ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.


Fig
. S1 Stability in culture medium. Aliquots of complete culture medium supplemented with ABMA were prepared, breathable sealed and placed in the incubator, 1, 2, 4 and 6 days before assessing cell protection activity against ricin by protein biosynthesis determination. The biological activity corresponds to the EC 50 ratio at each time point in comparison to the direct protection assay against ricin-induced cytotoxicity as shown in Fig 1B (100% activity for aliquot prepared 1 day before protein biosynthesis measurement).  AlamarBlue ® reagent was then added as 10% of the culture medium volume and the fluorescence was measured 2 h later with a Cytation 5 cell imaging multi-mode reader (BioTek). Results are from a single experiment. Fig. S4 ABMA has no effect on the binding of DT to its membrane receptor.
DTR8 is a recombinant DT receptor binding domain harboring a mutant in order to improve solubility but without affecting its affinity to receptors 1 . A fluorescent DTR8 was made by chemical coupling with Alexa488 (DTR A488 ). (A) Flow cytometry of Vero cells incubated at 4°C without (black) or with (gray) DTR A488 . (B) Cellular treatment with ABMA at 60 µM or DMSO for 1 h did not affect binding of DTR A488 on Vero cells at 4°C (p>0.05, n=3). Values from mean fluorescence intensity of three independent experiments.

Fig. S5 ABMA does not inhibit infection by CHIKV.
HEK293 cells were pre-treated with ABMA solubilized in DMSO for 1 h and then challenged with CHIKV for 1 h. Cells were washed and then incubated again with same concentrations of ABMA for 72 h. Virus induced-cytopathic effect (CPE) was measured by MTT assay. Following steps were performed as described in Figure 2A. The R factor was 4.7 and 5.2 for ABMA and Baf A1, respectively, and reached 63.9 when combined together. PC3 cells were incubated with vehicle alone (DMSO) or ABMA (120 µM) for 2 h at 37°C. Cells were fixed, permeabilized and labeled for the indicated compartment markers: PDI (Endoplasmic reticulum), TGN46 (Golgi apparatus). Images were obtained with confocal microscope (SP8X, Leica). Nuclei were stained with Hoechst 33342 (blue). Scale bar, 10 µm.      GAGs, Glycosaminoglycans.
The cells were plated in 96-well Cytostar-TTM scintillating microplates (Perkin-Elmer) with scintillator incorporated into the polystyrene plastic one day before. After incubation with various concentration of compounds (or carrier, DMSO) for indicated time at 37 °C, cells were challenged with increasing doses of ricin, diphtheria toxin or Stx2 for at least 16 h in continued presence of compounds. Subsequently, the medium was removed and replaced with DMEM without leucine (Gibco) containing 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U/mL penicillin -100 µg/mL streptomycin supplemented by 0.5 µCi/mL [ 14 C]-leucine. The cells were grown for an additional 3-6 hours at 37 °C in an atmosphere of 5% CO2 and 95% air. Protein biosynthesis was then determined by measuring the incorporation of radiolabeled leucine into cells using a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer).
The mean percentage of protein biosynthesis was determined and normalized from duplicate wells. All values are expressed as means ± SEM. Data were fitted with Prism v5 software (Graphpad Inc., San Diego, CA) to obtain the 50% inhibitory toxin concentration (IC 50 ) i.e. the concentration of toxin that is required to inhibit 50% protein synthesis of cells. IC50 values and protection factor R (R = IC 50 drug/IC 50 DMSO) were determined by the software's nonlinear regression "dose-response EC 50 shift equation". The goodness of fit for toxin alone (carrier) or with drug was assessed by r 2 and confidence intervals.
Determination of EC 50 values of ABMA against ricin, DT, Stx2. EC 50 was used to evaluate ABMA efficacy because it is more precise than R values and associated % protection. This is due to the fact that R values may fluctuate between cell experiments from different 96-wells plates corresponding to compound tested on different days. In contrast EC 50 value for a single compound is calculated from experimental data obtained on a single 96-well plate. Cell assays were performed with various concentrations of the compound. For each concentration, a percentage of protection was determined from R values calculated with Prism software with Rmax corresponding to the higher value of R of the series: Drug concentration was plotted against the corresponding percentage of protection of cells and the 50% efficacy concentration (EC 50 ) was calculated by non-linear regression using the Prism software package.

Anthrax toxin MEK cleavage Assay
Cell lysates from HUVECs intoxicated with LT (3 µg/ml protective antigen (PA) and 1µg/ml Lethal factor (LF)) in the absence or presence of ABMA were resolved on 12% SDS-PAGE gels using standard conditions and transferred to Immobilon-P PVDF membranes (Millipore). The antibodies used were as follows: an antibody directed toward the amino-terminal part of MEK2 (MEK2-N20, Santa Cruz), anti-beta-actin (Clone AC74, Sigma). The primary antibodies were revealed using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (DAKO) followed by chemiluminescence using Immobilon Western (Millipore). The chemiluminescent signals were recorded on a FUJIFILM LAS-3000, and the data were quantified using the MultiGauge V3.0 software.

BoNT/A SNAP25 cleavage assay
Primary cultures of rat cerebellar granule neurons (CGNs) were prepared from 6-days-old rats as previously described 2 . Briefly, cerebella were isolated, mechanically disrupted and then trypsinized. Cells were then collected and plated into 24 well plates, pre-coated with poly-Llysine (50 µg/ml), at a cell density of 5 × 10 5 cells per well. Cultures were maintained at 37 °C, 5% CO 2 , 95% humidity in neurobasal medium supplemented with B27, to which 20 mM KCl, 0.5 mM glutamine 100 U/ml penicillin and 100 U/ml streptomycin were added. To inhibit proliferation of non-neuronal cells, cytosine β-D-arabinofuranoside (5 µM) was added to the medium 24 h after plating. ABMA (30 µM) was added to cell medium 1 h prior to BoNT/A exposure (500 pM, 24h). Cell lysate were collected and SNAP-25 protein was detected by immune blot with antibody (SMI-81, Biolegend).

EBOV experiments
All experiments with replication competent EBOV were performed in the biosafety level 4 Image acquisition and analysis. Images were acquired on an automated spinning disk confocal microscope (Opera QEHS, Perkin Elmer Technologies) at the Imagopole (Institut Pasteur imaging platform). Briefly, 15 fields were acquired per well with a 10× air objective (NA 0.4) to reveal the nuclei stained with Hoechst (405 nm/450 nm) and to detect the RABV-RNP revealed by a fluorescein-coupled antibodies (488 nm/540 nm). The image analysis was performed using AcapellaTM (Perkin Elmer Technologies) as follows: (1) the nuclei were segmented in the Hoechst channel to estimate the number of cells imaged in each well (above 10,000 total nuclei -TN -in the non-infected control wells); (2) the segmentation mask was then increased to the limit of each individual cytoplasm, generating a "cell mask" in which the viral RNP (green dots) were segmented using a spot detection module of the software, allowing the reporting of the number of infected cells -InfC -i.e. containing at least 1 green spot. Finally, we calculated the ratio [InfC/TN*100], which reports the percentage of infection of each well.

DENV4 infection experiments
Vero cells (ATCC) were plated into 96-wells microplates for 30 min pre-treated with 6 concentrations of ABMA and one reference compound ribavirine, and infected with 125 TCID 50 per well of a DEN-4 serotype virus. Seven days later, cells were lysed using Igepal 2.5% in phosphate buffer (PBS) before quantifying viral replication using specific ELISA conditions (a specific serum from DENV-4-infected non-human primate and peroxidase-conjugated goat antimonkey IgG diluted in PBS, 5% milk, 0.5% Tween 20). Absorbance was measured using a spectrophotometer at 492 nm.
Previously, this viral strain was amplified and titrated by limit-dilution; the 50% tissue culture infectious dose (TCID 50 ) was calculated using Kärber's formula. subsequently viewed on a Leica DMIR or TCS SP5 microscope, respectively. Data were processed using FIJI (Image J). Antibodies against snHsp60 were prepared as previously described 3 .
To obtain progeny infection, compound-treated cells were lysed with glass beads 48 h post infection (hpi) and lysate was used to infect fresh HeLa229 cells. Progeny infection in absence of ABMA was lysed 24 hpi and analyzed together with primary infection samples by immunoblotting. Chlamydial growth was detected with antibodies against ctrHSP60 (clone A57-B9, Santa Cruz Biotechnology) and β-actin (clone AC-15, Sigma-Aldrich) was used as loading control. Immunofluorescence analysis of Progeny infection was also performed 24 hpi, cells were fixed and stained for DAPI. Ctr inclusions were detected by their GFP-expression signal.

In vitro antileishmanial evaluation
The experiments were performed by using the Leishmania infantum strain (MHOM/FR/2008/LEM5700). Antileishmanial activity on L. infantum axenic amastigotes was carried out as previously described 4 .