Short chain α-pyrones capable of potentiating penicillin G against Pseudomonas aeruginosa

Abstract

The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.

Introduction

Antibiotic resistance, which affects >2.5 million people in the U.S. per year, is a growing global threat that, if left unchecked, will be the number one cause of death with 10 million deaths globally per year by 2050.1, 2 Despite this crisis, antibiotic development has steadily declined between 1980 and 2010 due to the challenges associated with discovering and developing new classes of antibiotics without established resistance in bacteria. These challenges have led to the majority of pharmaceutical companies abandoning their antibiotic research and development programs over this same time period.³ This decline has resulted in a dearth of antibiotics coming through the U.S. Food and Drug Administration (FDA) approval pipeline, with only five4, 5 non-derivative antibiotics being FDA approved since 2000 and no new classes of antibiotics being approved for Gram-negative infections since the quinolone class of antibiotics in 1968.⁶

One pathogen that has been labeled by the Centers for Disease Control and Prevention (CDC) as a “Serious Threat” is multidrug-resistant Pseudomonas aeruginosa (MDRPA), a Gram-negative, biofilm-forming bacterium that causes approximately 32,600 infections and 2,700 deaths per year at an estimated $767 million per year healthcare cost.¹ Current treatment methods for non-resistant PA include β-lactams, aminoglycosides, cephalosporins, fluoroquinolones, and polymyxins.⁷ However, the majority of these treatments are ineffective against MDRPA due to intrinsically encoded or acquired genetic resistance for the most common antibacterial mechanisms of action (MOA) (i.e. inhibition of cell wall synthesis, protein synthesis, and DNA/RNA synthesis).⁷ Furthermore, there are two additional barriers to treating MDRPA and other resistant and non-resistant Gram-negative infections: slow diffusion across the bacterial outer membrane (OM) and promiscuous efflux pumps such as MexAB-OprM8, 9 that are able to remove a wide variety of molecules from the periplasmic and cytoplasmic space,. Together these lead to low levels of accumulation within the cell.¹⁰ Finally, MDRPA can also be less susceptible to antibiotics due to biofilm formation.¹¹

With the decline in novel antibiotics being developed and the increase in MDR infections, antibiotic adjuvants have emerged as one way to keep pace with evolving bacteria and to prolong the life of current antibiotics on the market. Antibiotic adjuvants are compounds that do not have the ability to kill bacterial cells on their own but instead are able to increase the activity of a known antibiotic by improving entry into the bacterial cell or inhibiting an active resistance mechanism that prevents a known antibiotic from being active.¹² Although β-lactams and their flagship penicillin G are one of the oldest classes of antibiotics, they are still widely used today. However, due to decades of use, there is widespread resistance to β-lactams, including methicillin-resistant Staphylococcus aureus (MRSA), which has increased production of β-lactamases and decreased binding of β-lactams to their target penicillin-binding proteins (specifically PBP2a),¹³ and carbapenem-resistance PA, which has decreased membrane permeability and multiple enzymatic mechanisms of antibiotic inactivation.⁷ β-lactam resistance has led to the development of adjuvant therapies to potentiate their activity including the first FDA approved β-lactam adjuvant therapy, Augmentin, that combines amoxicillin with clavulanic acid, which acts as a serine β-lactamase inhibitor.¹⁴ Recently, more β-lactam adjuvants have been found including inhibitors of BlaR1 phosphorylation in MRSA,¹⁵ tobramycin-cyclam conjugates, which potentiate meropenem against MDRPA,¹⁶ aryl amide 2-aminoimidazoles that suppress carbapenem resistance in Acinetobacter baumannii,¹⁷ and the antihistamine, Loratidine, which inhibits regulatory PASTA kinases Stk1 in MRSA.¹⁸

Our laboratory, like many others in academia and industry, has a large library of “inactive” synthetic analogs of various natural products. Although these molecules did not show activity in their original work, we should not let the effort and funds used to produce these analogs go to waste. There have been multiple examples of the utility of target-blind library screening19, 20 for finding unique adjuvant/antibiotic combinations due to the fact that adjuvant screening assays are quick, reliable, and highly amenable to a variety of pathogens. Additionally, natural product and derivative libraries tend to be structurally diverse, which allows for unique drug/adjuvant combinations to be found.

Herein, we describe the discovery and mechanism of action evaluation of α-pyrone adjuvants, compounds 1 and 2 (Figure 1), that potentiate penicillin G against PA. α-Pyrones 1 and 2 were developed during the structure activity relationship (SAR) evaluation of the pseudopyronine family of natural products, which are known antibiotics produced by Pseudomonas species of bacteria.21, 22, 23 In our original work, we found that longer alkyl chain α-pyrone analogs, like pseudopyronine A (3, Figure 1), were potent antibiotics against SA; however, the shorter alkyl chains proved only weakly active against SA or Escherichia coli (EC) at high concentrations.²³