxin-6-yl)-4’-methoxy-[1,1’-biphenyl]-3-yl)thiophene-2-carboxamide], as having strong anti-LCMV activity in cultured cells. KP-146 did not inhibit LCMV cell entry, but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop anti-arenaviral drugs. Key Words: arenavirus, Kröhnke pyridine library, Protein-Protein Interaction, antiLCMV activity Many emerging human viral diseases are caused by RNA viruses, whose inherent ability to diversify genetically and phenotypically endow them with the ability to cause frequent and unpredictable zoonotic events, some of them causing significant public health, social and economic burden. Implementation of safe and effective vaccine represents the most effective way to control viral diseases with large populations at risk of infection. However, the development of antiviral drugs can significantly contribute to mitigate the impact of zoonotic events. The use of non-peptidic, small-molecule combinatorial chemical libraries provides a powerful approach to identify lead compounds that inhibit virus multiplication by interfering with any of the vast array of viral and viral-host protein-protein interactions (PPIs) required for the completion of the virus life cycle. Small molecule mediated disruption of PPIs involving large and relatively shallow interfaces has been facilitated by the identification of hot spots that include specific amino acid residues that contribute significantly to the stability of the protein–protein complexACS Paragon Plus Environment

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es. These hot spots are typically lined with aromatic residues that engage in π-π and cation-π interactions.1-2 From a heuristic view, these hot spots can be seen as hydrophobic, slot-like regions or “card readers” wherein insertion of ridged scaffolds should disrupt the PPI. This conceptual approach led to the design of non-peptidic libraries containing compounds with planar, aromatic core structures elaborated with chemical diversity that are intended to function as inhibitors of PPIs.3 We have successfully implemented this concept to identify inhibitors of the MYC-MAX PPI4 within a Krönhke-Pyridine combinatorial library (KPL),5 and the fusogenic activation of HIV-1 gp41 protein using an Ugi-derived PPI library.6 In this work, we have explored whether our combinatorial KPL library could be further mined for the identification of compounds with anti-arenavirus activity. Arenaviruses are enveloped viruses with a bi-segmented negative strand RNA genome. Each genome segment, large (L, ca 7.3 kb) and small (S, ca 3.5 kb) uses an ambisense coding strategy to direct synthesis of two polypeptides.7-8 The S segment encodes for the viral nucleoprotein and the glycoprotein precursor GPC, which is co- and post-translationally processed by the signal peptidase and SKI-1/S1P cellular proteases, respectively, to produce the mature surface virion glycoproteins GP1 and GP2 and the stable signal peptide (SSP) that together form the GP1/GP2/SSP complex (GPcx) present at the surface of mature virions and that mediate receptor recognition and cell entry.7, 9 The L segment encodes for the viral RNA dependent RNA polymerase (L polymerase) and the matrix Z protein. Arenaviruses cause chronic infections of rodents with worldwide distribution, and human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose important public health problems in their endemic regions.7, 10-14 Thus, Lassa virus (LASV) infects several hundred thousand people yearly in West Africa that are associated with high morbidity and mortality, resulting in a high number of Lassa fever (LF) cases. Notably, increased travelling has resulted in cases of LF in non-endemic metropolitan areas including the US.15-16 Moreover, mounting evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance.17-20 In addition, several arenaviruses including LASV pose a credible biodefense threat.21 Concerns about arenavirus infections of humans are exacerbated due to the lack of FDA-licensed vaccines and current anti-arenaviral therapy being limited to an off-label use of ribavirin that is only partially effective and has a narrow therapeutic window.22-24 Several compounds have been reported to have anti-arenaviral activity in cultured cells, but their in vivo safety and efficacy remain to be determined.25 However, the broad-spectrum RNA-dependent RNA polymerase inhibitor favipiravir (a.k.a. T-705), as well as the GP-mediated fusion inhibitor ST-193, have shown very promising results in different animal models of arenaviral HF disease including protection of guinea pigs against a lethal LASV challenge.26-29 Nevertheless, the identification and characterization of additional safe and effective anti-arenaviral drugs can facilitate the implementation of combination therapy to combat human pathogenic arenaviruses, an approach known to counteract the emergence of drug resistant variants often observed with mono therapy strategies. Moreover, the observed sensitivity of a drug-resistant mutant to another drug with an independent mechanism of activity is usually further enhanced by the fitness cost often inflicted by a resistance mutation. Here, we document the screening of a 220-member KPL for compounds with anti-LCMV activity (Figure 1). This campaign has led to the identification of KP-146 [(5-(5-(2,3dihydrobenzo[b][1,4]dioxin-6-yl)-4’-methoxy-[1,1’-biphenyl]-3-yl)thiophene-2-carboxamide] as having strong anti-LCMV activity (EC50 = 0.37 µM, SI = 110). We used established cell-based assays30-36 to examine the effect of KP-146 on each step of the arenavirus life cycle, and found that KP-146 exerted its antiviral activity by interfering with the activity of both the LCMV vRNP, responsible for directing the biosynthetic processes of RNA replication and gene tranACS Paragon Plus Environment

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scription, and LCMV Z-mediated virus budding. Our attempts to select LCMV variants resistant to KP-146 were unsuccessful. KP-146 also inhibited multiplication of two other arenaviruses tested, Tacaribe (TACV) and Junin (JUNV). In contrast, KP-146 had only a very modest effect on multiplication of the paramyxovirus human parainfluenza virus type 3 (HPIV3) and the picornavirus coxsackievirus B3 (CVB3). Serial passages of LCMV in the presence of KP-146 (12.5 µM) did not result in selection of a viral population with increased resistance to KP-146. Our findings demonstrate how a simple ridged scaffold displaying three points of diversity can provide novel antiviral probes for the investigation of human pathogenic arenaviruses. RESULTS AND DISCUSSION Design and synthesis of KPL and KP-146: The KPL screened in this study was prepared as previously described.5 Briefly, a series of aldehyde components were coupled to JandaJel-NH2 resin wherein these resins were split and mixed followed by introduction of R2 substituents, which was accomplished via a Claisen-Schmidt condensation using a variety of methylketones. In a second parallel step, the R3 substituent was derived via a sequential Michael-type addition and ring closure in the presence of NH4OAc. The sum of this chemical sequence resulted in a library of 2, 4, 6-trisubstituted pyridine resins 4 as 55 batches (Supplementary Scheme 1). Synthesis of the identified “hit” KP-146 (Scheme 1) was accomplished as described in the Materials and Methods section.

Figure 1. Schematic of KPL screen for anti-LCMV activity. A549 cells were infected with a GFP-tagged rLCMV (moi = 0.01) and treated with each individual pool of the KPL. At 48 h p.i. GFP expression levels were determined. Pools with anti-LCMV activity were deconvoluted to identify individual compounds with anti-LCMV activity.

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Scheme 1: Synthesis of KP-146 O O

Br

O

1 a O

O N

O

O

Br

O OMe

O N

2

N

c

+

d, e, f

O

S

S

OMe

O

CO2Me CO2Me

MeO 5

6

S CONH 2 KP-146

b O OHC

S

+ OMe 4

CO2Me

3

a

Reactions and conditions: (a) Py, THF, r.t. overnight; (b) LiOH, MeOH, r.t. 30 minutes; (c) NH4OAc, DMF, 100 °C overnight; (d) LiOH, THF: H2O (8:2), r.t. overnight; (e) oxalyl chloride, DMF, DCM, r.t. overnight; (f) NH4OH, THF, r.t. Screen of the KPL to identify inhibitors of LCMV multiplication: The KPL was screened in 55 pools of four compounds (10 µM total pool compound concentration) to identify pools able to reduce levels of GFP in rLCMV/GFP-P2A-NP infected cells compared to vehicle treatment (see figure 1). We used ribavirin (Rib) (200 µM), a validated inhibitor of LCMV multiplication, as positive control. Selection of pools containing primary hits was based on the following criteria: 1) strong (> 50%) inhibition of GFP expression, and 2) low toxicity (cell biomass > 60%, as determined by DAPI staining) when compared to vehicle-treated and infected cells (Fig 2A). We identified pool #17 as able to significantly inhibit GFP expression levels in rLCMV/GFPP2A-NP infected A549 cells. We re-screened pool #17 using the same criteria and confirmed its LCMV inhibitory activity. We de-convoluted pool #17 by testing individually each of the compounds present in the pool for its ability to inhibit multiplication of LCMV/GFP-P2A-NP, as determined by reduced GFP expression levels in infected cells, in the absence of cytotoxicity. Compound KP-146 inhibited GFP expression levels by 40% when present at 27 µM (Fig 2B), whereas at this concentration KP-146 caused less than 20% reduction in levels of DAPI staining (not shown), that served as a surrogate to assess significant differences in cell numbers at the experimental endpoint. We observed similar results with compound KP-158, but resynthesis of this compound proved to be a cumbersome and a low-yield process, which led us to focus our efforts on the characterization of KP-146. We did not pursue further compound KP-143 as its anti-LCMV activity was associated with a significant decrease in cell biomass. Each compound in pool #17 was present at 2.5 µM, and at their combined concentration of 7.5 µM, KP-143, KP-146 and KP-158 appeared to exert a stronger anti-LCMV activity than each individual compound at 9 µM, suggesting that these compounds might exhibit some synergistic activity.


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Figure 2. Screening KPL pools. A. Identification of KPL pool compounds with anti-LCMV activity. A549 cells were infected (moi = 0.01) with rLCMV/GFP-P2A-NP. Virus was allowed to adsorb to cells (0.2 ml/well; M24 plate format) for 60 min and then treated with 55 different KPL pools (10 µM total compound concentration per pool; final volume of 0.5 ml/M24 well). At 48 h p.i. GFP expression levels were determined. Infectivity (%) was normalized to 100 % corresponding to GFP expression levels observed in vehicle treated infected cells. Each pool was tested in triplicate and variability among replicates was less than 10% (not shown). B. AntiLCMV activity of individual CC compound within KPL pool #17. Cells were infected as in A and treated with the indicated concentrations of each individual compound from KPL pool #17. At 48 h p.i. GFP expression levels were determined. Infectivity (%) was normalized to 100 % corresponding to GFP expression levels observed in vehicle treated infected cells. Data represent means ± SD of two independent experiments. Each independent experiment consisted of three replicates. C. Structures of KP-146 and KP-158. Dose-dependent effect of KP-146 on LCMV multiplication: To examine the dose-dependent effect of KP-146 on rLCMV/GFP-P2A-NP propagation and production of infectious progeny in A549, we infected cells with rLCMV/GFP-P2A-NP (moi = 0.01) in the presence of the indicated concentrations of KP-146. As a control we treated cells with compound KJ-Pyr-9 (KDJ9), from the same KPL and which was shown to interfere with MYC-MAX complex formation4 and was not present within pool #17 that exhibited anti-LCMV activity. At indicated h p.i. we determined the percentage of infected cells in the population, based on GFP expression (Fig 3A), production of infectious progeny (titers in TCS) (Fig 3B) and levels of viral RNA synthesis (Fig 3C). KP-146 exhibited a strong inhibitory effect on both virus propagation and production of infectious progeny in TCS as determined by immune focus forming assay (IFFA), which correlated with reduced levels of viral RNA synthesis. DAPI staining showed that at the highest concentration of 50 µM, KP-146 had only a modest effect in the cell number, whereas we did not observe differences between cells treated with 12.5 or 25 µM KP-146 and vehicle-treated control cells.


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Figure 3. Effect of KP-146 on virus propagation, production of infectious progeny and viral RNA synthesis. A. A549 cells were infected (moi = 0.01) with rLCMV/GFP-P2A-NP. After 60 min virus adsorption to cells (0.2 ml/well; M24 plate format), cells were treated (final volume of 0.5 ml/M24 well) with the indicated concentrations of either KP-146 or as control CC compound KJ-Pyr-9. A. At the indicated h p.i. cells were fixed (4% PFA) and GFP expression assessed by fluorescent microscopy. Nuclei were visualized by DAPI staining. B. TCS samples from A were collected and virus titers determined by immune focus forming assay (IFFA). Results for virus titers represent means ± standard deviation (SD) of two independent experiments; SD were within 10% and the corresponding bars have not been shown. Each independent experiment consisted of three replicates. Titers obtained at 0 h p.i. were in all cases < limited of detection (LoD). VC corresponds to vehicle-treated cells. C. At the indicated h p.i total cellular RNA was isolated and equal amounts (2 µg) from each sample analyzed by Northern blot hybridization using a LCMV NP-specific probe that hybridized to both the S genome RNA and NP mRNA species. Methylene Blue Stain (MBS) was used to assess transfer of equivalent amounts of RNA to the hybridization membrane. In contrast, KDJ9 did not exhibit anti-LCMV activity in any of these assays. To further assess the anti-LCMV activity of KP-146 we determined its CC50 and EC50 (Fig 4), which revealed a KP-146 selectivity index (SI = CC50/EC50) of 110 in A549 cells. To examine whether KP-146 had virucidal activity, we treated 2x105 ffu of rLCMV/GFP-P2A-NP with KP-146 at 25 µM, or DMSO vehicle control, for 30 min at 37oC/5%CO2 in DMEM/2% FBS. After treatment virus samples were diluted 1000-fold and titrated them by IFFA. Treatment with KP-146 did not affect significantly the infectivity of rLCMV/GFP-P2A-NP particles, indicating that KP-146 did not have virucidal activity.


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Figure 4. Determination of KP-146 EC50 and CC50 in A549 cells. EC50 and CC50 were determined as described in M&M. Data represent means (normalized) ± standard deviation (SD) of two independent experiments. Each independent EC50 and CC50 experiment consisted of three and four, respectively, replicates. Cells (1.8x104 cells/96-well) were infected (moi = 0.01; 50 µl/well) with rLCMV/GFP-P2A-NP and after 60 min adsorption treated with two-fold dilutions of KP-146 (range 100 µM to 0.10 µM) or vehicle treated (final volume of 100 µl/96well). At 48h p.i. TCS were collected and titers of infectious virus determined by IFFA and normalized (%) considering 100% titers determined in vehicle-treated and infected cells. Steps of the LCMV life cycle affected by KP-146: To gain insights about the mechanisms by which KP-146 exerted its anti-LCMV activity, we examined the effect of this compound on distinct steps of the LCMV life cycle. We first examined whether KP-146 affected a cell entry or post-cell entry step of the LCMV life cycle. To examine this, we performed a time of addition experiment (Fig 5A). Addition of KP-146 prior, at the time, or 2 h after initiation of virus adsorption to cells resulted in inhibition of LCMV multiplication. These results indicated that KP-146 affected a post-cell entry step of LCMV infection. To examine whether KP-146 affected RNA synthesis directed by the virus polymerase complex we used a LCMV minigenome (MG) assay that recapitulates the biosynthetic processes of RNA replication and gene transcription directed by the virus polymerase complex 32, 36. KP-146 exhibited a robust inhibitory effect on the activity of the LCMV MG (Fig 5Bi) at concentrations at which it did not significantly affect cell viability (Fig 5Bii). Likewise, we confirmed that KP-146, at the tested of concentrations, did not significantly affect levels of NP or GFP expression driven by a pol-II based expression plasmid (Fig 5Biii). We next examined the effect of KP-146 on LCMV budding, a process directed by the LCMV Z protein.7, 35, 37 For this we used a described assay based on transfection of cells with a Z-expressing plasmid and quantification of Z present in virus like particles (VLPs) collected from TCS of transfected cells.35 In cells treated with KP-146 at 33 µM, Z budding efficiency (VLP/VLP+WCL signal) was below detectable levels, whereas cell viability was > 70% (Fig 5C).


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Figure 5. Effect of KP-146 on different steps of the LCMV life cycle. A. Effect of KP-146 on LCMV cell entry. A549 cells were treated with the indicated KP-146 concentrations starting at 2 h prior infection, at the start of infection, or 2 h after infection (moi = 0.1) with rLCMV/GFPP2A-NP. At 4 h p.i. Ammonium Chloride (20 mM) was added to prevent secondary infection of cells by released virus. At 48 h.p.i. cells were fixed and infected cells identified based on GFP expression. Bi. Effect of KP-146 on the LCMV minigenome (MG) activity. BHK-21 cells were pre-treated with KP-146 for 2 h prior to transfection with the MG rescue plasmids. After 5 h, the transfection media was replaced with DMEM/2%FBS media containing the appropriate concentration of compound. At 72 h post transfection cells lysates were prepared and levels of GFP expression determined using lysates normalized for total amount of protein. Data represent means (normalized) ± standard deviation (SD) of two independent experiments. Each independent experiment consisted of two replicates and each GFP value determined was done in triplicate. Bii. Effect of KP-146 on BHK-21 cell viability. Cells were seeded at 1.8x104 in a 96-well plate, cultured overnight and then treated with the indicated compound concentrations for 48 h. Cell viability was determined using the CellTiter 96 AQueous One Solution reagent (Promega). Mean values obtained with vehicle (DMSO)-treated cells were set to 100%. Biii. Effect of KP-146 on NP and GFP expression mediated by a pol-II based expression plasmid in BHK-21 cells. Cells were transfected with pCAGGS expression plasmids for FLAGtagged NP or GFP and treated with KP-146 at the indicated concentrations. At 72 hours posttransfection cell lysates were prepared and levels of NP and GFP expression determined by Western blot using antibodies to FLAG and GFP. Ci. Effect of KP-146 on Z protein budding activity. 293T cells were transfected with pC-ZFLAG and treated with KP-146 at the indicated concentrations. After 72 h treatment, VLPs were collected by ultracentrifugation from TCS. Both VLP and whole cell lysate (WCL) samples were analyzed by Western blot using antibodies to FLAG and GAPDH. Cii. Effect of KP-146 on 293T cell viability. Cells were seeded at 1.8x104 in a 96-well plate, cultured overnight and then treated with the indicated compound concentrations for 48 h. Cell viability was determined as in Bii. Effect of KP-146 on multiplication of the arenaviruses JUNV and TCRV and the nonarenaviruses HPIV3 and CVB3: We examined the effect of KP-146 on multiplication of the arenaviruses Candid#1, a surrogate of JUNV, and TCRV. We used Candid#1 instead of JUNV because the use of live pathogenic strains of JUNV require BSL4 containment, whereas its live-attenuated vaccine strain Candid#1 can be used in BSL2 containment. For this experiment se selected a KP-146 concentration of 25 µM, as this concentration resulted in ca 3 logs reduction in production of infectious LCMV progeny (see Fig 3B). KP-146 25 µM strongly inhibited Candid#1 and TACV multiplication in Vero cells (Fig 6A) without affecting cell viability (Fig 6B). To assess the specificity of the anti-arenaviral activity of KP-146 we examined its effect on 8 ACS Paragon Plus Environment

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multiplication of two viruses unrelated to the arenaviruses, the paramyxovirus human parainfluenza virus type 3 and the picornavirus coxsackievirus B3. We used a recombinant form of HPIV3 that expressed GFP (rHPIV3-GFP) 38, which allowed us to determine production of infectious HPIV3 by FFUA, whereas titration of CVB3 was done by standard plaque assay 39. KP-146 had a rather modest inhibitory effect on rHPIV3-GFP and CVB3 multiplication in LLCMK2 and HeLa cells, respectively (Fig 6A), when present at 25 µM. Likewise, at 50 µM or lower concentrations, KP-146 did not affect Vero, LLCMK2 or HeLa cell viability (Fig 6B).

Figure 6. A. Effect of KP-146 on multiplication of the arenaviruses JUNV and TACV and the non-arenaviruses rHPIV3-GFP and CVB3. Vero cells were infected (moi = 0.1) with JUNV (Candid#1 strain) or TACV and treated with 25 µM KP-146 (symbol) or DMSO vehicle control (VC; symbol). At the indicated h p.i. TCS were collected and infectious virus titers determined by TCID50 (JUNV) or plaque assay (TACV) on Vero cells. Results for virus titers represent means ± standard deviation (SD) of two independent experiments. Titers obtained at 0 h p.i. were < limited of detection (LoD). LLC-CMK2 and HeLa cells were infected (moi = 0.1) with rHPIV3-GFP or CVB3, respectively, and at the indicated h p.i. TCS were collected and infectious virus titers determined by FFUA based on GFP expression (rHPIV3-GFP) or by standard plaque assay (CVB3). Results represent means ± standard deviation (SD) of two independent experiments. In all cases, virus was allowed to adsorb to cells for 60 min prior compound addition. B. Effect of KP-146 on viability of Vero, LL-CMK2 and HeLa cells. Cells were seeded at 1.8x104 in a 96-well plate, cultured overnight and then treated with the indicated compound concentrations for 48 h. Cell viability was determined using the CellTiter 96 AQueous One Solution reagent (Promega). Mean values obtained with VC-treated cells were set to 100%. Budding of TCRV Z protein was reported to use an ESCRT-independent budding, suggesting some mechanistic differences between LCMV and TCRV in Z budding, which could result in susceptibility differences to the inhibitory effect of KP-146. However, we found that KP-146 also inhibited TCRV Z budding (supplementary Figure 1). Production of infectious progeny during serial passages of LCMV in the presence of KP-146: To gain insights about potential viral gene products being targeted by KP-146, we attempted to select LCMV variants with increased resistance to KP-146 as their subsequent genetic characterization could uncover the viral genetic determinants responsible for increased resistance. For this we conducted serial passages (moi = 0.01) of rLCMV/GFP-P2A-NP in A549 cells in the presence of KP-146 at 12.5 µM, a compound concentration that reduced production of infectious progeny by ~ 1-log (Fig 3B). As control we conducted passages of rLCMV/GFP-P2ANP in the absence of KP-146. For each passage, we determined virus titers in TCS at 72 h p.i. Consistent with our previous results (Fig 3B), at P1 titers of rLCMV/GFP-P2A-NP were ~ 1 log lower in TCS of cells treated with KP-146. After 10 passages in the presence of KP-146 (12.5 ACS Paragon Plus Environment

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µM) we did not observe an increase in titers of infectious progeny at 72 h p.i. (Fig 7A). Moreover, LCMV present in TCS P10 exhibited a similar dose-dependent susceptibility to KP-146 as the parental virus used to initiate the serial passages (Fig 7B).

Figure 7. LCMV variants with increased resistance to KP-146 were not selected during serial passages in the presence of KP-146. A. To initiate the serial passages, A549 cells were infected with rLCMV/GFP-P2A-NP at moi = 0.01 in the presence (12.5 µM) or absence of KP-146 and at 72 h p.i. TCS were collected (P1). For each passage, TCS titers were determined and used to infect fresh monolayers of A549 at moi = 0.1 (2x105 cells/M12 well infected with 2x104 ffu) in the presence (12.5 µM) or absence of KP-146. Data represent means of two independent experiments where SD (not shown) was within 20% of the corresponding average virus titer in each experiment. B. Susceptibility to KP-146 of rLCMV/GFP-P2A-NP serially passed 10 times (P10) in the presence of KP-146 (12.5 µM). P10 from KP-146 treated (12.5 µM) serial passages was used to infect A549 cells in the presence of the indicated concentrations of KP-146. At the indicated h p.i. TCS were collected and titrated.

The completion of the arenavirus life cycle involves a complex network of PPIs including the participation of both viral and host cell proteins. Thus, cell entry of arenaviruses has been shown to involve the participation of a large number of host cell factors40-44 that ultimately facilitate the release of the vRNP into the cell cytoplasm where the vRNP directs virus RNA replication and gene transcription. In turn, the formation of a functional vRNP responsible for directing synthesis of arenavirus RNA, both RNA replication and gene transcription, involves NP-NP interaction required for the formation of the nucleocapsid template (genome RNA encapsidated by NP),45-46 and L-L interaction for the formation of a functional virus polymerase.47 Likewise, arenavirus budding involves the interaction of the viral matrix Z protein with different components of the multivesicular body pathway of the infected cell.48 Compounds that target the function of viral proteins, and their interactions, are expected to offer significant degree of specificity, but inhibitor-escape mutants have been isolated for virtually any virus for which a specific inhibitor has been developed, which poses a general problem in antimicrobial therapy 49 . Combination therapy has proven effective to reduce and delay the selection of escape mutants, but does not entirely solve this problem, as illustrated by the selection of HCV resistant variants in patients who experienced virologic failure with asunaprevir and daclatasvir 50. In contrast, intra-host virus evolution is unlikely to result in viral variants able to escape from inhibitors that disrupt cellular functions required for the completion of any of the steps of the virus live cycle. Moreover, related viruses are likely to rely on the same host machinery, thus providing an opportunity for the development of broad-spectrum antiviral therapeutics. Therefore, small molecules that disrupt virus-host cell protein interactions required for the completion of the virus life cycle provide us with valuable probes to uncover novel targets, as well as with lead candidate structures to develop anti-arenaviral drugs. ACS Paragon Plus Environment

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PPIs usually involve large and relatively shallow interfaces, which pose obstacles to their disruption by small molecules.51-52 However, the finding that PPIs are driven primarily by “hot spot” domains, usually rich in aromatic residues engaging in π-π and cation-π interactions, and the fact that the interface complementarity of many PPI is endowed with a significant degree of flexibility,53 makes it plausible that planar scaffolds are appropriate to access these hot spots and disrupt stable PPIs. Accordingly, we have developed a strategy to disrupt PPI using diversified ridged scaffolds. These structures contain aromatic core structures endowed with chemical diversity that are intended to function as inhibitors of PPI. In this study, we have documented the identification of compound KP-146 as a potent inhibitor of LCMV multiplication. Several distinct classes of compounds have been previously shown to inhibit arenavirus GPC-mediated membrane fusion by binding to a common site on GPC and stabilizing the prefusion GPC complex.54-58 These compounds specifically inhibited arenavirus cell entry. In contrast, results from our studies using cell-based functional assays for different steps of the life cycle of LCMV indicated that the KP-146 did not exhibit any noticeable virucidal activity or interference with the process of LCMV cell entry, but rather the anti-LCMV activity of KP-146 was related to its ability to interfere with the activity of the vRNP and the virus budding process mediated by the Z matrix protein. Whether KP-146 exerts its anti-LCMV activity by targeting viral, host or virus-host PPI required for the completion of the LCMV life cycle remains to be determined. Direct acting antivirals (DAAs) that target specific viral gene products and functions are likely to be well tolerated by the infected host cell but they suffer from the common problem in antiviral therapy posed by the emergence of drug resistant variants. On the other hand, the emergence of viral variants resistant to host-targeting antivirals (HTAs) is usually significantly reduced or entirely absent, but HTAs can be associated with significant side effects. However, side effects associated with the use of HTAs might be manageable in the case of acute infections, such as HF disease caused by arenaviruses, where the duration of the treatment would be rather short. Importantly, serial passages of LCMV in the presence of KP146 did not result in the emergence of a virus population with compound increased resistance. Together, our findings indicate that Kröhnke pyridine libraries represent a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop anti-arenaviral drugs for which the emergence of drug resistant viral variants may be highly unlikely. MATERIALS AND METHODS Cells and viruses. Vero E6 (ATCC CRL-1586), A549 (ATCC CCL-185), and 293T (ATCC CRL-3216) and HeLa (ATCC CCL2) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen CAT#: 10313-021) containing 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 µg/ml streptomycin, and 100U/ml of penicillin. BHK-21 (ATCC CCL-10) cells were maintained in DMEM with 10% FBS, 2mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml of penicillin and 5% tryptose phosphate broth solution (Sigma, CAT #: T8159-100ml). LLC-MK2 (ATCC CCL-7) cells were grown in DMEM with 5% FBS, 2mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml of penicillin. The live attenuated vaccine strain Candid#1 of Junin virus (JUNV), Tacaribe virus (TACV), rLCMV/GFP-P2A-NP, recombinant human parainfluenza virus type 3 expressing GFP (rHPIV3-GFP) 38 and coxsackievirus B339 have been described. Plasmids.T7MG-GFP, pCAGGS-NP, and pCAGGS-L,32, 59 as well as pCAGGS-LCMV-ZFLAG,60 have been described. Chemical library. Detailed description of the methods for the synthesis and preparation of the Krönhke-Pyridine combinatorial library (KPL) used in this work have been described.5 Briefly, the 220 members were synthesized on solid phase in 55 pools, each containing four individual compounds. Hit pools were subsequently de-convoluted to identify active single enti11 ACS Paragon Plus Environment


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ties. 1H and 13CNMR spectra were measured at 600 MHz on a Bruker DRX-600 spectrometer. HRMS spectra were measured using electrospray ionization (ESI). LC/MS (Hewlett Packard 1100 MSD) were performed using reversed-phase Discovery H18 column (3 m; 5 cm x 4.6 mm) with a gradient elution (30-100% solvent B; solvent A = water, solvent B = acetonitrile) and detection at 254 nm. The MS (Agilent 100 MSD) employed a scan range of 200-2000 MHz m/z positive mode; fragmentor = 100. Spray chamber conditions: dry gas flow = 12 L/min, nebulizer pressure 50 psig, dry gas temp of 350 °C, and a capillary voltage of 4000. Preparative thin-layer chromatography (prepTLC) was performed on glass plates coated with a 1 mm layer of silica gel 60 F-254. Reagents were purchased from Aldrich and Combi-Blocks and were used without any further purification. Synthesis of KP-146. To generate KP-146, a four step strategy was engaged using Kröhnke pyridine synthesis as the rate-determining step (Scheme 1). Commercially available thiophene 3 and methylketone 4 were used to obtain the -unsaturated ketone 5. Ketone 5 was treated with the previously synthesized pyridinium bromide salt 2 (from commercially available bromoketone 1) using Kröhnke reaction conditions to generate the corresponding pyridine 6. Subsequent saponification of the methyl ester, followed by acyl chloride formation and treatment with ammonia in water led to the formation of KP-146. Detailed descriptions of the synthesis of each of the intermediary compounds and final product KP-146 are provided, vide infra. 1-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)2-oxoethyl)pyridinium bromide (2). 6-bromo acetyl-1,4-benzodioxane 1 (2 g, 7.8 mmol) was dissolved in THF (100 mL) at room temperature. Then, pyridine (1.3 ml,15.6 mmol) was added and the resulting turbid solution was stirred overnight. The yellow precipitate formed is filtered, washed with ether and dried under vacuum to afford 2.5 g of the pyridinium salt 2. Methyl (E)-5-(3-(4-methoxyphenyl)-3-oxoprop-1-en-yl)thiophene-2-carboxylate (5). Methyl 5-formyl-2-thiophenecarboxylate 3 (1.1. g, 6.5 mmol.) and LiOH (160 mg, 6.5 mmol) were stirred in MeOH (100 mL) before methyl 4-methoxyacetophenone 4 (980 mg, 13.64 mmol) was added. After 30 min a thick yellowish precipitate was formed. The precipitate was filtered and dried under vacuum to yield 5 as a yellow solid (76%). No further purification was attempted. ESI (m/z) = (M+ H) 303. Methyl 5-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-methoxyphenyl)pyridin-4yl)thiophene-2-carboxilate (6). Thiophene 5 (225 mg, 0.744 mmol.), and NH4OAc (1.8 g, 22.32 mmol.) were dissolved in a mixture of acetic acid (3 mL) and DMF (5 mL). To it, pyridinium salt 2 (250 mg., 0.744 mmol.) was added and the reaction mixture was heated at 100 °C overnight. Then, the solvent was evaporated under vacuum and the remaining brown oil was dissolved in DCM (100 mL) and solid NaHCO3 was added until the gas release ceased. The organic phase was dried over MgSO4 and evaporated under reduced pressure. The product 6 was crashed out using dichloromethane-MeOH as a brown solid (320 mg, 94%). No further purification was attempted. ESI (m/z) = (M+ H) 460. Methyl 5-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-6-(4-methoxyphenyl)pyridin-4yl)thiophene-2-carboxamide (KP-146). 6 (200 mg, 0.436 mmol.) is dissolved in a mixture of THF: H2O (8:2) (20 mL) and lithium hydroxide (210 mg, 8.72 mmol.) is added. The solution is stirred overnight at room temperature. Then, it was evaporated to dryness and re-dissolved in dry dichloromethane. To it, an excess of oxalyl chloride (4 mL) is added followed by a drop of dry DMF. The reaction is stirred at room temperature overnight, and evaporated to dryness under vacuum. The brown solid is re-suspended in dry THF and added to a solution of NH4OH (50 mL). An orange precipitate is formed within 5 min, which was filtered and dried to generate KP-146. The solid was purified by preparative TLC using DCM:MeOH (9:1) as eluent. 1HNMR (600 MHz, DMSO-d6): = 8.25 (d, J = 6 Hz, 2H), 8.11 (brs, 1H), 8.06 (d, J = 6 Hz, 1H), 8.01 (s, 2H), 7.86 (s, 1H), 7.83 (s, 1H), 7.80 (d, J = 6 Hz, 1H), 7.54 (brs, 1H), 7.10 (d, J = 12 Hz, 12 ACS Paragon Plus Environment

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2H), 7.01 (d, J = 6 Hz, 1H), 4.33 (s, 4H), 3.85 (s, 3H); 13CNMR (150 MHz, DMSO-d6): = 162.54, 160.43, 156.27, 155.96, 144.98, 144.74, 143.58, 142.18, 141.42, 131.79, 130.86, 129.61, 128.24, 127.35, 119.99, 117.26, 115.51, 113.40, 113.55, 64.33, 64.09, 55.28; HRMS (ESI-TOF) : m/z calcd for C25H20N2O4S: 445.1216 (M + H)+; found: 445.1220. Virus titration. Titers of JUNV (Candid#1) were determined by TCID50 using the end-point dilution assay and the Reed-Muench calculation method. Briefly, 10-fold virus dilutions were used to infect VERO cell monolayers in quadruplicate in a 96-well plate. Three-day postinfection, cells were fixed with 4% formaldehyde in PBS and permeabilized in a 0.3% Triton X100 3% BSA PBS solution. Then, cells were stained using a mouse monoclonal antibody to NP (IC06-BE10) and an Alexa Fluor 568 labeled anti-mouse second stage antibody (Molecular Probes). Titers of TACV were determined by plaque assay using Vero cells 61. Titers of rLCMV/GFP-P2A-NP and rHPIV3-GFP were determined using an immune focus forming unit assay (IFFA). Briefly, 10-fold serial virus dilutions were used to infect Vero cells in a 96-well plate (2x104 cells/well) seeded 12-18 h prior infection. At 12-16 hours post infection (h p.i), cells were fixed with 4% paraformaldehyde (PFA) and infected cells identified based on GFP expression. Titers of CVB3 were determined by standard plaque assay using HeLa cells. Growth kinetics. Virus was added to cells at the indicated moi (250 µl/M24-well). After 90 min adsorption, the virus inoculum was removed, cells washed twice with DMEM/2% FBS, and the fresh appropriate complete medium added. At the indicated hours p.i. tissue culture supernatants (TCS) were collected and viral titers determined by IFFA or plaque assay. Determination of KP-146 EC50. Cells (1.8x104 cells/96-well) were infected (moi = 0.01) with rLCMV/GFP-P2A-NP and treated with two-fold dilutions of KP-146 (range 50 µM to 0.19 µM) or vehicle treated. At 48h p.i. TCS were collected and titers of infectious virus determined by IFFA and normalized (%) considering 100% titers determined in vehicle-treated and infected cells. Cytotoxicity assay. Cell viability was assessed using the CellTiter 96 AQueous One Solution reagent (Promega, CAT #: G3580). This method determines the number of viable cells based on the level of formazan product converted from MTS [3-(4,5-dimethylthazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolim] by NADPH or NADH generated in living cells. Cells were seeded at 1.8x104 in a 96-well plate and cultured overnight. Cells were treated with the indicated compound concentrations for 48 h before the addition of CellTiter 96 AQueous One Solution reagent. The assay was performed according to the manufacturer’s recommendations and absorbance values determined using an enzyme-linked immunosorbent assay (ELISA) reader (Spectra-Max Plus384; Molecular Devices). Mean values obtained with vehicle (DMSO)-treated cells were set to 100%. LCMV minigenome (MG) assay. LCMV MG assay was done as described 31. Briefly, BHK21 cells (1.25x105/24 well) were grown overnight and transfected for 5 h with the MG-encoding plasmid (T7MG-GFP, 0.25 µg/well) together with pCAGGS expression plasmids for NP (0.15 g/well) L (0.15 µg/well) and T7 RNA polymerase (0.2 µg/well) using the Lipofectamine 2000 reagent (Invitrogen). After 5 h, the transfection medium was replaced with fresh medium containing the compound and concentrations indicated. After 72 h of incubation, cell lysates were prepared (150 mM NaCl, 50 mM TRIS-HCl, 1mM EDTA, 0.5% NP-40) and levels of GFP expression determined using Bioteck Synergy H4 plate reader. GFP expression levels were normalized for total cell protein in the lysate (Pierce BCA Protein Assay Kit, Thermo Scientific, #23227). Budding assay. Budding assay was done as described.35 Briefly, 293T cells (3.5x105 cells/M12-well) were transfected with 0.4 µg of pC- LCMV-ZFLAG using Lipofectamine 2000. After 5 h the transfection medium was replaced with medium containing the compound at the indicated concentrations. After 72 h treatment, virus-like particle (VLP)-containing tissue culture supernatants (TCS) and cells were collected. After clarification from cell debris by centrif13 ACS Paragon Plus Environment


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ugation (1,500 X g /4oC /5 min) VLPs were collected by ultracentrifugation (230,000 X g /4oC/ 2 hours through a 20% sucrose cushion) and re-suspended in PBS. Cells were collected in lysis buffer (1% NP-40, 50mMTris-HCL [pH 8.0], 62.5mM EDTA, 0.4% sodium deoxycholate). Cell lysates and VLPs were analyzed by Western blotting. Western blot. Cell lysates and VLP were mixed with 4X SDS-loading buffer (50mM Tris [pH 6.8], 100mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 5 min. Clarified protein samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Immobilon P Transfer Membranes; Millipore). To detect FLAG or GAPDH, membranes were incubated with rabbit polycolonal antibody to FLAG (Cayman), or GAPDH (GAPDH AbI EMD Millipore), followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit immunoglobulin G 10antibody (Pierce). SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 (GE Healthcare Life Science).


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ASSOCIATED CONTENT SUPPORTING INFORMATION. This material is available free of charge via the Internet at http://pubs.acs.org. Scheme 1: schematic representation of 220-member KPL synthesis in 55 pools with 4 members per pool. Figure S1: Effect of KP-146 on TCRV Z budding activity. AUTHOR INFORMATION Corresponding Author: Juan C. de la Torre Department Immunology and Microbiology IMM-6 The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037 *E-mail: [email protected], Tel.: 858-7849462 Present address: P.O.M: Instituto de Productos Naturales y Agrobiología, CSIC, Avda. Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. † Authors contributed equally to this project. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was supported by NIH/NIAID grants AI047140 and AI077719 to JCT. POM was supported by a fellowship from the European Union’s Seventh Framework Program FP7/20072013 under REA Grant Agreement No. 623155. This is manuscript #29583 from The Scripps Research Institute. ABBREVIATIONS HF, hemorrhagic fever; LCMV, Lymphocytic choriomeningitis virus; PPI, protein-protein interactions; KPL, Kröhnke Pyridine Library.


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20. Jahrling, P. B.; Peters, C. J., Lymphocytic choriomeningitis virus. A neglected pathogen of man. Arch Pathol Lab Med 1992, 116 (5), 486-8. 21. Borio, L.; Inglesby, T.; Peters, C. J.; Schmaljohn, A. L.; Hughes, J. M.; Jahrling, P. B.; Ksiazek, T.; Johnson, K. M.; Meyerhoff, A.; O'Toole, T.; Ascher, M. S.; Bartlett, J.; Breman, J. G.; Eitzen, E. M., Jr.; Hamburg, M.; Hauer, J.; Henderson, D. A.; Johnson, R. T.; Kwik, G.; Layton, M.; Lillibridge, S.; Nabel, G. J.; Osterholm, M. T.; Perl, T. M.; Russell, P.; Tonat, K.; Working Group on Civilian, B., Hemorrhagic fever viruses as biological weapons: medical and public health management. JAMA 2002, 287 (18), 2391-405. 22. Damonte, E. B.; Coto, C. E., Treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy. Adv Virus Res 2002, 58, 125-55. 23. Moreno, H.; Gallego, I.; Sevilla, N.; de la Torre, J. C.; Domingo, E.; Martin, V., Ribavirin can be mutagenic for arenaviruses. J Virol 2011, 85 (14), 7246-55. 24. Parker, W. B., Metabolism and antiviral activity of ribavirin. Virus Res 2005, 107 (2), 165-71. 25. Lee, A. M.; Pasquato, A.; Kunz, S., Novel approaches in anti-arenaviral drug development. Virology 2011, 411 (2), 163-9. 26. Gowen, B. B.; Juelich, T. L.; Sefing, E. J.; Brasel, T.; Smith, J. K.; Zhang, L.; Tigabu, B.; Hill, T. E.; Yun, T.; Pietzsch, C.; Furuta, Y.; Freiberg, A. N., Favipiravir (T-705) inhibits Junin virus infection and reduces mortality in a guinea pig model of Argentine hemorrhagic fever. PLoS Negl Trop Dis 2013, 7 (12), e2614. 27. Mendenhall, M.; Russell, A.; Juelich, T.; Messina, E. L.; Smee, D. F.; Freiberg, A. N.; Holbrook, M. R.; Furuta, Y.; de la Torre, J. C.; Nunberg, J. H.; Gowen, B. B., T-705 (favipiravir) inhibition of arenavirus replication in cell culture. Antimicrob Agents Chemother 2011, 55 (2), 782-7. 28. Mendenhall, M.; Russell, A.; Smee, D. F.; Hall, J. O.; Skirpstunas, R.; Furuta, Y.; Gowen, B. B., Effective oral favipiravir (T-705) therapy initiated after the onset of clinical disease in a model of arenavirus hemorrhagic Fever. PLoS Negl Trop Dis 2011, 5 (10), e1342. 29. Safronetz, D.; Rosenke, K.; Westover, J. B.; Martellaro, C.; Okumura, A.; Furuta, Y.; Geisbert, J.; Saturday, G.; Komeno, T.; Geisbert, T. W.; Feldmann, H.; Gowen, B. B., The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset. Sci Rep 2015, 5, 14775. 30. Emonet, S. E.; Urata, S.; de la Torre, J. C., Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies. Virology 2011, 411 (2), 41625. 31. Emonet, S. F.; Garidou, L.; McGavern, D. B.; de la Torre, J. C., Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest. Proc Natl Acad Sci U S A 2009, 106 (9), 3473-8. 32. Lee, K. J.; Perez, M.; Pinschewer, D. D.; de la Torre, J. C., Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles. J Virol 2002, 76 (12), 6393-7. 33. Pinschewer, D. D.; Perez, M.; Sanchez, A. B.; de la Torre, J. C., Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein. Proc Natl Acad Sci U S A 2003, 100 (13), 7895-900. 34. Rojek, J. M.; Sanchez, A. B.; Nguyen, N. T.; de la Torre, J. C.; Kunz, S., Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses. J Virol 2008, 82 (15), 7677-87. 35. Urata, S.; Ngo, N.; de la Torre, J. C., The PI3K/Akt pathway contributes to arenavirus budding. J Virol 2012, 86 (8), 4578-85. 36. Vazquez-Calvo, A.; Martin-Acebes, M. A.; Saiz, J. C.; Ngo, N.; Sobrino, F.; de la Torre, J. C., Inhibition of multiplication of the prototypic arenavirus LCMV by valproic acid. Antiviral Res 2013, 99 (2), 172-9. 37. Perez, M.; Craven, R. C.; de la Torre, J. C., The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies. Proc Natl Acad Sci U S A 2003, 100 (22), 12978-83. ACS Paragon Plus Environment

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Mining a KrÃ¶hnke Pyridine Library for Anti-Arenavirus Activity - ACS

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