High-throughput screening identifies a novel natural product-inspired scaffold capable of inhibiting Clostridioides difficile in vitro

Clostridioides difficile is an enteric pathogen responsible for causing debilitating diarrhea, mostly in hospitalized patients. The bacterium exploits on microbial dysbiosis induced by the use of antibiotics to establish infection that ranges from mild watery diarrhea to pseudomembranous colitis. The increased prevalence of the disease accompanied by exacerbated comorbidity and the paucity of anticlostridial drugs that can tackle recurrence entails novel therapeutic options. Here, we report new lead molecules with potent anticlostridial activity from the AnalytiCon NATx library featuring natural product-inspired or natural product-derived small molecules. A high-throughput whole-cell-based screening of 5000 synthetic compounds from the AnalytiCon NATx library helped us identify 10 compounds capable of inhibiting the pathogen. Out of these 10 hits, we found 3 compounds with potent activity against C. difficile (MIC = 0.5–2 μg/ml). Interestingly, these compounds had minimal to no effect on the indigenous intestinal microbial species tested, unlike the standard-of-care antibiotics vancomycin and fidaxomicin. Further in vitro investigation revealed that the compounds were nontoxic to Caco-2 cell line. Given their potent anticlostridial activity, natural product-inspired scaffolds may suggest potential avenues that can address the unmet needs in preventing C. difficile mediated disease.

www.nature.com/scientificreports/ Following the first recurrence event, the risk of subsequent recurrences can increase by up to 50% 9 . The unprecedented challenges associated with the current treatment regime calls for an avant-garde drug scaffold that has the potential to treat CDI. An important component of modern drug discovery, high-throughput screening (HTS) is a keystone technology used to identify novel chemical entities that has the potential to become usable drugs 10,11 . De novo drug discovery, which focuses on the identification of innovative chemical scaffolds, integrates discovery based on either target-based HTS screening (screening to identify inhibitors of a specific enzyme target) or whole-cellbased phenotypic HTS screening (screening against a whole organism) 11 . In this study, a whole-cell-based high throughput screening of the AnalytiCon NATx library (consisting of 5000 natural product-inspired or natural product-derived synthetic compounds) was conducted with the goal of identifying novel scaffolds that have the potential to treat CDI. Unlike most of the complex natural products, the natural product-like synthetic compounds in the NATx compound library are prepared by reliable chemistry and are suitable for further medicinal chemistry optimization. Here, among the panel of hits identified that could inhibit the growth of C. difficile, we identified molecules with potent anti C. difficile activity. Minimum inhibitory concentration (MIC) of the hit compounds was determined against representative members of the human gut microflora. We also investigated the hit scaffolds for their cytotoxicity against human colorectal adenocarcinoma cell line (Caco-2).

Results
High-throughput screen of AnalytiCon NATx library and validation of hits using plate cherry-picking against C. difficile ATCC BAA 1870. The AnalytiCon NATx library containing 5000 natural product-like synthetic compounds was screened for possible inhibitors of C. difficile at a concentration of 3 µ M. In the initial screening, we obtained 34 compounds out of the 5000 that inhibited the growth of the pathogen (Fig. 1A). In order to confirm the anticlostridial activity of the hit compounds, the 34 hit compounds were cherry-picked from the plates and rescreened against C. difficile at the same concentration. The plate cherrypicking confirmed the anticlostridial activity of 10 compounds from the initially obtained 34 hits (Fig. 1B).
Antimicrobial activity of the hit scaffolds against gut microflora strains. Contrary to the standard-of-care antibiotics vancomycin and fidaxomicin, which inhibited the Gram-positive Bifidobacterial members at very low concentrations, NAT13-338148 was found to permit their growth even at concentrations > 8 µg/ ml. NAT18-355531 and NAT18-355768 inhibited the tested Bacteroides sp. and Bifidobacterium sp. at a slightly lower concentration (4 µg/ml) (Table 1B).
Cytotoxic potential of the hit scaffolds. To discern the cytotoxic effect of the natural product derived small molecules, the molecules were screened against Caco-2 cells using the MTS assay. Figure 2 represents the results garnered. All the three hit compounds (NAT13-338148, NAT18-355531, and NAT18-355768) were found to be nontoxic to Caco-2 cells at a concentration of 16 µg/ml. www.nature.com/scientificreports/

Discussion
C. difficile is a common cause of diarrhea, mainly affecting hospitalized patients, and is an increasing health threat worldwide 1,12 . A predisposing factor to CDI is the use of antibiotics for unrelated disease conditions that disrupts the intestinal microbiome causing a state permitting C. difficile growth and colonization 13,14 . In spite of antibiotics paving the way for CDI, the standard-of-care therapeutics are limited to antibiotics vancomycin and fidaxomicin, with metronidazole being recommended only in settings where there is limited access to the other two drugs 6 . A major limitation of these first-line antimicrobials is that they fail to assure sustained clinical cure and patients often suffer from recurrent CDI. The transplantation of fecal microbiota has recently been accepted www.nature.com/scientificreports/ as a potential intervention to tackle this problem of recurrence. However, the use of FMT is accompanied with an increased risk of exposure to organisms of concern 15,16 . The significant drawbacks of the current treatment repertoire necessitate an alternative paradigm for treating CDI with minimal effect on the indigenous intestinal microflora.
HTS is an enabling approach that can be exploited for the discovery of novel scaffolds and can be used as a starting point for drug discovery 17 . Herein, we used this tool to screen a library of 5000 natural product-like compounds against C. difficile. In our first screening, we found 34 out of the 5000 compounds with inhibitory activity against the pathogen. To validate this, we did a plate cherry-picking assay which confirmed the anticlostridial activity of 10 of the compounds out of the 34 hits obtained from the initial screening.
Based on the promising results, we sought to decipher the actual concentration of the compounds that can inhibit the pathogen. We did an MIC assay against a panel of 16 hypervirulent and clinically toxigenic C. difficile strains. Out of the 10 hits, we found 2 compounds with MIC 90 values (NAT18-355531 and NAT18-355768) comparable to the MIC 90 of the first-line drug vancomycin (1 µg/ml). The MIC 90 of the other compound, NAT18-338148, was only onefold greater than that of vancomycin.
A hall mark of CDI remains the recurrence of infection in spite of successful treatment of the initial episode. The absence of the healthy gut microbiome favors the colonization of the intestine by such enteric pathogens. Hence, it is crucial to seek a scaffold that has minimal effects on the human gut microbiome. Contrary to vancomycin and fidaxomicin, NAT13-338148 was found to have no inhibitory effect on the gut microbial species at the tested concentration (8 µg/ml). NAT18-355531 and NAT18-355768 were found to not inhibit the Bacteroides and Bifidobacterium sp. at a concentration of 4 µ g/ml.
Evaluating cytotoxicity of novel chemical entities is an approach adopted to increase the probability of success in preclinical animal studies 18 . In this study, we analyzed the cytotoxicity of the natural product-inspired small molecules against Caco-2 cells. The hit scaffolds had no deleterious effects on the Caco-2 cells when treated a concentration of 16 gµ/ml for 24 h.
Our results demonstrate three novel natural product-like compounds, NAT13-338148, NAT18-355531, and NAT18-355768, with potent in vitro anticlostridial activity. Further studies including synthesis of analogues, investigating their pharmacological parameters and determining the efficacy of the lead compounds and their synthesized analogues in a primary and recurrent CDI mice model will be needed to yield a therapeutic capable of reducing short-term diarrhea and long-term sequelae of recurrent CDI. Libraries and control antibiotics. The AnalytiCon NATx library containing 5000 natural productlike synthetic compounds was purchased from AnalytiCon Discovery (Potsdam, Germany) by the Chemical Genomics facility at the Purdue Institute of Drug Discovery. The compounds were provided in sixteen 384-well plates as 1 mM DMSO stock. Hit compounds were further purchased from AnalytiCon Discovery. Vancomy- Figure 2. Cytotoxicity assay of hit scaffolds against human colorectal adenocarcinoma (Caco-2) cell line. Percent viable Caco-2 cells measured as ratio of average absorbance relative to DMSO for analyzing cytotoxicity of the hit natural product-derived small molecules at 16 µg/ml using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. DMSO was used as a negative control to determine a baseline measurement for the cytotoxic impact of each compound. The absorbance values represent an average of a minimum of three samples analyzed for each compound. Error bars represent standard deviation values for the absorbance. MIC assay against gut microflora. The activity of the hit compounds (Table S1) was further verified against the 13-gut microflora (Table S3) as described in section "Minimum inhibitory concentration (MIC) assay against a panel of C. difficile strains". Briefly, the hit compounds (concentration = 8 gµ/ml) were added to the first row of the 96-well plate. Bacterial suspension of Bacteroides sp. and Bifidobacterium sp. in BHIS broth and Lactobacillus sp. in MRS broth were prepared to attain a bacterial concentration of 5 × 10 5 CFU/ml. The diluted bacterial suspension (in BHIS and MRS broth respectively) was used for serial dilution and plates were incubated at 37ºC for 48 h. MIC values were recorded following the incubation period.

Materials and methods
Cytotoxicity assay. To evaluate the potential toxic effect of the natural product-like synthetic compounds, cytotoxicity assay was performed against Caco-2 cells as has been described previously [27][28][29][30] . Caco-2 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% NEAA and incubated at 37 °C in presence of 5% CO 2 . Drugs were added at a starting concentration of 16 gµ/ml and the control wells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds in a 96-well plate for 24 h prior to addition of the assay reagent MTS. Absorbance readings at OD 490 were taken using a SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). Cell survival post treatment was plotted as percentage viability of drug-treated cells when compared to the DMSO-treated control cells using GraphPad Prism v 8.0.