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Two New Targets to Fight Drug-Resistant Bacteria

 

By: Carol A. Rouzer, VICB Communications
Published:  February 26, 2016

 

 

Compounds that activate heme biosynthesis and/or are selectively toxic to fermenting bacteria point the way to new therapeutic options against S. aureus.

 

The growing problem of antibiotic-resistant bacteria is one of our greatest global public health challenges, and Staphylococcus aureus is a particularly important pathogen in this regard. Causing primarily skin and soft tissue infections, S. aureus can also produce more serious illnesses, such as pneumonia and toxic shock. Thus, the facility with which this ubiquitous organism has acquired resistance to nearly every antibiotic renders it especially dangerous. The quest for new antibiotics to treat drug-resistant S. aureus led Vanderbilt Institute of Chemical Biology members Gary Sulikowski and Eric Skaar to search for probes that target novel bacterial metabolic pathways. Their search resulted in the discovery of Compound 1 (Figure 1), which stimulates heme biosynthesis and is toxic to bacteria growing under anaerobic conditions. The investigators originally thought that Compound 1’s two activities are linked, but new structure activity relationship (SAR) studies of a Compound 1 analog library reveal that these are distinct properties of the molecule [B. F. Dutter et al., (2016) ACS Chem. Biol., published online February 18, DOI:10.1021/acschembio.5b00934].




Figure 1
. Effect of Compound 1 on the HssRs heme sensor system. High concentrations of heme trigger the sensor protein HssS to undergo autophosphorylation at a specific histidine residue. Transfer of that phosphate to an aspartate of HssR enables HssR to bind to the promotor of the hrtAB gene, leading to increased expression of HrtAB. This protein is an efflux pump that alleviates heme toxicity. Compound 1 stimulates heme biosynthesis, increasing the intracellular heme concentration and triggering HssRS. Compound 1 also inhibits the growth of fermenting bacteria. Image reproduced by permission from B. F. Dutter et al., (2016) ACS Chem. Biol., published online February 18, DOI:10.1021/acschembio.5b00934. Copyright 2016, American Chemical Society.


 

As is the case for most organisms, S. aureus requires iron as a cofactor for multiple enzymes that catalyze normal metabolic processes. Iron also helps the bacteria to evade the host immune system, and it regulates the expression of many virulence factors. To satisfy its need for iron, S. aureus has evolved an efficient system that enables it to scavenge heme from host hemoproteins. However, high levels of heme can be toxic to the bacteria, so S. aureus also possesses the HrtAB efflux pump that alleviates heme toxicity. Expression of HrtAB is controlled by the HssRS heme sensor system (Figure 1) comprising two proteins, HssS and HssR. HssS is a sensor protein that is activated in the presence of excess heme. Activation of HssS results in autophosphorylation at a specific histidine residue. Then, transfer of this phosphate group to an aspartate of HssR enables binding of HssR to the promoter of the gene encoding HrtAB, triggering transcription. The discovery of Compound 1 resulted from a screen for molecules that activate HssRS. Follow-up studies revealed that Compound 1 triggers HssRS in S. aureus by activating endogenous heme synthesis, leading to unusually high levels of intracellular heme. This research also led to the unexpected finding that Compound 1 is toxic to bacteria grown under anaerobic conditions. In the absence of oxygen, bacteria must rely on fermentation to fulfill their requirement for energy. Thus, it appeared that Compound 1 may both modulate heme biosynthesis and interfere with anaerobic metabolic processes utilized by fermenting bacteria. The investigators’ original hypothesis that Compound 1’s two activities arise from a common mechanism led them to search for molecular features that are key to both.

 

To achieve their goal, the Sulikowski lab investigators synthesized a library of Compound 1 analogs, each bearing one or more modifications targeting selected regions of the molecule (Figure 2). They removed the B ring of the naphthyl group and then modified the resulting phenol by either altering the hydroxyl group or adding substituents at the meta- and/or para- positions. They next focused on the pyrazole ring, creating new molecules by N-alkylation. Finally, they replaced the furan ring with aryl, heteroaryl, or alkyl groups. The complete library contained 31 compounds.

 

 

 

Figure 2. Figure 2. A library of Compound 1 analogs was created by altering four principal sites. Removal of the B ring of the naphthol group yielded a phenol, which was then further modified at the hydroxyl group and by adding other substituents to the ring. The pyrazol ring was modified by addition of alkyl groups to the nitrogens, and the furan ring was replaced with other aromatic or alkyl groups. Image reproduced by permission from B. F. Dutter et al., (2016) ACS Chem. Biol., published online February 18, DOI:10.1021/acschembio.5b00934. Copyright 2016, American Chemical Society.

 

 

The Skaar lab investigators first tested the new compounds for their ability to activate HssRS using a strain of S. aureus that carries a plasmid encoding the xylE reporter gene fused to the hrt promoter. In these bacteria, activation of HssRS leads to hrt-dependent expression of xylE, a gene that encodes the XylE catechol oxidase. The reaction catalyzed by XylE yields a product that is readily measured spectrophotometrically, providing a convenient assay for HssRS-mediated gene regulation. The results of the screen showed that replacement of the naphthol group of Compound 1 with a phenol or 4-methoxyphenol led to only a modest reduction in activity. In contrast, O-methylation of the ortho-hydroxyl group or N-methylation of the pyrazole ring of Compound 1 completely eliminated activity. These results suggested that a hydrogen-bonding network is important for Compound 1’s efficacy, a hypothesis that was supported by further SAR results. Most attempts to replace the furan ring of Compound 1 with other substituents led to substantial loss of activity; however, an unsubstituted phenyl ring was well tolerated at that position. Complete dose-response curves of the six most active compounds revealed that none was substantially more potent than Compound 1, but they varied considerably in efficacy, the maximal achievable response. The investigators further evaluated the ability of the top six compounds to activate HssRS in wild-type S. aureus. This was easily accomplished by measuring the ability of bacteria pretreated with each compound to grow in the presence of high concentrations of heme. Any compound that activates the heme sensor system preadapts the bacteria to heme toxicity, thereby enabling them to grow in otherwise toxic concentrations of heme. All six compounds were active in this assay.

 

The Skaar lab next evaluated the compounds for selective toxicity to fermenting bacteria. Initial screening results demonstrated that the majority of the compounds was nontoxic in either aerobic or anaerobic conditions. In particular, removal of the B-ring of the naphthol group or O-methylation of Compound 1 eliminated toxicity. However, the investigators found that they could restore toxicity to compounds lacking the B-ring by making other changes, such as substitution of the furan ring with a phenyl ring or replacing the ortho-hydroxyl group with an aromatic amide. N-methylation and substitution of the furan ring with a large alkyl group also promoted toxicity.

 

These results suggested that the hydrogen bonding network required for HssRS activation is not required for toxicity to fermenting bacteria. Furthermore, modification at the 5-position with aromatic or heteroaromatic groups at the 5-position is required for HssRS activation, whereas substitution with large alkyl groups at this position promotes toxicity. The findings clearly demonstrate that toxicity and HssRS activation are separable but that there is overlap in the SAR, enabling the design of chemical structures that carry both activities. The findings pave the way for further SAR to optimize each activity individually. Perhaps more important, a search for the specific targets of molecules bearing each of the activities should provide critical information regarding the regulation of heme biosynthesis and anaerobic toxicity that may lead to novel antibiotics for the treatment of drug-resistant S. aureus.

 

 

Figure 3. Screening of the Compound 1 analog library revealed compounds that activated heme biosynthesis (blue sphere), compounds that were toxic to bacteria under anaerobic conditions (yellow sphere) and compounds that exhibited both activities (green intersection). Image reproduced by permission from B. F. Dutter et al., (2016) ACS Chem. Biol., published online February 18, DOI:10.1021/acschembio.5b00934. Copyright 2016, American Chemical Society.

 

 

 

View ACS Chemical Biology article: Decoupling Activation of Heme Biosynthesis from Anaerobic Toxicity in a Molecule Active in Staphylococcus aureus

 

 

 

 

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