Design, synthesis and biological evaluation of novel DNA gyrase inhibitors and their siderophore mimic conjugates

Highlights

• Siderophore mimics were conjugated with 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors.

• The most potent conjugate had an IC₅₀ of 58 nM against Escherichia coli DNA gyrase.

• Two conjugates displayed MICs of 14 µg/mL against E. coli ΔtolC strain.

• Most conjugates showed improved activities against wild-type Escherichia coli in iron-depleted medium.

Abstract

Bacterial DNA gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC₅₀ of 58 nM against Escherichia coli DNA gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA gyrase inhibitor – siderophore mimic conjugates.

Introduction

In recent decades, infectious diseases have been recognized as one of the most important global health issues, which is mainly attributed to the increasing problem of microbial resistance [1], [2]. According to the World Health Organization, the priority pathogens for which research and development of new treatment options are most urgently needed include 12 families of bacteria. Among these, Staphylococcus aureus, Pseudomonas aeruginosa and the Enterobacteriaceae family (including Escherichia coli) have been described as critical high-priority pathogens that are especially difficult to treat [3].

Unfortunately, the urgent need for development of new antibiotics is, however, accompanied by a lack of interest of the pharmaceutical industry for economic reasons and because of strict regulatory requirements [4]. Due to the problem of bacterial resistance, antibiotics with new mechanisms of action are needed, as they would then not be expected to be removed by the pre-existing resistance mechanisms, with the development of cross resistance also less likely to occur. In the last 10 years, some successful new drugs have been approved that have alternative mechanisms of action; e.g., antibiotics belonging to the class of oxazolidinones and lipopeptides [5]. In addition to overexpression of efflux pumps, reduced permeability of the outer membrane (OM) is recognized as a major mechanism that underlies resistance of Gram-negative bacteria and that can cause the failure of existing antibacterial therapies [6].

DNA gyrase is a validated target for antibacterial drug discovery [7], [8]. This enzyme belongs to the class of bacterial type IIa topoisomerases, and it is responsible for the introduction of negative supercoils during bacterial DNA replication. DNA gyrase is a heterotetrameric protein that is composed of two GyrA subunits and two ATP-binding GyrB subunits [7]. Many studies have already been carried out to develop clinically useful ATP-competitive GyrB inhibitors; however, none are currently available for therapeutic use [8], [9]. In 2015 we discovered structurally novel GyrB inhibitors based on the 4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine core (1–3, Fig. 1) [10], [11]. Compounds 1 and 2 showed low-nanomolar inhibition of E. coli DNA gyrase, but did not show activity against wild-type Gram-negative strains of E. coli and P. aeruginosa, which was most likely the consequence of cell-wall penetration issues and/or active efflux from the bacterial cells that is mediated by their efflux pump machineries. This latter explanation was supported by the findings that some of these compounds showed improved antibacterial activities when tested against the E. coli JW5503 strain that has a defective efflux pump, with MICs as low as 31 μM (e.g. 2, Fig. 1) [10]. Similar observations were also made for other structural classes of GyrB inhibitors, including benzo[d]thiazoles [12], N-phenylpyrrolamides [13] and ethyl ureas [14], [15].

One strategy to overcome the problem of low bacterial cell-wall penetration is conjugation of small molecular weight siderophore mimics to molecules that have antibacterial properties [16], [17]. Siderophores are high-affinity iron-chelating compounds that are secreted by microorganisms and are among the strongest known soluble iron-binding agents [18]. Bacteria use siderophores to exploit the very limited amounts of freely available iron in the host environment during infection, and hence to survive the proliferation process [19]. Iron–siderophore complexes are then transported across the bacterial membrane into the cytoplasm via iron-uptake machineries. Attaching a siderophore or a siderophore mimic to an antibacterial drug can therefore be used as a means to deliver antibiotics into bacteria [20], [21]. Siderophore mimics are small fragments of natural siderophores that have the similar structural features that are responsible for iron chelation. Commonly used siderophore mimics are shown in Fig. 2, and these include catechols, hydroxypyranones, hydroxypyridones and dihydroxypyridones. Some antibiotic–siderophore mimic conjugates have recently been taken to clinical trials, such as the monosulfactam BAL30072 [22] and cephalosporin cefiderocol [23] (Fig. 2). In November 2019 U.S. Food and Drug Administration approved cefiderocol for the treatment of complicated urinary tract infections [24].

In the present study, we designed new DNA gyrase inhibitors starting from compounds presented in Fig. 1. Moreover, we attached the siderophore mimics to selected DNA gyrase inhibitors to evaluate their impact on enzyme inhibition and antibacterial activities against Gram-negative bacteria.