Staphylococcus aureus resistance to albocycline can be achieved by mutations that alter cellular NAD/PH pools

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Abstract

Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.

Introduction

Antibacterial drug development is susceptible to the onset of antibiotic resistance, an increasingly common problem exacerbated by the widespread use of antibiotics in healthcare and animal agriculture.1, 2 The demand for new drugs that directly address bacterial resistance can be accomplished by (1) identifying novel small molecules for existing targets; (2) discovering novel bacterial targets to screen compound collections; or (3) revisiting established antibiotics and exploring their attendant mechanism of action.3 The albocycline (ALB) scaffold is interesting in that it displays promising antibacterial profile against methicillin- and vancomycin-resistant isolates.4 In 2017, we reported a concise total synthesis of ALB; however, currently the natural product is isolated in significant quantities by culturing the producing organism, Streptomyces maizeus, to enable the efficient semi-synthesis of analogs.5, 6 In tandem with synthetic efforts, we sought to uncover albocycline’s mechanism of action by taking biochemical and genomic approaches (see Fig. 1, Fig. 2).

Currently, approaches to determine the mode of action of drugs can be categorized into two main types.7 First, affinity-based methods are used to detect proteins displaying high binding affinity to the drug.8 These strategies are most successful when the active form of the drug and the targets co-exist in vivo.9 On the other hand, such approaches require the physiological target (drug-binding protein) to rely on correlations between binding/activity assays in vitro and protein knockdown phenotypes. However, these correlations can be deceptive due to differences in chemical inhibition; which can be critical, or the protein knockdown phenotypes that can be indirect or resulting from cumulative effects.10 Second, other tactics may target model organisms that are companionable with genetic manipulations.11 The failure of such approaches occurs if the drugs were inactive in these organisms as a result of multi-drug resistance mechanisms or target divergence.12

Although ALB represents a promising lead candidate against antibiotic resistant S. aureus, further work is required in order to identify and validate its biological target. To date, there are three hypotheses regarding its mechanism of action. The structural similarity of ALB with the aglycones of known macrolide antibiotics (e.g., erythromycin) raises the possibility of inhibiting protein synthesis; however, studies by Tomoda in 2013 found no evidence to support this notion, and concluded instead that ALB functions to inhibit the synthesis of peptidoglycan (PG), as they observed a buildup of PG intermediates.4 Yet, prior to this work, in 1969, Reusser at Upjohn performed studies of ALB in Gram-positive Bacillus subtilis cells to suggest it reversibly inhibited nicotinamide biosynthesis.13

Previously, we reported computational and biochemical studies accompanied with biological findings for MurA (i.e., the enzyme responsible for catalyzing the first step in the peptidoglycan biosynthesis), illustrating that MurA is not the target of albocycline.14 Herein, we present the latest findings relying on both biochemical approaches [i.e., fluorescence polarization (FP) assay and kinetic studies] along with genomic studies (i.e., sequencing of resistant strains) in order to better understand albocycline’s cellular target(s).

Section snippets

FP method

5 nM BODIPY-ERY was incubated with 500 nM 70S (10 nM active 70S as determined by binding assays) in buffer (20 mM HEPES pH 7.5, 50 mM NH4Cl, 10 mM MgCl2, 0.05% Tween 20) in a total volume of 20 µL in the wells of a 384-well plate for 30 min at room temp. 2 µL of 10X solithromycin (1% DMSO final) or blank was added incubated at room temperature for 1.5 h. The 384-well plate was read on a Clariostar (BMG Ltd) plate reader (485 nm excitation/535 nm emission) to determine fluorescence polarization

Biochemical assays (Fluorescence polarization: Determination of dissociation constants with bacterial ribosomes)

To confirm Tomoda’s finding that ALB does not function analogously to macrolides inhibiting translation via radiolabeling experiments, we used a direct assay to assess translation inhibition via blockage of the exiting peptide chain. We determined the dissociation constants (Kd) via FP assays with bacterial ribosomes using BODIPY-functionalized erythromycin.21 Accordingly, a titration curve was plotted for determining FP as a function of the [70S] ribosomes at a fixed probe concentration

Conclusion

In conclusion, we employed biochemical and genetic assays in order to identify the target of ALB. For the biochemical assays, we were able to calculate the dissociation constants (Ki) for ALB against bacterial ribosome (Ki > 100 μM) and MurA (Ki = 23.6 μM). The data strongly support the conclusion that neither protein or peptidoglycan are the primary targets of ALB. Furthermore, genetic sequencing we were able to identify 14 highly-resistant mutants, exhibiting mutants in a non-essential gene

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Dr. David P. Labeda (National Center for Agricultural Utilization Research, United States Department of Agriculture) for kindly providing S. maizeus. We thank Prof. Ann Valentine for optimizing the protocol for culturing S. maizeus, as well as Drs. Robert Stanley and Carol Manhart for kindly providing autoclave facilities.

Funding Information

The present study was supported by the fund from NIH (GM126221).

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