Screening of an FDA-Approved Library for Novel Drugs against Y. pestis

Yersinia pestis is a Gram-negative pathogen that causes plague, a devastating disease that kills millions worldwide. Although plague is efficiently treatable by recommended antibiotics, the time of antibiotic therapy initiation is critical, as high mortality rates have been observed if treatment is delayed for longer than 24 h after symptom onset. To overcome the emergence of antibiotic resistant strains, we attempted a systematic screening of Food and Drug Administration (FDA)-approved drugs to identify alternative compounds which may possess antibacterial activity against Y. pestis. Here, we describe a drug-repurposing approach, which led to the identification of two antibiotic-like activities of the anticancer drugs bleomycin sulfate and streptozocin that have the potential for designing novel antiplague therapy approaches. The inhibitory characteristics of these two drugs were further addressed as well as their efficiency in affecting the growth of Y. pestis strains resistant to doxycycline and ciprofloxacin, antibiotics recommended for plague treatment.


Introduction
Yersinia pestis is a Gram-negative pathogen that causes plague, a devastating disease that has killed millions throughout human history. The two main forms of the disease are the bubonic and the pneumonic plague. Bubonic plague develops following transmission of the pathogen to humans by infected fleas. Primary pneumonic plague results from the inhalation of bacteria-containing droplets or aerosols and is capable of spreading from person to person [1], leading to the rapid progression of a contagious and fatal disease. Left untreated, it usually leads to death within 3-4 days after exposure [2]. Most recently, the 2017 epidemic of Y. pestis infection on the island of Madagascar had an unusual predominance of pneumonic cases (1878 out of 2414 clinically suspected plague cases), and strict infection and prevention control measures had to be taken to stop the propagation of the infection [3]. Plague is treatable by recommended antibiotics; however, high mortality rates have been observed if treatment is delayed [4]. The antibiotics recommended to treat plague include aminoglycosides, fluoroquinolones, tetracyclines, and chloramphenicol [2]. However, the isolation of naturally occurring antibiotic-resistant Y. pestis strains [5,6] emphasizes the urgent need for the development of additional countermeasures. In addition, the possibility that such strains may be maliciously used as bioweapons represents a high-priority public health concern.
In recent years, health officials have warned that antimicrobial resistance is a major threat to global health. In response, efforts are focusing on exploring the potential use of existing drugs, an approach known as "drug repurposing" [7,8]. Finding new uses for existing drugs can be achieved either on the basis of a detailed understanding of the chemistry of individual compounds and their potential targets from in silico modeling followed by in vitro cell culture and in vivo animal studies or by directly screening a large library of compounds in disease-relevant phenotypic assays, thus allowing an unbiased approach to obtain successful drug candidates. In this study, we screened 1363 compounds of an FDA-approved drug library (APExBIO DiscoveryProbe) for their ability to reduce the growth of Y. pestis and identified a couple of candidates that exhibited potent activity against Y. pestis growth in vitro, suggesting that they may potentially be used to treat plague infection.

Design of the Study
In this study, we employed the APExBIO DiscoveryProbe library of FDA-approved drugs (https://www.apexbt.com/discoveryprobetm-fda-approved-drug-library.html) to screen for inhibitory molecules that reduce the growth of Y. pestis and may serve for designing novel antiplague therapeutic strategies. The library included 1363 compounds belonging to a variety of drug classes, not only those known for the treatment of infectious diseases, such as antibiotics, but also groups of drugs recommended for the treatment of noninfectious pathological conditions, such as cancer, cardiovascular and metabolic diseases. According to the rationale of the survey, compounds were distinguished as potential inhibitors and selected for subsequent interrogation if they significantly affected the in vitro bacterial growth, as manifested by a reduced turbidity of the culture in comparison to that of untreated bacteria (see Section 2.2). For compound screening, Y. pestis bacteria were cultured in 96-well microtiter plates at 37 • C, conditions considered to mimic those encountered in the mammalian host. In preliminary experiments, heart infusion broth (HIB) medium was used to afford significantly superior growth of Y. pestis compared to that in the standard medium Mueller−Hinton broth (MHB), as stipulated by the clinical and laboratory standards institute [9] for antimicrobial susceptibility assays, and was therefore used for the screening processes.

Identification of Novel Potential Antiplague Drugs
All compounds were first tested at a concentration of 10 µM, and the potential candidate drugs considered were those that exhibited a ten-fold or higher inhibition index (90% reduction of bacterial growth compared to that of untreated bacteria) after 24 h and a five-fold or higher inhibition index (80% reduction) after 48 h. Indeed, in some cases, after 48 h, the inhibitory effect of some of the putative drugs may have occurred due to their degradation in the conditions employed for screening. Treatment with the positive control antibiotic ciprofloxacin (0.05 µg/mL) resulted in total inhibition of bacterial growth. Fifty-five compounds meeting the inhibitory criterion were selected after the primary screen and retested in a secondary screen, resulting in confirmation of 45 compounds exhibiting an inhibitory effect on Y. pestis growth (3.3% confirmation rate for the 1363 compounds). These 45 compounds, depicted in Table 1, included 36 antibiotics that are known to inhibit Y. pestis or belong to categories of drugs known to possess antimicrobial activity against Y. pestis. Mitomycin C, in spite of its observed inhibitory effect, was excluded from the subsequent analysis due to its reported phage induction ability, which may have complicated the interpretation of the results [10,11]. Eight additional drugs (out of 45) have not been previously reported as potential growth inhibitory agents against Y. pestis. Based on their previously known clinical indications, the novel antiplague compounds (Table 2) included (i) the three antiseptic drugs cetrimonium bromide, cetylpyridinium chloride and chlorquinaldol, (ii) the two antiparasite (vermifuge) drugs closantel and closantel sodium, (iii) the two anticancer drugs streptozocin and bleomycin sulfate, and (iv) the antifungal drug zinc pyrithione.

Characterization of the Anti-Y. pestis Inhibitory Activity of Novel Drugs
The eight novel, potentially antiplague drugs were further interrogated for in vitro efficacy by determining their Y. pestis growth inhibitory concentration 50% (IC 50 ) and inhibitory concentration 90% (IC 90 ). These values were quantified in HIB and in MHB according to the CLSI guidelines ( Figure 1 and Table 2). In addition, their minimal inhibitory concentration (MIC) were calculated in HIB ( Table 2).
Six of the eight drugs were not further investigated because they were either used in the past solely for veterinary purposes (closantel and closantel sodium) or reportedly employed for antiseptic external therapy only (cetrimonium bromide, cetylpyridinium chloride, chlorquinaldol and zinc pyrithione). The two anticancer compounds bleomycin sulfate and streptozocin, which were previously reported to serve as human treatments, were selected for further analysis. Notably, both bleomycin and streptozocin were shown to efficiently inhibit the growth of other Gram-positive and Gram-negative bacteria, including Staphylococcus epidermidis, Klebsiella pneumonia and Escherichia coli [12,13].

Characterization of the Anti-Y. pestis Inhibitory Activity of Novel Drugs
The eight novel, potentially antiplague drugs were further interrogated for in vitro efficacy by determining their Y. pestis growth inhibitory concentration 50% (IC50) and inhibitory concentration 90% (IC90). These values were quantified in HIB and in MHB according to the CLSI guidelines ( Figure 1 and Table 2). In addition, their minimal inhibitory concentration (MIC) were calculated in HIB (Table 2). Six of the eight drugs were not further investigated because they were either used in the past solely for veterinary purposes (closantel and closantel sodium) or reportedly employed for antiseptic external therapy only (cetrimonium bromide, cetylpyridinium chloride, chlorquinaldol and zinc pyrithione). The two anticancer compounds bleomycin sulfate and streptozocin, which were previously reported to serve as human treatments, were selected for further analysis. Notably, both bleomycin and streptozocin were shown to efficiently inhibit the growth of other Gram-positive and Gram-negative bacteria, including Staphylococcus epidermidis, Klebsiella pneumonia and Escherichia coli [12,13].
Bleomycin is a radiomimetic compound which forms complexes with iron that reduce molecular oxygen to superoxide and hydroxyl radicals inflicting DNA damage (single and double-strand breaks) that results in preferential inhibition of DNA synthesis in Bleomycin is a radiomimetic compound which forms complexes with iron that reduce molecular oxygen to superoxide and hydroxyl radicals inflicting DNA damage (single and double-strand breaks) that results in preferential inhibition of DNA synthesis in cancer cells [14,15]. This compound is used to treat hematologic (Hodgkin's and non-Hodgkin's lymphomas) as well as solid malignancies (testicular, ovarian and cervical cancers). The standard dose of bleomycin (adjusted based on body surface area, BSA) for the treatment of cancer patients is 15 mg/m 2 (intravenous administration), resulting in a reported circulatory concentration (Cmax) of 706 µM and exhibiting a circulatory half-life (T1/2) of 4 h [16]. This bleomycin dose is more than two orders of magnitude higher than the IC 90 of 3.77 µM observed for inhibition of Y. pestis growth ( Table 2), suggesting that an effective anti-Y pestis treatment with bleomycin may be well tolerated. Streptozocin is an alkylating antineoplastic agent which inhibits synthesis of DNA both in microorganisms and mammalian cells by cross-linking the DNA strands [17]. It is used to treat metastatic cancer of pancreatic islet cells. The recommended dose for oncologic treatment is 1.5 g/m 2 by intravenous administration, resulting in an effective circulatory concentration (Cmax) of 1438 µM with a circulatory T1/2 of 0.22 h [16]. This concentration is 300 times higher than the concentration required for inhibition of Y. pestis growth (IC 90 = 4.75 µM, Table 2), suggesting, as in the case of bleomycin, that efficient treatment of Y. pestis infection requires well-tolerated doses. Of note, several anticancer compounds possessing DNA-damaging activity (such as lomustine, irinotecan, topotacan and busulfan) which were screened in the current study, were not found to inhibit the growth of Y. pestis.
As new drugs are needed against antibiotic-resistant strains, bleomycin sulfate and streptozocin were tested for their ability to inhibit the growth of antibiotic-resistant strains of Y. pestis. In view of the fact that ciprofloxacin and doxycycline represent the antibiotics of choice for postexposure prophylaxis and treatment of plague infections in the event of an outbreak, we used nonvirulent Y. pestis strains that are resistant to ciprofloxacin and doxycycline [18,19]. These strains are derived from the fully virulent Y. pestis Kim53 strain. The IC 90 calculated for the inhibition of growth promoted by bleomycin sulfate in the ciprofloxacin (3.8 µM) or doxycycline (7.2 µM) resistant strains clearly demonstrated that bleomycin sulfate may effectively prevent the growth of Y. pestis antibiotic-resistant strains. Similarly, for streptozocin, the IC 90 = 4.6 µM for the ciprofloxacin-resistant strain and IC 90 = 26 µM for the doxycycline-resistant strain indicated that streptozocin may serve as a new candidate to combat drug-resistant Y. pestis strains. Of note, the initial screen was conducted under BL2 laboratory conditions using the EV76 attenuated strain (see above). On the basis of previous studies from our laboratory, the results obtained using this strain may be extrapolated to fully virulent strains. While this aspect will require future confirmation, at least in the case of susceptibility to bleomycin and streptozocin, the EV76 and Kim53 derived attenuated-strains are very similar.
Bleomycin and streptozocin, similar to other drugs employed for the treatment of cancer, are cytotoxic, and their administration may be accompanied by severe side effects, although the effective concentration for Y. pestis growth inhibition determined in this study appears to be considerably lower than the concentration required for effective cancer chemotherapy. Furthermore, the treatment regimen is expected to be much shorter than that required for cancer treatment. Repurposing anticancer drugs for infectious diseases has been reported despite potential side effects [20]. For example, miltefosine was approved to treat leishmaniasis [21], and the implementation of treatments based on gallium compounds to control P. aeruginosa in patients with cystic fibrosis has completed phase II clinical trials [22]. Concerns regarding the clinical use of bleomycin and streptozocin in the treatment of bacterial infections may be mitigated by considering their therapeutic benefit in the case of severe infection, especially those caused by strains resistant to the antibiotics of choice. Such strains resistant to individual antibiotics or exhibiting multidrug resistance have become frequent in recent decades, and treatment options for patients with lifethreatening resistant infections have become alarmingly limited [23,24]. Furthermore, the COVID-19 pandemic seems to have rapidly increased the use of antibiotics worldwide [25], accelerating the threat of antimicrobial resistance. Therefore, drugs such as bleomycin sulfate and streptozocin or the other compounds identified in the present study may represent an important basis for designing novel therapeutic strategies. The implementation of the novel drugs for the treatment of plague will involve future studies addressing their in vivo efficiency as well as the possibility of mitigating their possible adverse effects [26]. It is worth noting that previous studies established that bleomycin sulfate inhibits the growth of Klebsiella pneumonia at an IC 50 very similar to the value calculated in the present study for Y. pestis [27]. Further studies will address the applicability of the drugs identified in the current survey for the treatment of other infections caused by multidrug-resistant Gram-negative pathogens of public health concern.