Metal coordinating inhibitors of Rift Valley fever virus replication.

Rift Valley fever virus (RVFV) is a veterinary and human pathogen and is an agent of bioterrorism concern. Currently, RVFV treatment is limited to supportive care, so new drugs to control RVFV infection are urgently needed. RVFV is a member of the order Bunyavirales, whose replication depends on the enzymatic activity of the viral L protein. Screening for RVFV inhibitors among compounds with divalent cation-coordinating motifs similar to known viral nuclease inhibitors identified 47 novel RVFV inhibitors with selective indexes from 1.1-103 and 50% effective concentrations of 1.2-56 μM in Vero cells, primarily α-Hydroxytropolones and N-Hydroxypyridinediones. Inhibitor activity and selective index was validated in the human cell line A549. To evaluate specificity, select compounds were tested against a second Bunyavirus, La Crosse Virus (LACV), and the flavivirus Zika (ZIKV). These data indicate that the α-Hydroxytropolone and N-Hydroxypyridinedione chemotypes should be investigated in the future to determine their mechanism(s) of action allowing further development as therapeutics for RVFV and LACV, and these chemotypes should be evaluated for activity against related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, Crimean-Congo hemorrhagic fever virus.

1. Introduction

Bunyavirales is a large order of enveloped viruses with a segmented, negative-polarity, single-stranded RNA genome and includes multiple members that pose a significant risk to public health: Rift Valley fever virus (RVFV), La Crosse virus (LACV), Hantaan virus, severe fever with thrombocytopenia virus (SFTSV), and Crimean-Congo hemorrhagic fever virus (CCHFV) [1, 2]. RVFV is an arbovirus transmitted to animals by mosquito vectors. It is traditionally endemic in eastern and southern Africa but has recently expanded its range throughout sub-Saharan Africa and parts of the Middle East. RVFV is a serious veterinary pathogen, causing Rift Valley fever in domestic animals including cattle, horses, sheep, goats, and camels. Rift Valley fever is characterized by fever, hemorrhage, diarrhea, death, and nearly complete spontaneous abortions in infected animals. Veterinary outbreaks of RVFV infections can reach epidemic proportions, particularly in rainy years [3, 4]. Often, RVFV has a severe economic impact in regions with many affected herds.

Humans can also be infected by RVFV via contact with infected animal body fluids or tissues, by breathing droplets contaminated with RVFV, or less frequently via mosquito bites; with human to human transmission of RVFV being rare [2]. Most human infections are either asymptomatic or cause mild fever with hepatic involvement. However, 8–10% of infections become severe, where symptoms can include lesions to the eye which cause blindness in 50% of ocular cases, encephalitis, gastrointestinal dysfunction, jaundice, joint/muscle pain, hemorrhagic fever, disorientation/hallucination, and partial paralysis ([5], Reviewed in [2]). Hemorrhagic fever is rare (~1% of cases) but has a ~50% fatality rate in cases where it occurs. Human RVFV infections can be diagnosed by ELISA or RT-PCR assays, but treatment is limited to supportive care [2]. Currently, there is a live-attenuated veterinary vaccine approved for RVFV (MP12), which has undergone a phase I clinical trial for use in humans (NCT00415051).

In addition to RVFV, LACV is an arbovirus found throughout the midwestern United States, with childhood infections commonly underdiagnosed due to a lack of available diagnostics and therapeutics [6, 7]. Currently there are 50–150 cases of neuroinvasive disease reported annually [8]. Since 2011, LACV has continued to spread beyond the Midwest and into northeastern, mid-Atlantic and southern states, resulting in 700 cases of neuroinvasive disease since 2011 [9].

All viruses of the order Bunyavirales replicate within the cytoplasm, facilitated by the L protein which is comprised of the RNA-dependent RNA polymerase (RdRp), cap binding domain and viral endonuclease. These activities can make Bunyavirales a target for both direct acting antivirals as well as host direct antivirals [10-12]. The virally encoded enzymes requires either Mg++ or Mn++ ions for catalysis and hence viral replication [1, 13]. This is analogous to the Mg++-binding motif of the Influenza Virus PA cap-snatching endonuclease [14] and bears significant similarity to the D‥E‥D‥D or D‥D‥E motifs found in ribonucleases H and viral integrases [15]. Inhibiting viral enzymatic function by altering Mg++ or Mn++ ions can block viral replication, as has been shown for the HIV integrase [16], the HIV ribonuclease H [17], the Hepatitis B Virus (HBV) ribonuclease H [18], and the influenza virus cap-snatching enzyme [19, 20]. The most common mode of inhibition is for small molecules to chelate the Mg++ ions in viral enzymatic active sites, with specificity and affinity modulated by additional contacts between the inhibitors and the enzymes [21-23], and sometimes also by contacts with the nucleic acid substrate [24]. This metal-chelating mechanism is used by the HIV integrase inhibitors Bictegravir [25], Dolutegravir [26, 27], Elvitegravir [28], and Raltegravir [28], and the Influenza Virus PA cap-snatching inhibitor Baloxavir marboxil [29]. The US Food and Drug Administration has approved 62 drugs that act by coordinating active-site cations in metalloenzymes as of 2017 [30], making active site metal ion chelation a well-established drug mechanism.

In these studies, we hypothesized that metal chelating compounds similar to inhibitors of the HIV and HBV ribonucleases H, the HIV integrase, and the Influenza Virus PA endonuclease would inhibit RVFV and LACV replication. This hypothesis is based on i) the inhibitory mechanism employed by metal chelating compounds against metalloenzymes, ii) the essential nature of the L protein enzymatic function for viral replication [31], iii) the structural similarities of Mg++-dependent viral endoribonucleases, even between the Phenuiviridae and Peribunyaviridae families [15, 31, 32], and iv) the successes in developing drugs for HIV and Influenza virus that act by chelating the catalytic Mg++ ions. In order to evaluate metal chelating compounds, we developed and validated assays to quantify live virus growth in the presence of potential antiviral compounds. Using these assays, we screened 397 compounds to identify chemotypes with antiviral potential in both non-human primate and human cell lines. We then further identified compounds which had activity against the Phlebovirus RVFV and the closely related Orthobunyavirus, LACV. To determine specificity against bunyaviruses versus RNA viruses, we measured the in vitro efficacy of the best compounds against the positive strand RNA flavivirus, Zika. Additionally, we developed live virus assays to quantify antiviral compound activity validated these assays using known antivirals.

2. Materials and methods

2.1. Compound acquisition and synthesis

Commercially acquired compounds are indicated by the vendor’s name and catalog number in S1 Table. Thiotropolones (TTP) compounds were synthesized as described in [33]. α-Hydroxytropolones (αHT) compounds were synthesized as described in [34]. For 265, 308, and 311 see [35]. For 169 and 362 see [36]. For 385, 694, 696, 698, 700, 702, 704, 703, 838, and 840 see [37]. For 321, 336, 358, and 359 see [38]. For 388, 389, 390 and 539 see [39]. For 330 and 331 see [40]. For 113, 118 and 120 see [41]. For 260 see [42]. For 111 see [34]. For 335 see [43].

The novel N-hydroxypyridinediones (HPD) compounds were synthesized following a three-step synthetic procedure (S1 Data, Scheme 1). The key structure 5-acetyl-1-(benzyloxy)-6-hydroxy-4-methylpyridin-2(1H)-one (ZEV1) was synthesized with an improved yield of 75% by refluxing a mixture of O-benzyl hydroxylamine (1 eq) and diketene (2 eq) in the presence of triethylamine (1 eq) in dry toluene. Subsequently, the benzyl group was cleaved by catalytic hydrogenation over 10% palladium on carbon to afford the target compound ZEV2 almost quantitatively. 5-Acetyl-1,6-dihydroxy-4-methylpyridin-2(1H)-one (ZEV2) was coupled with the appropriate substituted aniline using sulfuric acid as catalyst in absolute ethanol at reflux. The desired compounds were obtained in good yields ranging from 60% to 70%, with the only exception being 668 (ZEV-V5) which was isolated in an overall yield of 25%. HPD compounds not published previously include 515–518, 668 and 670; preparation procedures and characterization data of compounds are in S1 Data. Nucleoside analogues ribavirin and β-D-N4-Hydroxycytidine N4-Hydroxycytidine were purchased from Cayman Chemical. Compounds were diluted to 10 mM in DMSO and stored in single-use aliquots in opaque tubes at -20 °C.

2.2. Cells and viruses

RVFV strains MP-12 and ZH501, as well as LACV strain original (BEI NR-540) were passaged in Vero E6 cells (ATCC® CRL-1586™) before clarification by centrifugation at 3,000 rpm for 30 minutes and stored at -80°C until further use. RVFV isolates were a kind gift of Drs. M. Buller (Saint Louis University) and A. Hise (Case Western Reserve University). MP-12 is a BSL-2 vaccine strain of RVFV and is not classified as a select agent allowing easier assay development. The ZIKV strain, PRVABC59 was a kind gift of Robert Lanciotti (CDC). Virus inhibition assays and toxicity assays, described below, were completed in both Vero E6 and A549 cells (ATCC CCL-185). Unless otherwise specified, all cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma- D5796-500ML) containing 1% HEPES (Sigma- H3537-100ML) and 5% FBS (Sigma- F0926) at 37°C, 5% CO2. Studies with infectious RVFV-ZH501 viruses were approved by the SLU IBC and were conducted in our select agent registered A/BSL-3 laboratory.

2.3. Focus Forming Assay (FFA)

FFAs are used to quantify infectious virus and are the basis for the antiviral compound inhibition assay. Briefly, 100μL of Vero E6 cells at a concentration of 3x105 cells/ml were plated in a 96-well flat bottom plate resulting in a confluency of 90–95% the day prior to the assay. To quantify viral stocks, ten-fold serial dilutions of virus supernatants were then made in a 96-well round bottom plate containing 5% DMEM media before being added to the Vero cell monolayer and allowed to adsorb for one hour in an incubator with 37°C, 5% CO2. Following virus adsorption, a solution of 2% methylcellulose (Sigma-M0512-250G) was diluted 1:1 in 5% DMEM and warmed to room temperature. The methylcellulose-media mixture overlay was added to the plate by adding 125 μL of overlay media to each well and returned to an incubator with 37°C, 5% CO2 for 24 hours. Plates were then fixed in a solution of 5% paraformaldehyde (PFA) diluted in tissue culture grade 1X PBS, then washed in 1X PBS for 15 minutes. Foci were visualized by an immunostaining protocol using anti-nucleocapsid protein antibody (1D8) diluted 1:5000 to detect RVFV and anti-Gc protein antibody (4C12A1) diluted 1:5000 to detect LACV with FFA staining buffer (1X PBS, 1mg/ml saponin (Sigma: 47036)) as a primary detection antibody overnight at 4°C. The anti-nucleocapsid protein antibody (1D8) was obtained from Joel Dalrymple and Clarence J Peters (USAMRIID) via BEI resources. The secondary antibody consisted of goat anti-mouse conjugated horseradish peroxidase (Sigma: A-7289) diluted 1:5,000 in FFA staining buffer and allowed to incubate for 2 hours at room temperature. Foci were visualized using KPL TrueBlue HRP substrate and allowed to develop for 10–15 minutes, or until blue foci are visible. The reaction was then quenched by washing with Millipore water. RVFV ZH501 foci assays were measured within the BSL3 facility, while RVFV MP12 and LACV viral foci were quantified with an automated ELISPOT machine (CTL universal S6) using the Immunospot software suite.

2.4. Antiviral compound efficacy assay

For RVFV (MP-12 and ZH501), Vero cells were plated at 3x105 cells per mL in a flat bottom 96 well plate. Twenty-four hours later they were infected with RVFV at a multiplicity of infection of 0.005. Compound was added and plates were incubated for 1 hour at 37°C and then cells were overlayed with methylcellulose. After 24 hours plates were fixed, RVFV infectious foci were stained and quantified as described for the FFA above. Data is normalized to PBS, using the average number of foci in each individual assay and presented as Focus Forming Units (FFU). All assays required the range of foci to be 70–90 foci per control well for the assay to pass quality control.

For RVFV (MP-12)- A549 cells, a human lung epithelial cell line, were plated at 3x105 cells per mL in 96 well plates and incubated at 37°C for 24 hours. Cells were then infected with RVFV strain MP-12 at a multiplicity of infection of 0.005. Plates were incubated for 1 hour at 37°C and then cells were overlayed with methylcellulose. After 24 hours plates were fixed, RVFV infectious foci were stained and quantified as described for the FFA above.

For LACV (original), Vero cells were plated at 2x105 cells per mL in a flat bottom 96 well plate. Twenty-four hours later they were infected with LACV at a multiplicity of infection of 0.01. Compound was added and plates were incubated for 1 hour at 37°C and then cells were overlayed with methylcellulose. After 24 hours plates were fixed, LACV infectious foci were stained using the murine anti-LACV Gc antibody, detected with an anti-mouse HRP secondary antibody and quantified as described for the FFA above.

For Zika virus (ZIKV) we have used our previously validated assay [44, 45], briefly Vero cells were plated at 4x105 cells per mL in a flat bottom 96 well plate. Twenty-four hours later they were infected with ZIKV at a multiplicity of infection of 0.01. Compound was added and plates were incubated for 1 hour at 37°C and then cells were overlayed with methylcellulose. After 48 hours plates were fixed, ZIKV infectious foci were stained using the ZIKV cross-reactive antibody (4G2), detected with an anti-mouse HRP secondary antibody and quantified as described for the FFA above.

Fifty percent effective concentrations (EC50) for key hits were determined by screening for suppression of viral growth using an eight point, 2.5-fold dilution series of the compounds starting at 100 μM. EC50 values were calculated by non-linear curve fitting in GraphPad Prism v8.

2.5. Compound cytotoxicity

Initial compound cytotoxicity was estimated in Vero cells in the primary antiviral compound experiments by staining the virally infected, compound treated cells with crystal violet after viral foci had been quantified. After cells were stained with crystal violet wells were washed 2x with water. The crystal violet dye was then extracted using 50% ethanol and absorbance was measured at 570nm in an ELISA plate reader. Estimated 50% cytotoxic concentration (CC50) values were derived by non-linear curve fitting in GraphPad Prism of the four data points derived from the primary screen.

Quantitative CC50 values were measured in two systems. Compound cytotoxicity in uninfected A549 cells was determined using the CytoTox-GloTM Cytotoxicity Assay (Promega) according to the manufacturer’s instructions. Briefly, A549s were seeded at 3x105 cells per mL in 96 well plates and incubated for 24 hours at 37°C. An eight point, 3-fold dilution series of compounds was added to the cellular monolayer starting with the highest concentration, 600 μM, in addition to DMSO and PBS as controls. Plates were incubated for 48 hours at 37°C, then the AAF-Glo reagent was added to the cells for 15 minutes and luminescence was measured to determine the number of dead cells. Lysis buffer was added next for 15 minutes, then luminescence was measured to determine the total cell number. The dead cell number was then subtracted from the total cell number to generate the viable cell number.

Second, cytotoxicity was measured in the HepG2-derived hepatoblastoma cell line, HepDES19 [46]. Cells were treated with a range of compound concentrations in a final DMSO concentration of 1% for three days and mitochondrial function was measured by MTS assays as described [47]. CC50 values were then calculated by non-linear curve fitting in GraphPad Prism v8.

2.6. RNaseH inhibition reactions

Activity of human RNaseH was measured using a FRET assay in which the RNA:DNA heteroduplex was formed by annealing an 18 nucleotide-long RNA oligonucleotide with a fluorescein label at the 3’ end to a complementary DNA oligonucleotide with an Iowa Black quencher at the 5’ end. RNaseH activity cleaves the RNA, permitting the fluorescein to diffuse away from the quencher, increasing fluorescence. The oligonucleotides employed were:

DNA: 5’-IABkFQ-AGC TCC CAG GCT CAG ATC-3’ (IABkFQ: Iowa Black quencher)

RNA: 5’- GAU CUG AGC CUG GGA GCU FAM-3’ (FAM: Fluorescein fluorophore).

Recombinant human RNaseH 1 was purified from E. coli as described in [48]. Enzyme and substrate (12.5 nM) were combined in 100 mM NaCl, 50 mM HEPES pH 8.0, 2 U RNase OUT (ThermoFisher), and test compound in a final concentration of 1% DMSO. Reactions were started by adding MgCl2 to 5 mM and incubating at 37°C for 90 min. with detection of fluorescence every 2 min. in a plate reader. The initial rate was determined for each compound concentration, and IC50 values were determined from the reaction rates by non-linear curve fitting in GraphPad Prism.

3. Results

3.1. Development and validation of a live virus bunyavirus antiviral compound efficacy assay

Based on our previous work optimizing focus forming assays (FFAs) for Zika virus (ZIKV) and SARS-CoV-2, [45, 49, 50] we developed an antiviral compound efficacy assay for RVFV and LACV. We chose to develop an FFA because it is a high-throughput cell-based assay that can capture a range of information about viral growth and replication, such as number of infection foci and foci morphology. Virally induced foci data serve as critical information for the development of antiviral compounds. To establish and validate the assay, we identified an appropriate cell line, defined the primary antibodies capable of detecting viral antigen, and determined an optimal cell concentration for seeding the wells of 96 well plates. Previous work from a number of laboratories demonstrated that Vero E6 cells, a non-human primate African green monkey kidney epithelial cell line, are highly sensitive to RVFV infection and widely available [51].

To identify an appropriate primary antibody to detect viral antigen, we stained a serial dilution of virally infected Vero E6 cells. We evaluated 5 total monoclonal antibodies (mAbs), with 2 antibodies recognizing the Gc glycoprotein (4B6, 3D11), 2 antibodies recognizing the Gn glycoprotein (7B6, 3C10), and one antibody recognizing the nucleocapsid protein (1D8) from the Joel M. Dalrymple and Clarence J. Peters (USAMRIID antibody collection) (Fig 1A). The murine anti-flavivirus mAb 4G2 was used as a negative control. The mAb 1D8 had the best signal to noise ratio and was thus used throughout the rest of these studies.

Fig 1

Development and validation of RVFV antiviral screen.

A. Identification of optimal mAb for the detection of RVFV by mAb staining of a serial dilution of RVFV strain MP-12 in an FFA. B. Impact of cell number on the sensitivity of antiviral compound screen. C. Evaluation of the sensitivity of the antiviral compound screen based upon the evaluation of ribavirin and β-D-N4-Hydroxycytidine N4-Hydroxycytidine (NHC/EIDD-1931), a known antiviral for RVFV. Data is presented as focus forming units. These data are the cumulation of three independent experiments with technical duplicates.

In order for RVFV to form distinct foci and for the assay to have the highest level of sensitivity, it is critical to plate cells at an optimal density. We examined the impact of cell density on foci formation by plating identical dilutions of RVFV virus stocks on 96-well plates seeded with differing numbers of E6 cells (7.5 × 104, 1.5 × 105 or 3 × 105 cells/mL). At these concentrations, the monolayers were ~70, 80 and 90 percent confluent, respectively, and the same virus dilution resulted in 1 × 103 FFU/mL of RVFV MP-12. We observed the highest sensitivity when either 1.5 × 105 or 3 × 105 cells/mL were seeded in comparison to 7.5 × 104 cells/mL (Fig 1B). We have previously tested higher cell densities for FFAs measuring flavivirus and coronavirus replication, and we have noted that cell concentrations higher than 3 × 105 cells/mL results in an overly confluent monolayer with more cells than can adhere to the wells, which can lead to highly variable viral titer information [45].

To validate the assay design, we completed an antiviral compound inhibition assay by plating Vero E6 cells at 3x105 cells/ml in a 96 well flat bottom plate. These wells were infected with sufficient virus to form ~70–80 foci per well, and cells were treated with serial dilutions of ribavirin or β-D-N4-Hydroxycytidine N4-Hydroxycytidine (NHC/EIDD-1931) starting at 100μM as positive controls. Further, additional wells served as negative controls by being treated with 100μM DMSO as a vehicle control or PBS. The ratio of foci forming units (FFU) in comparison to PBS was measured and the effective concentration 50 (EC50) was calculated. Ribavirin had an EC50 of 22.0 μM, similar to work by other groups [52, 53], while the EC50 value for NHC was 5.16 μM (Fig 1C).

3.2. Primary screening for antiviral activity

Primary screens were conducted that evaluated 397 compounds either with known metal-chelating motifs or motifs similar to metal-chelating ones. The most common chemotype among the compounds screened was the troponoids (tropones, tropolones (TRP), thiotropolones (TTP), and α-hydroxytropolones (αHTs)), but the compound set also included a wide range of other chemotypes such as the N-hydroxypyridinediones (HPD), flavonoids, N-hydroxynapthyridinones, dihydronapthalenes (DHN), dioxobutanoic acids, hydroxyxanthanones, thienopyrimidinones, pyridinepiperazinthieonpyrimidins, N‐biphenyltrihydroxybenzamides, and aminocyanothiophenes. Almost half of the compounds screened were αHTs as the library from which they were drawn was assembled in support of anti-HBV ribonuclease H antiviral development and the αHTs are a leading chemotype in that effort [38, 54, 55].

To screen these compounds for antiviral activity, FFAs were used. Briefly, cells were infected with RVFV MP-12, treated with 60, 20, 6.7, or 2.2 μM of compound, and the number of RVFV foci was determined 24 hours later. Antiviral efficacy was calculated as an estimated 50% effective concentration (EC50) from the number of RVFV foci detected in comparison to vehicle control (S1 Table). Following detection of the RVFV foci, cytotoxicity concentrations (CC50) were estimated by qualitatively assessing monolayer integrity by staining the cell monolayers with crystal violet and measuring the optical density of crystal violet staining, which is proportional to the number of viable cells. Screening hits were defined as compounds that i) had an estimated EC50 determined from the four-point screening assay of <20 μM and ii) by measuring monolayer integrity using crystal violet, as commonly done for cell-based bioassays.

Forty-seven screening hits were identified (S1 Table). Thirty-nine of 174 troponoids screened (22%) were hits, with 34 of them being αHTs, three being TRPs, and two being TTPs. In contrast, only eight of 223 non-troponoids (3.6%) were hits. Seven of these eight hits were HPDs. The hit rate among the 24 HPDs screened (29%) was similar to that of the αHTs. The remaining screening hit was a DHN.

3.3. Quantification of antiviral activity against RVFV

Based upon the activity of the primary screen of αHTs and HPDs and because their antiviral activity against RVFV is novel, we defined the dose-response curve for 25 αHTs, 4 HPDs, and 6 additional compounds selected to broaden the chemical diversity. This also served to spot-check compounds with poor estimated EC50 values. In these studies, Vero cells were treated with 8 different concentrations ranging from 60 to 0.024 μM at the time of infection and their ability to prevent virus replication was measured by comparing the number of viral infection foci to wells treated with vehicle control. EC50 ranged from 1.2 to 40.8 μM (Table 1), with 21 of the top 30 compounds being αHTs. Interestingly, two closely related compounds AG-II-18-P (308), a thiophene substituted αHT and the closely related furan counterpart AG-I-183-P (309), had an EC50 of 1.2 μM and 1.6 μM respectively, and were the two best compounds identified.

Table 1

Top antiviral hits.

Compound number	Name 1	Chemotype 2	EC50 (μM)	CC50 (μM)3	SI	Chemist
α-Hydroxytropolones
308	AG-II-18-P	αHT	1.2	111	92	Murelli
309	AG-I-183-P	αHT	1.6	>120	75	Murelli
362	DS-I-69	αHT	2.5	80	32	Murelli
694	NBA-I-127 Bis	αHT	3.7	>120	32	Murelli
359	AG-II-108-C	αHT	5.1	>120	24	Murelli
696	NBA-I-128 Bis	αHT	6.0	118	20	Murelli
1017	AL-23	αHT	8.0	>120	15	Murelli
867	DS-1-124	αHT	8.7	>120	14	Murelli
702	NBA-I-159 Mono	αHT	8.8	>120	14	Murelli
1039	AB-3-45	αHT	8.9	>120	13	Murelli
336	YA-I-78	αHT	9.0	70	8	Murelli
330	NBA-I-14	αHT	10.4	>120	12	Murelli
698	NBA-I-150	αHT	11.7	>120	10	Murelli
311	AG-II-3-P	αHT	11.8	115	10	Murelli
210	MolMoll 19617	αHT	11.8	>120	10	Purchased
390	AB-2-70	αHT	11.9	>120	10	Murelli
838	NBA-I-130	αHT	12.7	>120	9	Murelli
704	NBA-I-160	αHT	14.3	>120	8	Murelli
331	NBA-I-31	αHT	15.8	80.4	5	Murelli
703	NBA-I-159 Bis	αHT	16.1	>120	7	Murelli
539	AB-2-91	αHT	18.6	71	4	Murelli
840	NBA-I-155-Mono	αHT	31.6	>120	4	Murelli
320	NBA-I-13	αHT	34.0	>120	4	Murelli
335	DH-2-60	αHT	40.8	>120	3	Murelli
Thiotropolones
680	BE1105	TTP	6.3	>120	19	Elgendy
686	BE1111	TTP	6.2	>120	19	Elgendy
Tropolones
341	Specs AP-355/40802214	TRP	25.2	>120	5	Purchased
340	Specs AP-355/40633884	TRP	27.3	70	4	Purchased
342	Specs AP-355/40633885	TRP	55.5	>120	2	Purchased
N-Hydroxypyridinediones
670	ZEV-V7	HPD	14.0	>120	9	Zoidis
668	ZEV-V5	HPD	19.2	>120	6	Zoidis
518	ZEV-V3	HPD	19.5	>120	6	Zoidis
515	ZEV-E2	HPD	24.3	>120	5	Zoidis
Dihydronapthalene
327	Aldrich Select CNC_ID 444085867	DHN	39.7	44	1.1	Purchased
Nucleoside Analogue
EIDD-1931	β-D-N4-HydroxycytidineN4-Hydroxycytidine	Nuc	5.2	>120	23	Purchased
	Ribavirin	Nuc	22.0	>120	5	Purchased

1 Chemist’s name, common name, or vendor catalog number.

2 αHT, α-Hydroxytropolone; TRP, tropolone; TTP, thiotropolone; DHN, dihydronapthalene; HPD, N-Hydroxypyridinedione; FLV, flavenoid; DOB, dioxobutanoic acid; HXT, hydroxyxanthanone; TPD, thieopyrimidinone; ACT, aminocyanothiophene.

3 Values of 120 indicate the data were at or above the upper limit of quantification in the assay.

To better understand potential cellular cytotoxicity in the context of infection, crystal violet staining of the cell monolayers was quantified by absorbance and CC50 values were calculated after foci were counted. The CC50 ranged from 44 to >120 μM in Vero cells, with 24 compounds having a CC50 of >120 μM. Second, cytotoxicity was assessed in the HepG2 derivative HepDES19 [46] to model longer-duration compound exposure in hepatocytes. Cells were treated with a range of compound concentrations for three days and mitochondrial function was measured using MTS assays. CC50 values ranged 1.8 to >100 μM in the hepatoblastoma cells (S2 Table).

To validate compound efficacy and cytotoxicity, we completed additional dose response curve experiments in A549 cells (Table 2 and Fig 2). We selected A549 cells, a human alveolar basal epithelial cell line, for two reasons, the potential for respiratory exposure of humans via aerosol or droplets and the epithelial nature of A549s because of the evidence that natural infection of RVFV leads to infection of epithelial cells within the kidney, liver, and spleen [5, 56, 57]. There was a concordance for the majority of compounds between the EC50 value defined in Vero cells and the EC50 concentration defined in A549 cells. The values from the corresponding compounds in Vero cells is provided next to the data for A549s. In A549 cells, both compound AG-II-18-P (308), a thiophene substituted αHT, and its furan counterpart (309) had the lowest EC50 values of 8.4 and 5.9 μM, respectively. To quantify cytotoxicity independent of viral infection, cellular toxicity was quantified by measuring intracellular protease release. In this manner, we were able to quantify cytotoxicity in the same cell lines which were used to determine efficacy. In these assays, A549 cells were cultured and plated as for the efficacy assays, and cells were incubated with compound for two days and cell viability measured. For the αHTs, CC50 values ranged from 43 to >240 μM, and they ranged from 16.5 to > 240 μM for the HPDs.

Table 2

EC50 against RVFV replication.

			A549			Vero
Compound number	Compound name	Chemotype	EC50, μM	CC50 μM	SI	EC50, μM	CC50 μM	SI
309	AG-I-183-P	αHT	5.9	98.1	16.8	1.6	>120	75.0
686	BE1111	TTP	7.9	67.6	8.6	6.3	>120	19
308	AG-II-18-P	αHT	8.4	119.0	14.2	1.2	>120	100.0
390	AB-2-70	αHT	9.4	224.2	23.9	11.9	>120	10.1
680	BE1105	TTP	11.6	>240	20.7	6.3	>120	19.0
670	ZEV-V7	HPD	15.3	>240	15.7	14	>120	8.6
840	NBA-I-155-Mono	αHT	15.4	141.6	9.2	31.6	>120	3.8
1039	AB-3-45	αHT	15.5	>240	15.5	8.9	>120	13.5
518	ZEV-V3	HPD	17.2	16.5	1.0	20.0	>120	6.0
331	NBA-I-31	αHT	17.7	>240	13.6	15.8	80.4	5.0
327	Aldrich Select CNC_ID 444085867	DHN	19.6	27.5	1.4	39.7	42.9	1.1
704	NBA-I-160	αHT	20.0	43.0	2.2	14.3	>120	8.4
867	DS-1-124	αHT	33.0	>240	7.3	8.7	>120	13.8
668	ZEV-V5	HPD	35.7	82.0	2.3	19.2	>120	6.3
330	NBA-I-14	αHT	42.6	>240	5.6	10.4	>120	11.5
320	NBA-I-13	αHT	90.3	154.1	1.7	33.96	>120	3.5
517	ZEV-V2	HPD	95.8	71.8	0.7	>120	>120	-
515	ZEV-E2	HPD	>120	93.6	0.6	24.3	>120	4.9
335	DH-2-60	αHT	>120	96.2	0.3	40.8	>120	2.9

Fig 2

In vitro dose-response and cytotoxicity of compounds against RVFV (MP12).

A549 cells were infected with RVFV MP12 then treated with decreasing concentrations of compound. The reduction in virus concentration was measured by FFA at twenty-four hours post infection. Data is representative of three individual experiments with two biological replicates. Error bars represent standard deviation.

3.4. Efficacy of compounds against wild type RVFV and LACV

In order to determine efficacy for wild type isolates of RVFV, we measured the efficacy of the αHT AG-II-18-P (308) and the nucleoside analogues NHC and ribavirin as a positive control against the highly pathogenic strain ZH501 in Vero cells (Fig 3A). Both NHC and ribavirin are small molecule inhibitors, with ribavirin having demonstrated activity against RVFV and NHC a nucleoside analog with activity against a broad array of viruses. The assay design is identical to Fig 1, with the exception that foci were measured at 18 hours post infection because of the increased rate of replication. In this assay, compound 308 had an EC50 of 54 μM, while NHC and ribavirin had EC50 values of 30.5 and 73.6 μM. Prior investigations of ribavirin restriction of RVFV has demonstrated similar results in Vero cells, showing the validity of the assay, and acting as a reference point for the novel compounds identified [53].

Fig 3

Antiviral effect of compounds on bunyavirus replication.

Vero cells were infected with either RVFV ZH501 (A) or LACV (B) then treated with decreasing concentrations of antiviral compound. Viral growth was measured by FFA. Data represents three independent experiments completed with biological replicates. Error bars represent standard deviation.

To determine if the metal chelating compounds including the αHTs AG-II-18-P (308), AG-I-183-P (309) and 16 related compounds were specific to RVFV or would have a broader activity, we investigated efficacy of these compounds against the bunyavirus LACV, as well as an unrelated flavivirus, ZIKV in Vero E6 cells. All three viral pathogens require viral metalloproteases for viral replication, while RVFV and LACV also encode a viral endonuclease used for cap-snatching [10–12, 58]. We observed a dose-dependent decrease in viral titers in the antiviral efficacy assays, with 6 of the 18 metal chelating compounds against LACV virus resulting in EC50’s ranging between 12.7 and 89.15 μM, while the nucleoside analogue β-D-N4-Hydroxycytidine had an EC50 of 3.6 μM (Fig 3B and Table 3). Of the 6 compounds that had activity against LACV, only 2 had activity against ZIKV. The αHT compound 704 and the TTP compound 680 had EC50’s of 23.0 and 19.5 μM respectively, with the nucleoside analogue β-D-N4-Hydroxycytidine having a EC50 of 1.7 μM. The ability of a subset of compounds to inhibit the growth and replication of both RVFV and LACV speaks to the potential of these metal chelating compounds to be developed as potential bunyaviridae therapeutics, or potentially as phlebovirus (RVFV)-or orthobunyavirus (LACV)-specific antivirals.

Table 3

EC50 against LACV and ZIKV replication.

Compound number	Compound name	Chemotype	LACV EC50, μM	ZIKV EC50, μM
308	AG-II-18-P	αHT	15.1	>120
309	AG-I-183-P	αHT	14.6	>120
320	NBA-I-13	αHT	>120	>120
327	Aldrich Select CNC_ID 444085867	DHN	12.7	>120
330	NBA-I-14	αHT	>120	32.7
335	DH-2-60	αHT	>120	>120
390	AB-2-70	αHT	>120	>120
515	ZEV-E2	HPD	>120	>120
517	ZEV-V2	HPD	>120	>120
518	ZEV-V3	HPD	>120	>120
668	ZEV-V5	HPD	>120	>120
670	ZEV-V7	HPD	>120	>120
680	BE1105	TTP	31.3	23
686	BE1111	TTP	45.4	>120
704	NBA-I-160	αHT	89.15	19.5
840	NBA-I-155-Mono	αHT	>120	>120
867	DS-1-124	αHT	>120	>120
1039	AB-3-45	αHT	>120	>120
NHC	β-D-N4-Hydroxycytidine	NUC	3.6	1.74

3.5. Selective indexes

Selectivity indexes (SI or CC50/EC50) were calculated for all compounds with EC50 values based on cytotoxicity in both Vero cells after one day compound treatment and in A549 cells after two days of treatment. SIs ranged from <1 to 402 in Vero cells (Table 1 and S2 Table) and <1 to 23.9 in A549 cells (Table 2). Confirmed hits were defined as compounds with SIs > 5 in Vero cells (n = 34) because this indicates that the reduction in RVFV foci was not a result of non-specific killing of the Vero cells in which the assays were completed (Table 1). Structures of all compounds in Tables 1 and 2 are in S2 Data, and structures of all confirmed hits are in S3 Data.

4. Discussion

Most primary hit compounds against RVFV were either αHTs or HPDs, but TRP, TTP, and DHN hits were also found. This distribution of hits is partially due to sampling bias owing to the disproportionate number of αHTs in the compound collection screened. Sampling bias, however, does not fully explain the hit distribution because only a minority of the αHTs screened were active, and because there were many chemotypes in the compound collection where hits were not found. These included dioxobutanoic acids, hydroxyxanthanones, thienopyrimidinones, pyridinepiperazinthieonpyrimidins, N‐biphenyltrihydroxybenzamides, and aminocyanothiophenes. EC50 values of the 62 compounds for which quantitative data were obtained ranged from 0.1 to >120 μM (S1 and S2 Tables). Selective indexes for these compounds in Vero cells in which the screening was conducted ranged from 1.1 to 1200. Twenty-eight compounds were confirmed hits based upon their selective index (SIs > 5 in Vero cells, 7.5% of the compounds screened) indicating that they were due to bona fide inhibition of RVFV rather than secondary effects of cytotoxicity. However, the increased CC50 values in the A549 (16.5 to >240 μM) and HepDES19 cells (<1 to 48 μM) indicate that compounds active against RVFV replication can have cytotoxicity in human lung and liver-derived cells, which must be addressed during subsequent hit-to-lead medicinal chemistry campaigns. One avenue for reducing cytotoxicity, at least for the αHTs, may be to reduce the lipophilicity and number of aromatic rings in the molecules because these parameters correlate with αHT toxicity [38]. RVFV infections proceed rapidly in vivo, so optimizing these hits to achieve toxicity profiles suitable for a one- to two-week treatment regimen in RVFV-infected patients or animals will likely be enough to yield usable drugs.

Identification of primary screen hits among the αHT, HPD, TRP, TTP, and DHN chemotypes indicates that a range of compounds can inhibit RVFV replication, but the lack of hits among the other chemotypes screened implies that there is specificity to RVFV inhibition (Fig 4). This is further supported by the efficacy of a subset of αHT class of compounds against LACV, with two of the compounds (308, 309) having activity against LACV, but not the unrelated flavivirus, Zika virus. The potential for specific inhibition of RVFV was confirmed by the wide range of inhibition patterns observed during counter-screening (S2 Table). For example, compound 362 had an EC50 of 2.6 μM vs. RVFV, an IC50 against human ribonuclease H1 of 212 μM, was inactive against E. coli growth, and was only modestly effective against the pathogenic fungus C. neoformans (minimum 80% inhibitory concentration (MIC80) = 24 μM). This indicates that although these chemotypes which can bid to the active sites of metalloenzymes and can have broad anti-microbial activity, individual compounds can be selective through specific interactions with their various targets (reviewed in [59]).

Fig 4

Representative structures of RFVF inhibitors.

(A) Inactive and active troponoid natural products, illustrating preference for oxygen triad, along with common nuclease inhibition mode for αHTs. (B) Synthetic αHTs with activity under 10 μM against RVFV, demonstrating broad substitution tolerance. (C) Representative examples of alternative scaffolds with activity against RFVF.

The 174 troponoids (αHTs, TRPs, TTPs) evaluated yielded 28 confirmed hits with TI values ≥ 5 (Table 1). Inhibition of RFVF by tropolones appears to require an intact metal ion chelating trident on the compound, which implies an interaction with two closely-spaced divalent cations on the target molecule due to the compounds’ known mechanisms of metal chelation (Fig 4a) [59]. For example, while the tropolones β- and γ-thujaplicin were inactive against the virus at concentrations upwards of 120 μM, the αHT β–thujaplicinol (46) was active, with an EC50 of 13.8 μM (Fig 2A). These trends extended to additional αHTs, of which 11 different molecules had EC50 values under 10 μM (Table 1), the most potent of which had an EC50 of 1.2 μM (308), (S3 Data). These more potent molecules had a broad range of appendages (Fig 4B), such as ketone (308, 309, 359, 362), amide (867, 1017, 1039), mono- (702) and bis (694, 696) thioethers, sulfoxide (336), and included a 3,7-dihydroxytropolone (362). Thus, there is tolerance to a variety of functional groups (Fig 4c). Apart from the αHTs, four additional tropolones had measurable EC50 values (TRPs 340, 341, 342 and 359, EC50 = 5.1–55.5 μM), each of which had a carbonyl appendage α to the tropolone oxygens that could provide an alternative third cation contact point. However, only 341 had an EC50 under 10 mM (S2 Data). Intriguingly, both acylated thiotropolones (680, 686) had sub-mM activity despite the lack of any tridentate cation binding motif. It seems possible that these thioester linkages could undergo cleavage in the cell, and that the active component in both instances is the free thiotropolone, as has been postulated previously for their potent anti-Cryptococcus neoformans activity [37].

Seven HPD primary hits were found among the 24 HPDs screened. Four of these were confirmed hits, 515, 518, 668, and 670, with EC50 values ranging from 14.0–24.3 μM (Fig 2). This is insufficient to generate a meaningful structure-activity relationship, but trends can be inferred. The oxygen trident of the HPD scaffold is essential for its activity, as the loss of any one of these oxygens results in inactive compounds. In each case it is presumed based on data with other HPDs against HBV [47] that strong ionic interactions, along with charge-assisted hydrogen bonds potentially anchor the chelator moiety of HPDs (N-Hydroxyimide group) to a cellular or viral metalloenzyme ensemble comprised of the two positively charged Mg++ ions. Lastly, aromatic substitutions at the imine nitrogen are tolerated that carry modifications including halogen electron withdrawing groups and an alkyl electron donating group.

The mechanism(s) of action of these RVFV inhibitors are unknown and could involve inhibition of viral and/or cellular proteins. However, inhibiting one or more mono- or di-metalloenzymes needed for viral replication by chelating their active site cations is a potential mechanism. The rationale for implicating metal chelation comes from the compound structures and their known activities against HIV and HBV [16-18]. The αHTs and HPDs have metal chelating tridents suitable for binding to the Mg++ ions in di-metalloenzyme active sites, and the αHTs are known to work by this mechanism against the HIV ribonuclease H and/or integrase [17, 21]. However, the failure of many compounds with known ability to inhibit divalent cation containing metalloenzymes (~70% of the αHTs did not inhibit RVFV growth) indicates that metal chelation by itself is insufficient, presumably because additional compound:target interactions are needed to provide sufficient binding affinity to inhibit viral replication. One potential target for these inhibitors is the RVFV L protein, where either the viral RNA-dependent RNA polymerase (RdRp) activity or the cap-snatching endonuclease activity of the viral L protein could be affected [32, 60]. This is because both are di-metalloenzymes that catalyze a reaction required for viral replication.

5. Conclusions

Screening for RVFV replication inhibitors among compounds selected for their similarity to inhibitors of viral nucleases identified 47 novel RVFV inhibitors. The frequent efficacy of the αHT and HPD compounds screened against RVFV replication indicates that these two scaffolds are promising candidates for optimization into anti-RVFV drugs that target metalloenzymes for use against human and/or veterinary infections. Cytotoxicity was observed in human hepatoblastoma cells, indicating that identifying and mitigating the causes of cytotoxicity will be key to optimizing these hits. The conserved features of viral replication among Bunyavirales, and the activity of these compounds against RVFV and a subset of these compounds having activity against LACV implies that these hits hold potential for development into treatments for related pathogens, including Hantaan virus, severe fever with thrombocytopenia syndrome virus, and Crimean-Congo hemorrhagic fever virus.

Primary screening data.

(XLSX)

Click here for additional data file.

EC50 value against RVFV.

(XLSX)

Click here for additional data file.

Experimental methods for compound generation and validation of compounds.

(PDF)

Click here for additional data file.

Structures of all compounds in Tables 1 and 2.

(PDF)

Click here for additional data file.

Structures of all confirmed hit compounds.

(PDF)

Click here for additional data file.

3 Jun 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Kylene Kehn-Hall

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Thank you for stating the following in the Competing Interests section:

"I have read the journal's policy and the authors of this manuscript have the following competing interests: pending patent application.

AP, GZ, JB, JT, and RM are inventors on a pending US patent application covering use of these compounds to treat Bunyavirus infections."

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Geerling et al. have exploited the fact that divalent cations play essential roles in catalysis and/or substrate binding of many enzymes, including those encoded by viruses. The authors cite notable examples of the use of small molecules to chelate active site divalent cations to inhibit metalloenzymes, including numerous FDA approved drugs. The authors have used these observations as the basis for screening known chelator molecules and structurally similar molecules for inhibitory activity against a potent viral pathogen, RVFV.

Thus, in the primary screen 375 (line 101) or 397 (line 264) compounds with known or plausible metal chelation ability were tested in a live-cell assay for the ability to reduce focus forming units (visualized by staining with antibody against the RVFV nucleoprotein) after 24 hours. Cytotoxicity was estimated by subsequent staining of the treated cells with crystal violet and visual inspection of the integrity of the cell monolayers in the cell culture wells.

The compounds with the highest potency and least cytotoxicity were retested in dose response curves in liver cells (HEP DES19) for cytotoxicity and lung cells (A549) for antiviral activity. Cross-testing for antiviral activity against a highly pathogenic strain of RVFV and against LACV from another family of Bunyaviruses was used to validate the initial hits.

Overall the results of the screening are interesting and give some optimism for further development of these compounds toward antiviral drugs. The primary screening method using low MOI infection of VeroE6 cells, methylcellulose overlay followed by immunostaining looks solid. Follow up of best compounds using different cell types and viruses is good but could be expanded slightly to support mechanistic assertions.

The only obvious weakness of the manuscript as presented is in the lack of mechanistic analysis. Although this might not be essential for an initial screening study, it is a little puzzling that there is rather extensive discussion of a specific chelation mechanism, and that the nuclease/ cap-snatching activity of the L protein may be the target. It would certainly be significant to identify inhibitors of cap-snatching activity for the bunyaviruses. However, there are no data to support these assertions.

One relatively easy experiment to preliminarily test this hypothesis would be to assay antiviral activity of select compounds against an RNA virus that does not employ cap-snatching (in addition to the demonstration of activity against LACV). If activity is lost, this is one data point that supports the hypothesis. If activity is retained, then the hypothesis (and discussion) would require some modification.

The authors use the observation that some of the compounds display activity against LACV as well as RVFV as an indication of specificity, but orthobunyaviruses and phleboviruses are fairly divergent. Although there may not be structural models available, it could be useful to indicate a percent amino acid conservation of the L proteins between these two families, or some other indication that the divalent ion binding sites of these two enzymes are similar.

The authors do a pretty good job of informally describing the potential SARs of identified a-HTs and HPDs, but a figure summarizing the gain/loss of activity corresponding to the changes to the scaffold would be helpful.

Minor point:

The explanation for why A549 cells were used in the dose response assays is a little curious, since it is noted that RVFV pathology is most pronounced in kidney, liver, spleen, not lung.

Reviewer #2: Geerling and colleagues describe the screening of metal coordinating antiviral compounds for Rift Valley fever virus (RVFV) and La Crosse (LACV) bunyavirus. Compounds were selected based on previous data for these compound classes to be effective for other viruses, such as HIV, HBV, and influenza. Currently, no approved therapeutics exist for RVFV and LACV and there is need for identification and development.

The authors developed focus forming assays for RVFV and LACV to perform antiviral screens in 96 well format. Compounds with antiviral activity were further characterized in dose-response studies against both the vaccine strain MP-12 for RVFV and the pathogenic RVFV ZH501 isolate. Polymerase inhibitors were identified with activities in the low to mid uM range. Identified compounds belong to the classes of alpha-Hydroxytropolones and N-Hydroxypyridinediones.

Overall, this is a fairly straight forward study, however, with limited compound characterization. Future studies will be necessary to further characterize individual compounds. The following comments should be addressed to improve the manuscript:

The abstract refers to 47 identified compounds and the Conclusion to 27.

Line 55: Please add references.

Lines 67 and 70: The same information about the lack of therapeutics is provided.

Lines 75 and 81: Repeat information is listed.

Line 91: Please add references for the listed antiviral compounds.

Line 92: Are more recent data for approved drugs available (after 2017)?

Line 133: Please add the institution for Drs. Buller and Hise.

Line 179: The information for LACV antibodies has already been described in paragraph 2.3

Compound cytotoxicity: Why did the authors not determine CC50 values in un-infected Vero cells?

Lines 239-243: Please add NHC to the figure legend

Line 251: If a reference is available for NHC testing against RVFV, please add.

Lines 307-309: The authors should add that aerosol transmission of RVFV has been described, which would justify the use of A549 cells.

Lines 309-311: While there is concordance in EC50 values for multiple compounds in both cells lines tested, there are also multiple compounds with differences (e.g., 308, 327, 867, 330, 515).

Why did the authors only test compound 308 against ZH501 and not 309 as well? Both compounds have the highest SI values (and 309 was tested against LACV).

Figure 1: The legend in B can be removed, since the same information is listed on the x-axis.

Figure 2: For multiple compounds the dose-response curves are not complete. Listed EC50 values might not be accurate.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Aug 2022

We have attached a document which has all of the responses to the reviewers comments, that maybe easier to read then the information below.

The information below is the exact text as in the attached Response to Reviewers document.

Response to Reviewer’s Comments

Review Comments to the Author

Reviewer #1: Geerling et al. have exploited the fact that divalent cations play essential roles in catalysis and/or substrate binding of many enzymes, including those encoded by viruses. The authors cite notable examples of the use of small molecules to chelate active site divalent cations to inhibit metalloenzymes, including numerous FDA approved drugs. The authors have used these observations as the basis for screening known chelator molecules and structurally similar molecules for inhibitory activity against a potent viral pathogen, RVFV.

Thus, in the primary screen 375 (line 101) or 397 (line 264) compounds with known or plausible metal chelation ability were tested in a live-cell assay for the ability to reduce focus forming units (visualized by staining with antibody against the RVFV nucleoprotein) after 24 hours. Cytotoxicity was estimated by subsequent staining of the treated cells with crystal violet and visual inspection of the integrity of the cell monolayers in the cell culture wells.

The compounds with the highest potency and least cytotoxicity were retested in dose response curves in liver cells (HEP DES19) for cytotoxicity and lung cells (A549) for antiviral activity. Cross-testing for antiviral activity against a highly pathogenic strain of RVFV and against LACV from another family of Bunyaviruses was used to validate the initial hits.

Overall the results of the screening are interesting and give some optimism for further development of these compounds toward antiviral drugs. The primary screening method using low MOI infection of VeroE6 cells, methylcellulose overlay followed by immunostaining looks solid. Follow up of best compounds using different cell types and viruses is good but could be expanded slightly to support mechanistic assertions.

The only obvious weakness of the manuscript as presented is in the lack of mechanistic analysis. Although this might not be essential for an initial screening study, it is a little puzzling that there is rather extensive discussion of a specific chelation mechanism, and that the nuclease/ cap-snatching activity of the L protein may be the target. It would certainly be significant to identify inhibitors of cap-snatching activity for the bunyaviruses. However, there are no data to support these assertions.

We appreciate the reviewer’s comments and have eliminated the discussion of a specific chelation mechanisms within the results and discussion sections of the manuscript.

One relatively easy experiment to preliminarily test this hypothesis would be to assay antiviral activity of select compounds against an RNA virus that does not employ cap-snatching (in addition to the demonstration of activity against LACV). If activity is lost, this is one data point that supports the hypothesis. If activity is retained, then the hypothesis (and discussion) would require some modification.

We have investigated a panel of 18 compounds that were effective against RVFV, against Zika virus and LACV and quantified compound efficacy. Of the 18 compounds, 6 compounds that had activity against RVFV also had activity against LACV, with only two of those 6 compounds having activity against ZIKV.

The authors use the observation that some of the compounds display activity against LACV as well as RVFV as an indication of specificity, but orthobunyaviruses and phleboviruses are fairly divergent. Although there may not be structural models available, it could be useful to indicate a percent amino acid conservation of the L proteins between these two families, or some other indication that the divalent ion binding sites of these two enzymes are similar.

We agree with the reviewers that within the genus bunyaviridae that orthobunyaviruses such as LACV and phleboviruses such as RVFV have divergent amino acid sequences. However, there are the number of

structural commonalities between the L proteins of both viral families. The enzymatic domains of the L proteins of both are co-linear with the endonuclease domain at the 5’ end of the protein and the cap binding domain at the 3’ end of the protein (1-3), while the four tunnels which lead to the catalytic center are structurally similar in size (2, 3). Specifically, the 5’ endonuclease for both LACV and RVFV are classified as His+, and contain PD(E/D)K motif (reviewed in (4)). The biochemical and structural similarities of IAV endonucleases and LACV/RVFV endonuclease has led to the proposal that IAV endonuclease inhibitors could be used against the viruses as well.

The authors do a pretty good job of informally describing the potential SARs of identified a-HTs and HPDs, but a figure summarizing the gain/loss of activity corresponding to the changes to the scaffold would be helpful.

We have added figure four and text describing the SAR of the αHTs and HPDs. This will allow the readers to understand the gain and loss of activity corresponding to the changes to the compounds scaffold.

Minor point:

The explanation for why A549 cells were used in the dose response assays is a little curious, since it is noted that RVFV pathology is most pronounced in kidney, liver, spleen, not lung.

We chose to use A549 cells for three key reasons: A549 are human cells commonly used in virological assays; they represent lung epithelial cells which can be a potential target of aerosol/droplet transmission of RVFV, a route which can occur during the processing of wild animals for food; and A549 cells, although derived from the lung, are epithelial cells, which is the cell type infected in the kidney, liver and spleen.

Reviewer #2: Geerling and colleagues describe the screening of metal coordinating antiviral compounds for Rift Valley fever virus (RVFV) and La Crosse (LACV) bunyavirus. Compounds were selected based on previous data for these compound classes to be effective for other viruses, such as HIV, HBV, and influenza. Currently, no approved therapeutics exist for RVFV and LACV and there is need for identification and development.

The authors developed focus forming assays for RVFV and LACV to perform antiviral screens in 96 well format. Compounds with antiviral activity were further characterized in dose-response studies against both the vaccine strain MP-12 for RVFV and the pathogenic RVFV ZH501 isolate. Polymerase inhibitors were identified with activities in the low to mid uM range. Identified compounds belong to the classes of alpha-Hydroxytropolones and N-Hydroxypyridinediones.

Overall, this is a fairly straight forward study, however, with limited compound characterization. Future studies will be necessary to further characterize individual compounds. The following comments should be addressed to improve the manuscript:

We greatly appreciate the comments and criticism provided. We have addressed every point thereby improving the text of the manuscript, including adding references or by providing a clear response with the rationale.

The abstract refers to 47 identified compounds and the Conclusion to 27.

The text within the manuscript has been clarified to describe the 47 novel inhibitors identified.

Line 55: Please add references.

References were added to support our statement.

Lines 67 and 70: The same information about the lack of therapeutics is provided.

Lines 75 and 81: Repeat information is listed.

We have removed the repeat information and have edited the text to make sure it is easy to read and understand.

Line 91: Please add references for the listed antiviral compounds.

References for each antiviral compound were added to the manuscript.

Line 92: Are more recent data for approved drugs available (after 2017)?

This reference is a review that is highly focused on mechanisms of metalloenzymes that has not been updated since 2017, so we cannot provide a newer reference. The reference continues to support the underlying argument that metal chelation is a viable drug mechanism.

Line 133: Please add the institution for Drs. Buller and Hise.

Their respective institutions were added to the text of the manuscript.

Line 179: The information for LACV antibodies has already been described in paragraph 2.3

Compound cytotoxicity: Why did the authors not determine CC50 values in un-infected Vero cells?

In the Vero cell assays we focused on the outcome of virally infected compound treated cells to permit rigorous interpretation of the efficacy of the compounds. Vero cells are a non-human primate cell line, our experimental plan was to investigate compound toxicity in HepDES19 cells and A549 cells, two human cells lines from tissues relevant to systemic drug administration.

Lines 239-243: Please add NHC to the figure legend

We have added NHC to the figure legend.

Line 251: If a reference is available for NHC testing against RVFV, please add.

No reference is available at this time.

Lines 307-309: The authors should add that aerosol transmission of RVFV has been described, which would justify the use of A549 cells.

We agree with the reviewer that aerosol transmission to humans is one possible route of exposure, which justifies the use of A549 cells. We have added a statement and supporting literature in the text of the manuscript.

Lines 309-311: While there is concordance in EC50 values for multiple compounds in both cells lines tested, there are also multiple compounds with differences (e.g., 308, 327, 867, 330, 515).

We agree with the reviewer that the level of concordance does vary depending on the individual compound. This is not uncommon in early-stage primary screening assays as different cell lines can have different phase I and/or phase II metabolic enzymes and/or different transporters/efflux pumps. Differences in these enzymes can alter transport and stability of compounds differentially in different cell lines, causing quantitative differences in EC50 values. Such issues are routinely examined in follow up studies after primary hit compounds are identified.

Why did the authors only test compound 308 against ZH501 and not 309 as well? Both compounds have the highest SI values (and 309 was tested against LACV).

We understand and appreciate the reviewer’s question. In this case it was due to logistics. The federal permit which controls our select agent laboratory requires that ZH501 is used in isolation and requires decontamination of the room by gas sterilization. We had a small window of opportunity to evaluate compounds against the ZH501 in our select agent laboratory and compound 309 was not available at the time.

Figure 1: The legend in B can be removed, since the same information is listed on the x-axis.

The legend has been updated accordingly.

Figure 2: For multiple compounds the dose-response curves are not complete. Listed EC50 values might not be accurate.

We agree with the reviewer’s observation that some compounds do not have a complete dose-response curve. The EC50 values are determined by non-linear curve fitting, which takes into account the upper and lower concentration limits of the compounds. Based upon the concentrations used in our assays for efficacy, any compound which did not decrease viral infection were categorized as having an EC50 of >120 μM. EC50 values above this level are biologically meaningless.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

25 Aug 2022

Metal coordinating inhibitors of Rift Valley fever virus replication

PONE-D-22-12188R1

Dear Dr. Brien,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kylene Kehn-Hall

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have addressed the comments raised by the reviewers. The manuscript has improved and is now acceptable for publication..

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

7 Sep 2022

PONE-D-22-12188R1

Metal coordinating inhibitors of Rift Valley fever virus replication

Dear Dr. Brien:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kylene Kehn-Hall

Academic Editor

PLOS ONE