(M1030-05-25) Screening of FDA-Approved Drug Library against Vancomycin Resistant Enterococci

Purpose: Emergence of Vancomycin Resistant Enterococci (VRE) which is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, is a serious public health concern. It is ranked as the world’s highest priority pathogen according to World Health Organization. To identify and validate new therapeutic targets against VRE, we have screened National Cancer Institute (NCI) library of 1600 compounds. The chemical diversity of the library was further enhanced by metabolizing the library with human liver microsomes. We screened the original and in-vitro metabolized libraries to identify active parent compounds and metabolites, respectively. We also identified synergistic interactions of library drugs with vancomycin (Vm) when screened in presence and absence of vancomycin. For instance, compound NSC227186 showed high level of synergy in presence of vancomycin. Moreover, the metabolite obtained from this compound after metabolism with human liver microsomes gave potent activity against VRE. In summary, this study demonstrates a multidimensional screening approach to identify novel clinical agents and targets.

Methods: Library replication of the original library: The original or the un-metabolized (UM) library was replicated in 384 well plates with the help of liquid handling workstation to prepare the pre-metabolized library (PM). Control antibiotics were also added in the last column of the plates. Microsomal Metabolism to prepare PM library: Microsomal reaction mixture (human liver microsomes, potassium phosphate, MgCl_2, NADP) were added to the drugs and the plates were incubated for a day followed by the extractions of the drugs with DMSO. UM/PM vs -/+ Vancomycin library screen against VRE: Four sets of library screens were prepared (UM-Vm, UM+Vm, PM-Vm, PM+Vm) and screened against VRE. The concentration of vancomycin used to screen the plates was 16 µg/ml. This was later followed by the addition of resazurin salt dye which differentiated between the growth and no growth with the help of blue and pink colour. Hitpicking and Minimum Inhibitory Concentration determination: A list of each active well between (UM-Vm, UM+Vm, PM-Vm, PM+Vm) sets were generated and the Minimal Inhibitory Concentrations (MIC) were determined. Spectrum of Activity of active compounds: MICs were determined for active compounds against 8 strains of VRE, 7 strains of Methicillin Resistant Staphyloccocus aureus (MRSA) and 1 strain of E. coli. Checkerboard Assays to confirm synergy with Vancomycin: Checkerboard assay was done in 96 well plates with the following drugs (NSC227186, NSC343256, NSC305780, NSC133114, NSC325014, NSC8675) which showed synergistic activity with vancomycin.

Results: Analysis of Hits: After the library screening a pooled hit list of any active/potent compound was made. MICs for all the compounds were determined under the four screening conditions (UM-Vm, UM+Vm, PM-Vm, PM+Vm). The table for MICs is attached in the figure section. We observed that the most interesting compounds were NSC227186, NSC343256, NSC305780, NSC133114, NSC325014, NSC8675. Spectrum of Activity Determination: To access the potential active compounds in the library spectrum of activity assay was done against 8 strains of VRE, 8 strains of MRSA and 1 strain of E. coli. It was observed that few strains of VRE and E. coli showed no activity in presence of these compounds. Whereas, few strains of VRE did show activity against the selected compounds. However, high activity against MRSA with a low MIC was observed in presence of these compounds. Checkerboard Assay showed that there exist a high level of synergy between vancomycin and the active drugs. The Fractional Inhibitory value is below 0.5 which is considered as a good synergy between two compounds. Metabolite determination: Compound NSC227186 of the drug library gave an active metabolite against VRE after the metabolism with human liver microsomes and this metabolite was further determined by LC-MS/MS.

Conclusion: This study initially aimed to evaluate the potential of in-vitro metabolism of NCI approved drug library to enhance a drug repurposing screening effort by allowing active metabolites to be identified. To further enhance the ability of this approach to reveal novel and interesting activities, it was combined with a -/+ resistant to antibiotic (cefoxitin) screen. This provided a 2-dimensional screening approach (2x2 experimental design). The results from this approach substantially exceeded our expectations. We were able to discover a number of synergistics compounds with vancomycin. Moreover, we also found a potent metabolite against VRE. This study illustrates the utility of a comparative library screening approach to identify new antibacterial agents and approaches

References: 1) Ayon, N. J., and Gutheil, W. G. (2019) Dimensionally Enhanced Antibacterial Library Screening, ACS Chemical Biology 14, 2887-2894.
2) Ventola, C. L. (2015) The antibiotic resistance crisis: part 1: causes and threats, P T 40, 277-283.
3) Silver, L. L. (2011) Challenges of antibacterial discovery, Clin Microbiol Rev 24, 71-109.
4) Frieden, T. (2013) Antibiotics resistance threats in the United States, 2013, CDC Publishing Web
5) Brown, E. D., and Wright, G. D. (2016) Antibacterial drug discovery in the resistance era, Nature 529, 336-343.
6) Butler, M. S., Blaskovich, M. A., and Cooper, M. A. (2017) Antibiotics in the clinical pipeline at the end of 2015, J Antibiot (Tokyo) 70, 3-24.