DABMA: A Derivative of ABMA with Improved Broad-Spectrum

Letter Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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DABMA: A Derivative of ABMA with Improved Broad-Spectrum Inhibitory Activity of Toxins and Viruses Yu Wu,† Valeŕ ie Pons,‡ Romain Noël,‡ Sabrina Kali,§ Olena Shtanko,⊥ Robert A. Davey,# Michel R. Popoff,∥ Noël Tordo,§ Daniel Gillet,*,† Jean-Christophe Cintrat,*,‡ and Julien Barbier†

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Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette, France ‡ Service de Chimie Bio-organique et de Marquage (SCBM), CEA, Université Paris-Saclay, LabEx LERMIT, 91191 Gif-sur-Yvette, France § Antiviral Strategies Unit, Virology Department, Institut Pasteur, 75015 Paris, France ⊥ Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, Texas 78227, United States # Department of Microbiology, NEIDL, Boston University, Boston, Massachusetts 02118, United States ∥ Bactéries anaérobies et Toxines, Institut Pasteur, 75015 Paris, France S Supporting Information *

ABSTRACT: The small molecule ABMA has been previously shown to protect cells against multiple toxins and pathogens including virus, intracellular bacteria, and parasite. Its mechanism of action is directly associated with host endolysosomal pathway rather than targeting toxin or pathogen itself. However, the relationship of its broad-spectrum anti-infection activity and chemical structure is not yet resolved. Here, we synthesized a series of derivatives and compared their activities against diphtheria toxin (DT). Dimethyl-ABMA (DABMA), one of the most potent analogs with about 20-fold improvement in protection efficacy against DT, was identified with a similar mechanism of action to ABMA. Moreover, DABMA exhibited enhanced efficacy against Clostridium dif f icile toxin B (TcdB), Clostridium sordellii lethal toxin (TcsL), Pseudomonas Exotoxin A (PE) as well as Rabies and Ebola viruses. The results revealed a structure−activity relationship of ABMA, which is a starting point for its clinical development as broad-spectrum drug against existing and emerging infectious diseases. KEYWORDS: Structure−activity relationship study, broad-spectrum inhibitor, endolysosomal pathway, toxin, virus

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selectively acts at host cell late endosomes, an essential compartment for toxin translocation as well as entry and replication of pathogens.10,16−18 Its safety and efficacy against ricin has been validated in vivo.10 Moreover, ABMA was found to protect cells against four bacterial toxins (DT, LT, TcdB, and TcsL toxins), four viruses (Ebola, rabies, dengue-4 virus, and herpes simplex virus-2), two species of Chlamydiales intracellular bacteria, and the parasite Leishmania infantum at micromolar level.10,19 ABMA bears a hydrophobic adamantane and a substituted aromatic moiety (Scheme 1). Here, we have evaluated the structure−activity relationship of this novel scaffold to develop more potent derivatives with broad spectrum therapeutic potential against infectious diseases. We focused on three main sites available for diversity screening (Scheme 1): amino group (1), benzene ring (2), and the adamantane moiety (3). A cell-based assay was used to detect the cytotoxicity of DT by measuring protein synthesis inhibition in human cells and thus ranking the potency of analogs. DT is a single pathway-orientated toxin, which

utbreak of emerging pathogens without licensed treatments and drug resistant pathogens has long been a major public health concern.1,2 A promising strategy in drug discovery is identifying therapies targeting host components, which are indispensable for pathogen to enter and replicate in host cells.3−11 Repurposing already approved drugs targeting host function as antimicrobial agents has made some progress.11 Amodiaquine, a clinically used antimalarial drug, selected from a cell-based multiplex approach against anthrax lethal toxin (LT) and DT, protects cells against others toxins (TcdB) and several viruses (including Ebola, SARS coronavirus, Venezuelan equine encephalitis virus, rabies, Junin, and chikungunya virus) by inhibiting host cathepsin B.6 Moreover, Several new chemicals were identified as host-targeted inhibitors by modulating intracellular trafficking of toxins and pathogens. EGA, an active molecule against LT, blocks trafficking of various toxins (DT, PE, cytolethal distending toxin (CDT) from Haemophilus ducreyi, Clostridium toxins) and viruses (influenza virus and lymphocytic choriomeningitis virus) in early endosomes.9,12−14 Previously, we performed a cell-based high-throughput screen against ricin toxin and identified three inhibitors.15 Retro-1 and -2 that selectively block retrograde trafficking of toxins and pathogens at the early endosome-TGN (trans-Golgi Network) interface without apparent toxicity.15 Another hit, ABMA, © XXXX American Chemical Society

Received: April 4, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acsmedchemlett.9b00155 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

Letter

Scheme 1. Three Highlighted Regions Were Explored for Studying SAR of ABMA

Table 2. Analogs of ABMA (Benzene Ring Modification)

Table 1. Analogs of ABMA (Modification on Amino Group)

Scheme 2. Adamantane-Derived Drugs and Their Protection Fold against DT

of 30 μM on cells with increasing concentrations of DT. ABMA was used as a positive control to calculate the protection fold (see Supporting Materials and Methods). Half-maximal effective concentrations, EC50 values, were calculated for compounds displaying better activity relative to ABMA.

translocates its enzymatic subunit into the cell cytoplasm exclusively from acidified endosomes.20 In the first round of experiments, compounds were evaluated at a fixed concentration B



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Table 3. Analogs of ABMA (Adamantane Replacement and Modification)

All modifications of the amino group (imino, methylation, amide or acetylation or benzamide; see Table 1, 1a−1e) were all less potent against DT than ABMA indicating importance of the amino group moiety for ABMA anti-DT activity. Next, the importance of the phenyl ring was evaluated. As summarized in Table 2, replacement of the phenyl ring by indole (2a) or 2-adamantane (2b) reduced potency. In contrast, moving the bromide to the para position on the ring (2d) or substituting by iodine (2e) improved activity by 2.3 or 6. Moreover, the methoxy group (2f) is not mandatory for effect if halogen is introduced in the para position (2d and 2e) instead of the meta. Similarly, nitryl in the meta position and without the methoxy group (2c) also increased its activity by 5.7-fold. Finally, we focused on the adamantane part of ABMA. Compounds containing adamantane moieties are used as both antiviral and antiparkinsonian medications.21,22 Scheme 2 compared activity of known adamantane-derivatives (amantadine, 1-(1-adamantyl) ethylamine hydrochloride, and mem-

antine) against DT. Each has negligible inhibitory activity against DT (see protection fold in Scheme 2). Apparently, an amine bearing an adamantane substituent is not sufficient for sustaining activity. We continued to examine the importance of the adamantane group. We noted there was no marked difference in activity between 1-substituted amino-adamantane and the 2-amino isomer (compare 2a and 2b). Then, when the adamantyl moiety was replaced, respectively, by cyclohexyl, phenyl, and benzyl groups (compounds 3a−3c), activity was lost (Table 3). A longer methylene chain (three CH2 between the phenyl ring and the amine) was also inactive (3d). A bulky t-butyl chain (C8, 3f), naphthyl (3g), or trimethylbicyclo (3h) each resulted in lowered activity compared to ABMA, while a 5-methyl-2-(propan-2-yl) cyclohexan-1-amine derivative showed a slight increase in bioactivity (3i). A dimethyl adamantane (3j) greatly improved protection by more than 18-fold. Combining the findings from the previous series of analogs (Tables 1 and 2), we synthesized C



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Table 4. Anti-DT Activity and Toxicity of Selected ABMA Analogs

Figure 1. Activity and toxicity of DABMA. (A) ABMA and DABMA at 30 μM inhibit cytotoxicity of DT on A549 cells. (B) Cells were incubated with indicated concentration of DABMA and then challenged with increasing concentrations of DT. (C) EC50 of DABMA. (D) Toxicity of DABMA measured by Alamar Blue assay. Data are shown as mean values of duplicate wells ± SD from one of representative experiments (n = 3). (E) Colonyforming assay of DMSO (a), DABMA (b, 60 μM), DABMA (c, 30 μM), and Paclitaxel (d, 5 μM) in 24-well plate, with clones produced by A549 cells. Images are from one representative experiment (n = 2). Paclitaxel is an apoptosis inducer, used as positive control here.

related compounds with iodine replacement of bromide on the

Among the 23 new derivatives, we selected compounds 3j, 3k, and 3l with 10−35-fold improvement in protection for EC50 (50% of maximal effective concentration) and CC50 (50% cytotoxicity concentration) determination. CC50 was evaluated by Alamar Blue assay. Among them, compound 3j, dimethyl-1-

phenyl ring (3k and 3l) and found both compounds exhibited strongly increased protection factors of 35-fold and 12-fold, respectively. D



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Figure 2. DABMA induces accumulation of lysotracker and cholesterol in endosomes, distinct from Baf A1 action. (A) LysoTracker Deep Red (50 nM, 30 min) staining of A549 cells pretreated with DMSO or compounds (DABMA, ABMA at 30 μM, compound 1b at 60 μM) for 2 h. Nuclei were stained with Hoechst 33342 (blue). Fluorescence intensities (10,000 cells/well) from 3 × 3 fields of each well were determined by imaging reader (Cytation 5, Biotek), shown as mean ± SEM (n = 4). (B) Cells were respectively treated with DMSO or compounds for 18 h, then fixed and stained with the cholesterol-avid fluorophore Filipin III. (C) Immunostaining of Rab7 from DMSO or DABMA 6 h pretreated cells. Nuclei were stained with DAPI (blue). Histograms show quantification of fluorescence (mean ± SEM, n > 30 cells for B and C) by Image J. A.U. means arbitrary unit. Statistical significance was tested using unpaired t tests, compared to DMSO control (n.s., p > 0.05; *p < 0.05; **p < 0.01). Representative images were obtained by confocal microscope (SP8X, Leica). Scale bar, 10 μm. (D) DABMA together with Baf A1 largely improved inhibitory effect on DT. A549 cells were respectively incubated with Baf A1 (0.15 nM), DABMA (15 μM), both compounds, and vehicle alone (DMSO) before addition of DT in the continued presence of the compounds. Following steps were performed as described in Supporting Materials and Methods.

fluorescent puncta, compared with ABMA at the same concentration (30 μM). Noteworthy, other analogs, compound 1c (1a and 1d, data not shown) lacking antitoxin activity, had similar cell staining as the untreated/DMSO treatment control (Figure 2A). These analogs each had modifications of the amino group, thus losing the protonation site and not being able to penetrate the lysosomes (lysosomotropic effect). This strongly suggested having protonatable site(s) is required for antitoxin activity. Previous work indicated ABMA-induced cholesterol accumulation similar to U18666A, a known cholesterol transport inhibitor.10 Thus, we investigated intracellular cholesterol distribution in compound-treated A549 cells by filipin III, a fluorescent probe with high affinity for cholesterol. Here we also observed increased filipin III signals in the presence of DABMA, but not in the presence of compound 1c (Figure 2B). Enhanced endosome volumes probed by lysotracker staining in the presence of DABMA was further confirmed by increased Rab7 immunostaining (Figure 2C) rather than Lamp1 staining (see Figure S3), suggesting that DABMA targets late endosomes. In addition, other cellular organelles were not influenced by DABMA, such as Golgi apparatus and endoplasmic reticulum (stained by Cytopainter) or mitochondria (stained by Mito Tracker, see Figure S4). Moreover, as we observed with ABMA,10 the combination of DABMA (EC50 = 25.7 μM) and bafilomycin A1 (Baf A1, EC50 = 0.6 nM) at concentrations lower than their respective EC50s

adamantyl (5-bromo-2-methoxybenzyl) amine (DABMA), showed the best selectivity index (Table 4) with some toxicity (>100 μM, see also Figure S1 for HeLa and HUVEC cells) but was ranked as the most potent inhibitor (Figure 1). For compound 3k, even though exhibiting the highest protection factor, its CC50 and consequent selectivity index were less than those of DABMA. The ClogP and pKa were calculated for each using Chemdraw (PerkinElmer). We found that enhanced activity was associated with increased ClogP, indicating that hydrophobicity is favorable for compound bioactivity. Moreover, colony-forming assay of DABMA at high concentration (60 μM) and low concentration (30 μM) indicated that DABMA, even 2-fold exceeding its EC50, did not influence cell viability (Figure 1E), suggesting it has a neglectable long-term-toxicity. We further evaluated DABMA using other cell lines including Vero, PC3, A431, and DLD1 (data not shown) as well as primary HUVEC cells (human umbilical vein endothelial cells, see Figure S2) against DT and found similar activity indicating that its inhibitory action on DT is not cell type-specific. To show that DABMA prevented the cytotoxicity effect of DT by acting on the endolysosome pathways as reported for the parent molecule, we compared the morphology of acidic endosomes of cells exposed to ABMA and DABMA. We first checked lysotracker Deep Red staining on A549 cells after incubation with DABMA for 2 h. Images obtained by confocal microscopy showed more intensely labeled and enlarged E



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Figure 3. DABMA inhibits TcdB, TcsL, and PE with improved efficacy. Vero cells were treated with the indicated doses of TcdB for 4 h (A and B) or with TcsL for 16 h (C) in the presence of DMSO, ABMA, or DABMA at the same concentration (30 μM), and their morphological changes were imaged by an inverted microscope (Ti−U, Nikon). Rounded cells were counted and normalized to total cells (>100 cells for each treatment) counterstained with Hoechst 33342 (A−C). (D) L929 cells were intoxicated with increasing doses of PE in the presence of DMSO or compounds (at 30 μM) for 18 h, and their protein synthesis was determined as in Figure 1A. Data are shown as mean values of duplicate wells ± SD from one of representative experiments (n = 3).

Figure 4. DABMA inhibits rabies and Ebola virus with improved efficacy. (A) BSR cells were pretreated for 4 h with indicated concentrations of compounds in 96-well plate, then challenged with RABV (MOI = 14) for 1 h. Cells were washed and incubated again with compounds for 24 h. Infected cells were fixed and detected by immunostaining of the RABV ribonucleocapsid (RNP). Images of nuclei and RNP staining from 15 fields of each well (more than 10,000 cells) were used to calculate the percentage of inhibition. (B) Compounds preincubated for 1 h with HeLa cells (20,000/ well, 96-well plate) were challenged with EBOV-eGFP in the presence of compounds for 24 h. Cells were fixed and stained with DAPI, and numbers of nuclei and eGFP-positive (infected) cells were counted. The relative infection efficiencies were calculated by dividing the number of infected cells by the number of nuclei. Data are shown as mean values of triplicate wells ± SD, from one of representative experiments (n = 3).

Since ABMA has broad-spectrum anti-toxin and pathogen activity, we tested if DABMA could inhibit other toxins and viruses with improved efficacy. Toxin B from Clostridium dif f icile (TcdB) and lethal toxin from Clostridium sordellii (TcsL) are both acidic endosome-dependent toxins, and are sensitive to ABMA. They inactivate small GTPase by their glucosyltransferase activity and induce cell rounding.24,25 Vero cells were respectively treated with same concentrations of ABMA and DABMA, and then challenged with increasing concentrations of TcdB or TcsL for the indicated time. We observed that at the

substantially improved the protection factor (R) against DT compared to individual applications. The R factor was 6.3 and 5.2, respectively, for DABMA (15 μM) and Baf A1 (0.15 nM) and reached more than 300 when combined (Figure 2D) without additive toxicity (Figure S5). Baf A1, a potent inhibitor of vacuolar H+ ATPase (V-ATPase), blocks DT translocation by inhibiting endosome acidification.23 Our findings indicate DABMA has a similar action to ABMA impacting the host late endosome, which is not linked to the increase in endosome acidification; otherwise, it would be counterbalanced by Baf A1. F



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same concentration (30 μM), ABMA partially reversed cell rounding by TcdB, while DABMA reduced cell rounding more efficiently (Figure 3A,B). Similarly, TcsL-induced cell rounding can be inhibited by DABMA with 5.6-fold improvement compared to ABMA (Figure 3C). EC50s of DABMA for TcdB and TcsL were, respectively, 38.6 and 15.8 μM, and the corresponding selectivity indexes were 6.34 and 7.38. Besides these toxins, we tested inhibitory activities of ABMA and DABMA on Pseudomonas aeruginosa exotoxin A (PE). The intracellular intoxication pathway of PE is more complex than previously tested toxins (DT, TcdB, and TcsL), as it utilizes the retrograde transport pathway (from early endosomes to the trans-Golgi network (TGN)) as well as Rab9-dependent movement from late endosomes to TGN.26,27 As shown in Figure 3D, both ABMA and DABMA protected protein synthesis of L929 cells intoxicated by PE. Importantly, DABMA had an improved protection factor of 4.8-fold compared to ABMA. Its EC50 and selectivity index were 20.77 μM and 4.35. Knowing that DABMA protected cells from several toxins with enhanced efficacy compared to ABMA, we tested whether DABMA was able to inhibit cell infection by viruses that were previously shown sensitive to ABMA. Ebola virus (EBOV) and rabies (RABV) both rely on acidic-endosomes to fuse with their surface glycoprotein, after which their capsid is released from endosomes to initiate viral gene expression and genome replication.28 Also, EBOV relies on the fusion of NPC1, an endosomal cholesterol transporter, and infection is inhibited by U18666A.29 We previously showed that ABMA inhibited RABV infection in baby hamster kidney (BSR) cells with a similar efficacy to ribavirin, an antiviral drug inhibiting viral RNA synthesis and viral mRNA capping. Figure 4A showed DABMA inhibited RABV more efficiently than ABMA as well as ribavirin. EBOV has been declared as a Public Health Emergency of International Concern by WHO because of high infectivity/ fatality and no approved therapeutics. Encouragingly, 1 hpretreatment with DABMA at submicromolar level inhibited more than 50% infection of a replication competent recombinant EBOV encoding GFP as an infection marker in HeLa cells (Figure 4B, EC50: 908 nM), with 6.6-fold improvement on EC50 compared to ABMA. DABMA CC50 in the same human cells was 116.7 μM (see Figure S1), indicating a selectivity index superior to 120. One recently identified inhibitor against EBOV with submicromolar EC50 is tetrandrine.30 Tetrandrine disrupts two-pore channels (TPCs) from host lysosomes, thereby restricting virus trafficking.30 We examined that ABMA and tetrandrine have apparent synergistic effect against DT (data not shown, submitted), suggesting distinct inhibitory mechanisms. We anticipate that further SAR exploration of ABMA and more detailed investigation of its target by new techniques31,32 may led to new anti-EBOV drug candidate. Altogether, our results suggested that DABMA inhibits four toxins as well as two viruses with improved efficacy compared to ABMA. Twenty-three new molecules were synthesized, based on three major moieties of ABMA, to clarify functional significance of each moiety for its activity. The adamantane moiety is optimal to sustain its activity but not predominant, whereas the amino group as the protonated site is indispensable for activity. The aromatic moiety with minor modification can modulate activity as well as toxicity. From these analogs, we identified a structureoptimized ABMA derivative, DABMA, which exhibited broadspectrum anti-toxin and anti-virus activity with significantly

improved efficacy. We demonstrated that mode of action of DABMA is similar to that of ABMA, selectively acting on late endosomes followed by cholesterol accumulation. DABMA represents a promising starting point for the development of drug against diverse infectious diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00155. Materials and methods (pp S2−S5); evaluation of DABMA cytotoxicity on HeLa and HUVEC cells by Alamar Blue (Figure S1); DABMA inhibits cytotoxicity of DT on HUVEC cells (Figure S2); immunostaining of Lamp1 from DMSO or DABMA 24 h pretreated cells (Figure S3); DABMA does not affect the morphology of the Golgi apparatus, the endoplasmic reticulum, and the mitochondrial (Figure S4); combination of DABMA and Baf A1 does not induce additive toxicity (Figure S5) (PDF)

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Copies of spectra (ZIP)

AUTHOR INFORMATION

Corresponding Authors

*(D.G.) Phone: +33 (0)1 69 08 76 46. E-mail: daniel.gillet@cea. fr. *(J.-C.C.) Phone: +33 (0)1 69 08 21 07. E-mail: [email protected]. ORCID

Yu Wu: 0000-0002-8250-7571 Jean-Christophe Cintrat: 0000-0001-5758-8077 Author Contributions

D.G., J.B., and J.-C.C. conceived and designed the experiments; Y.W., V.P., R.N., S.K., O.S., R.-A.D., and N.T. performed the experiments; M.-R.P. prepared clostridial toxins; Y.W., J.-C.C., and J.B. analyzed the data; Y.W., J.-C.C., J.B., and D.G. wrote the paper. Funding

This work has been funded by the joint ministerial program of R&D against CBRNE risks, CEA and under Contract ANR-18CE18-0016. This work was also supported by U.S. National Institutes of Health and National Institute of Allergy and Infectious Diseases grant R01AI063513. SIMOPRO and SCBM are members of the Laboratory of Excellence LERMIT supported by a grant from the Agence Nationale de la Recherche (ANR-10-LABX-33) and RetroLeishma project R3. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS DT, diphtheria toxin; LT, lethal toxin from Bacillus anthracis; PE, Pseudomonas exotoxin A; TcdB, toxin B from Clostridium dif f icile; TcsL, lethal toxin from Clostridium sordellii; CDT, cytolethal distending toxin; TGN, trans-Golgi network; SAR, structure−activity relationship; HUVEC, human umbilical vein endothelial cells; Baf A1, bafilomycin A1; EBOV, Ebola virus; RABV, rabies virus; BSR, baby hamster kidney cells; TPCs, twopore channels G



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