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. 2024 Apr:102:105073.
doi: 10.1016/j.ebiom.2024.105073. Epub 2024 Mar 22.

High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae

Benjamin Blasco  1 , Soojin Jang  2 , Hiroki Terauchi  3 , Naoki Kobayashi  3 , Shuichi Suzuki  3 , Yuichiro Akao  4 , Atsuko Ochida  4 , Nao Morishita  4 , Terufumi Takagi  4 , Hiroyuki Nagamiya  4 , Yamato Suzuki  5 , Toshiaki Watanabe  5 , Hyunjung Lee  2 , Sol Lee  2 , David Shum  2 , Ahreum Cho  2 , Dahae Koh  2 , Soonju Park  2 , Honggun Lee  2 , Kideok Kim  2 , Henni-Karoliina Ropponen  1 , Renata Maria Augusto da Costa  1 , Steven Dunn  6 , Sunil Ghosh  7 , Peter Sjö  8 , Laura J V Piddock  9
Affiliations

High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae

Benjamin Blasco et al. EBioMedicine. 2024 Apr.

Abstract

Background: The current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections.

Methods: We developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using "eNTRy rules" criteria associated with increased likelihood of intracellular accumulation in Escherichia coli.

Findings: We identified 72 compounds with confirmed activity against K. pneumoniae and/or drug-resistant A. baumannii. Two new chemical series with activity against XDR A. baumannii were identified meeting our criteria of potency (EC50 ≤50 μM) and absence of cytotoxicity (HepG2 CC50 ≥100 μM and red blood cell lysis HC50 ≥100 μM). The activity of close analogues of the two chemical series was also determined against A. baumannii clinical isolates.

Interpretation: This work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K. pneumoniae and A. baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects.

Funding: BMBF and GARDP.

Keywords: Acinetobacter baumannii; Antibacterial drug discovery; High-throughput screening; Klebsiella pneumoniae; Multidrug-resistance.

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Conflict of interest statement

Declaration of interests Small-molecule libraries were provided by Takeda Pharmaceutical Company Ltd, Eisai Co., Ltd and Daiichi Sankyo Co., Ltd. Yuichiro Akao, Atsuko Ochida, Nao Morishita, Terufumi Takagi, Hiroyuki Nagamiya are employees of Takeda Pharmaceutical Company Ltd. Yamato Suzuki and Toshiaki Watanabe are employees of Daiichi Sankyo Co., Ltd. HKR performed her contributions while employed at GARDP but is now employed by AMR Action Fund. SD received payments as part of a consultancy agreement with GARDP to carry out tasks including genomic data curation and analysis, figure generation, and data analysis. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Hit identification workflow. The generic high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii consists of the following steps: (A) a primary screen at a single concentration of compound in duplicate (Takeda, Daiichi Sankyo) or two concentrations in singlicate (Eisai) against the three primary screening strains; (B) confirmation of activity with the primary screening sample in dose–response mode. Following structure disclosure (‘unblinding’) of confirmed active compounds and clustering analysis by partner pharmaceutical companies, active compounds related to known antibacterial scaffolds or with problematic chemotype are removed. New batches of the prioritised confirmed active compounds are purchased from commercial vendors or re-synthesised and quality checked to help confirm chemical structure identity and high purity. Close structural analogues can also be purchased at the same time to develop an early SAR understanding. These compounds are then retested against the active primary screen strains in dose–response mode and, in parallel, counter-screened for cytotoxicity (HepG2 cell line) and haemolytic activity (red blood cells), both in dose–response mode. The main checkpoint criteria for confirmed hits are i) whole-cell potency with EC50 ≤50 μM and MIC ≤100 μM (or ≤32 mg/L) against at least one of the two MDR/XDR primary screening strains; and ii) low/no cytotoxicity; compounds with HepG2 CC50 <100 μM or red blood cell lysis HC50 <100 μM are ‘flagged’ rather than discarded outright; ideally there should be a greater than 5-fold selectivity window between the CC50/HC50 and the EC50 of the compound against K. pneumoniae and/or A. baumannii primary screening strains. Confirmed hits are progressed to hit validation and profiling assays. Confirmed hits and close analogues are screened in dose–response mode against a panel of clinical isolates selected to represent globally prevalent sequence types and drug resistance genes/phenotypes associated with human infections. This step ensures that the activity of the hits from the primary screen is not specific for one or more of the primary screening strains, and that the hits are not cross-resistant to any classes of antibiotics used to treat infections with Enterobacterales and/or Acinetobacter spp. In addition to potency of compounds in these antibacterial assays, other properties such as in vitro metabolic stability (measured in human and rodent microsomes) and aqueous solubility are also measured as these are important to evaluate the potential for systemic dosing. We note that the order and details of each operation can vary depending on multiple factors (e.g., available resources, nature of and pre-existing knowledge on the chemical compounds and project timeline). Each box represents a process colour-coded by drug discovery discipline: blue, microbiology; yellow, chemistry; orange, safety and toxicology; green, ADME. Key criteria for progression to the next step are indicated beside each box using the same colour-coding, and the number of compounds that remained after each step are shown in greyed circles. HTS, high-throughput screening.
Fig. 2
Fig. 2
Key physicochemical properties of the Eisai, Daiichi Sankyo and Takeda compound sets. Composition of the libraries in terms of a) molecular weight, b) AlogP and clogD, c) rotatable bonds, d) globularity (Glob) and fraction sp3 (Fsp3) and e) number of ionizable amines. In box-and-whisker plots (a–d), the central horizontal line of each box shows the median value of the property distribution, and the top and bottom horizontal lines of each box indicate the first and third quartile values, respectively. Whiskers are calculated according to the Tukey method.
Fig. 3
Fig. 3
Spectrum of activity and properties of confirmed active compounds. Venn diagrams indicating the overlap of confirmed active compounds (EC50 ≤50 μM) against A. baumannii NCTC 13424, K. pneumoniae NCTC 13438 and K. pneumoniae Ecl8 for the A) Takeda set and B) Eisai set. The three confirmed active compounds that were followed up are highlighted in green font. The range of physicochemical properties as indicated in Fig. 2 (i.e., molecular weight (MW), AlogP, clogD, rotatable bonds (RB), globularity (Glob) and fraction sp3 (Fsp3)) for confirmed active compounds are indicated beside each strain circle.
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