Development of phenyl-urea-based small molecules that target penicillin-binding protein 4.

Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.

Synthesis, Stereochemical Resolution, and Analogue Synthesis of Variabiline, an Aporphine Alkaloid That Sensitizes Acinetobacter baumannii and Klebsiella pneumoniae to Colistin.

Increasing antimicrobial resistance, coupled with the absence of new antibiotics, has led physicians to rely on colistin, a polymyxin with known nephrotoxicity, as the antibiotic of last resort for the treatment of infections caused by Gram-negative bacteria. One approach to increasing antibiotic efficacy and thereby reducing dosage is the use of small-molecule potentiators that augment antibiotic activity. We recently identified the aporphine alkaloid (±)-variabiline, which lowers the minimum inhibitory concentration of colistin in Acinetobacter baumannii and Klebsiella pneumoniae. Herein, we report the first total synthesis of (±)-variabiline to confirm structure and activity, the resolution, and evaluation of both enantiomers as colistin potentiators, and a structure-activity relationship study that identifies more potent variabiline derivatives. Preliminary mechanistic studies indicate that (±)-variabiline and its derivatives potentiate colistin by targeting the Gram-negative outer membrane.

Identification of a 2-Aminobenzimidazole Scaffold that Potentiates Gram-Positive Selective Antibiotics Against Gram-Negative Bacteria.

The development of novel therapeutic approaches is crucial in the fight against multi-drug resistant (MDR) bacteria, particularly gram-negative species. Small molecule adjuvants that enhance the activity of otherwise gram-positive selective antibiotics against gram-negative bacteria have the potential to expand current treatment options. We have previously reported adjuvants based upon a 2-aminoimidazole (2-AI) scaffold that potentiate macrolide antibiotics against several gram-negative pathogens. Herein, we report the discovery and structure-activity relationship (SAR) investigation of an additional class of macrolide adjuvants based upon a 2-aminobenzimidazole (2-ABI) scaffold. The lead compound lowers the minimum inhibitory concentration (MIC) of clarithromycin (CLR) from 512 to 2 µg/mL at 30 µM against Klebsiella pneumoniae 2146, and from 32 to 2 µg/mL at 5 µM, against Acinetobacter baumannii 5075. Preliminary investigation into the mechanism of action suggests that the compounds are binding to lipopolysaccharide (LPS) in K. pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, assembly, or transport in A. baumannii.

Sensitization of Gram-Negative Bacteria to Aminoglycosides with 2-Aminoimidazole Adjuvants.

In 2019, five million deaths associated with antimicrobial resistance were reported by The Centers for Disease Control and Prevention (CDC). Acinetobacter baumannii, a Gram-negative bacterial pathogen, is among the list of urgent threats. Previously, we reported 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide activity against A. baumannii. In this study, we identify several of these adjuvants that sensitize A. baumannii to aminoglycoside antibiotics. Lead compounds 1 and 7 lower the tobramycin (TOB) minimum inhibitory concentration (MIC) against the TOB-resistant strain AB5075 from 128 µg/mL to 2 µg/mL at 30 µM. In addition, the lead compounds lower the TOB MIC against the TOB-susceptible strain AB19606 from 4 µg/mL to 1 µg/mL and 0.5 µg/mL, respectively, at 30 µM and 15 µM. The evolution of resistance to TOB and 1 in AB5075 revealed mutations in genes related to protein synthesis, the survival of bacteria under environmental stressors, bacteriophages, and proteins containing Ig-like domains.

Spectrum of activity of Salmonella anti-biofilm compounds: Evaluation of activity against biofilm-forming ESKAPE pathogens.

The ESKAPE pathogens are a group of bacteria that are a leading cause of health-care associated infections and are known to be agents of chronic, biofilm-mediated infections. These chronic bacterial infections often respond poorly to antibiotics and in some cases may require surgical intervention in order to cure the infection. As biofilms are often the critical mediator of a chronic infection, it is essential to develop therapies that target bacteria within the biofilm state. Herein, we report the development of a rapid, 96-well plate-based assay that employs conditions specific for each species to optimize biofilm production and allow for easy identification of differences in biofilm mass after treatment with anti-biofilm candidates. We used these ESKAPE-specific biofilm assays to test our previously identified Salmonella anti-biofilm small molecule compounds, JG-1 and M4, for anti-biofilm activity. The results demonstrated that JG-1 and M4 have anti-biofilm activity against Enterobacter spp., S. aureus, E. faecium, P. aeruginosa, and A. baumannii. In addition, we identified that M4 has significant antimicrobial activity against S. aureus and E. faecium at concentrations >10 µM (X µg/mL). These findings support the claim that JG-1 and M4 have broad-spectrum anti-biofilm activity, while M4 has antimicrobial activity against the Gram-positive members of the ESKAPE pathogens. Thus, these compounds have the potential to have a significant impact on treating multiple types of commonly encountered biofilm-mediated infections.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Pyochelin biotransformation by Staphylococcus aureus shapes bacterial competition with Pseudomonas aeruginosa in polymicrobial infections.

Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated bacterial species from polymicrobial infections of patients with cystic fibrosis and chronic wounds. We apply mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S. aureus is equipped with an elegant mechanism to inactivate pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase). Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular reactive oxygen species (ROS) production in S. aureus. In a murine wound co-infection model, an S. aureus mutant unable to methylate pyochelin shows significantly lower fitness compared with its parental strain. Thus, Spm-mediated pyochelin methylation is a mechanism to increase S. aureus survival during in vivo competition with P. aeruginosa.

A structure activity relationship study of 3,4'-dimethoxyflavone for ArlRS inhibition in Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to many ß-lactam antibiotics, and their highly coordinated excretion of virulence factors. One way in which MRSA accomplishes this is by responding to environmental stimuli using two-component systems (TCS). The ArlRS TCS has been identified as having a key role in regulating virulence in both systemic and local infections caused by S. aureus. We recently disclosed 3,4'-dimethoxyflavone as a selective ArlRS inhibitor. In this study we explore the structure-activity relationship (SAR) of the flavone scaffold for ArlRS inhibition and identify several compounds with increased activity compared to the parent. Additionally, we identify a compound that suppresses oxacillin resistance in MRSA, and begin to probe the mechanism of action behind this activity.

Small Molecule Attenuates Bacterial Virulence by Targeting Conserved Response Regulator.

Antibiotic tolerance within a biofilm community presents a serious public health challenge. Here, we report the identification of a 2-aminoimidazole derivative that inhibits biofilm formation by two pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. In S. mutans, the compound binds to VicR, a key response regulator, at the N-terminal receiver domain, and concurrently inhibits expression of vicR and VicR-regulated genes, including the genes that encode the key biofilm matrix producing enzymes, Gtfs. The compound inhibits S. aureus biofilm formation via binding to a Staphylococcal VicR homolog. In addition, the inhibitor effectively attenuates S. mutans virulence in a rat model of dental caries. As the compound targets bacterial biofilms and virulence through a conserved transcriptional factor, it represents a promising new class of anti-infective agents that can be explored to prevent or treat a host of bacterial infections. IMPORTANCE Antibiotic resistance is a major public health issue due to the growing lack of effective anti-infective therapeutics. New alternatives to treat and prevent biofilm-driven microbial infections, which exhibit high tolerance to clinically available antibiotics, are urgently needed. We report the identification of a small molecule that inhibits biofilm formation by two important pathogenic Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus. The small molecule selectively targets a transcriptional regulator leading to attenuation of a biofilm regulatory cascade and concurrent reduction of bacterial virulence in vivo. As the regulator is highly conserved, the finding has broad implication for the development of antivirulence therapeutics that selectively target biofilms.

Dimeric 2-aminoimidazoles are highly active adjuvants for gram-positive selective antibiotics against Acinetobacter baumannii.

The Centers for Disease Control and Prevention (CDC) reports that hospital acquired infections have increased by 65% since 2019. One of the main contributors is the gram-negative bacterium Acinetobacter baumannii. Previously, we reported aryl 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide antibiotics against A. baumannii. Macrolide antibiotics are typically used to treat infections caused by gram-positive bacteria, but are ineffective against most gram-negative bacteria. We describe a new class of dimeric 2-AIs that are highly active macrolide adjuvants, with lead compounds lowering minimum inhibitory concentrations (MICs) to or below the gram-positive breakpoint level against A. baumannii. The parent dimer lowers the clarithromycin (CLR) MIC against A. baumannii 5075 from 32 µg/mL to 1 µg/mL at 7.5 µM (3.4 µg/mL), and a subsequent structure activity relationship (SAR) study identified several compounds with increased activity. The lead compound lowers the CLR MIC to 2 µg/mL at 1.5 µM (0.72 µg/mL), far exceeding the activity of both the parent dimer and the previous lead aryl 2-AI. Furthermore, these dimeric 2-AIs exhibit considerably reduced mammalian cell toxicity compared to aryl-2AI adjuvants, with IC50s of the two lead compounds against HepG2 cells of >200 µg/mL, giving therapeutic indices of >250.

6-Bromoindirubin-3'-oxime derivatives are highly active colistin adjuvants against Klebsiella pneumoniae.

Multidrug resistant (MDR) bacterial infections have become increasingly common, leading clinicians to rely on last-resort antibiotics such as colistin. However, the utility of colistin is becoming increasingly compromised as a result of increasing polymyxin resistance. Recently we discovered that derivatives of the eukaryotic kinase inhibitor meridianin D abrogate colistin resistance in several Gram-negative species. A subsequent screen of three commercial kinase inhibitor libraries led to the identification of several scaffolds that potentiate colistin activity, including 6-bromoindirubin-3'-oxime, which potently suppresses colistin resistance in Klebsiella pneumoniae. Herein we report the activity of a library of 6-bromoindirubin-3'-oxime analogs and identify four derivatives that show equal or increased colistin potentiation activity compared to the parent compound.

Re-sensitization of Multidrug-Resistant and Colistin-Resistant Gram-Negative Bacteria to Colistin by Povarov/Doebner-Derived Compounds.

Colistin, typically viewed as the antibiotic of last resort to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, had fallen out of favor due to toxicity issues. The recent increase in clinical usage of colistin has resulted in colistin-resistant isolates becoming more common. To counter this threat, we have investigated previously reported compounds, HSD07 and HSD17, and developed 13 compounds with more desirable drug-like properties for colistin sensitization against 16 colistin-resistant bacterial strains, three of which harbor the plasmid-borne mobile colistin resistance (mcr-1). Lead compound HSD1624, which has a lower LogDpH7.4 (2.46) compared to HSD07 (>5.58), reduces the minimum inhibitory concentration (MIC) of colistin against Pseudomonas aeruginosa strain TRPA161 to 0.03 µg/mL from 1024 µg/mL (34,000-fold reduction). Checkerboard assays revealed that HSD1624 and analogues are also synergistic with colistin against colistin-resistant strains of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae. Preliminary mechanism of action studies indicate that HSD1624 exerts its action differently depending on the bacterial species. Time-kill studies suggested that HSD1624 in combination with 0.5 µg/mL colistin was bactericidal to extended-spectrum beta-lactamase (ESBL)-producing E. coli, as well as to E. coli harboring mcr-1, while against P. aeruginosa TRPA161, the combination was bacteriostatic. Mechanistically, HSD1624 increased membrane permeability in K. pneumoniae harboring a plasmid containing the mcr-1 gene but did not increase radical oxygen species (ROS), while a combination of 15 µM HSD1624 and 0.5 µg/mL colistin significantly increased ROS in P. aeruginosa TRPA161. HSD1624 was not toxic to mammalian red blood cells (up to 226 µM).

Overcoming intrinsic resistance in gram-negative bacteria using small molecule adjuvants.

Gram-negative bacteria are intrinsically resistant to many classes of antibiotics, predominantly due to the impermeability of the outer membrane and the presence of efflux pumps. Small molecule adjuvants that circumvent these resistance mechanisms have the potential to expand therapeutic options for treating Gram-negative infections to encompass antibiotic classes that are otherwise limited to treating Gram-positive infections. Adjuvants that effect increased antibiotic permeation, either by physical disruption of the outer membrane or through interference with synthesis, transport, or assembly of membrane components, and adjuvants that limit efflux, are discussed as potential avenues to overcoming intrinsic resistance in Gram-negative bacteria.

2-Aminoimidazole Analogs Target PhoP Altering DNA Binding Activity and Affect Outer Membrane Stability in Gram-Negative Bacteria.

Multidrug-resistant bacteria cause immense public health concerns as once effective antibiotics no longer work against even common infections. Concomitantly, there has been a decline in the discovery of new antibiotics, and the current global clinical pipeline is woefully inadequate, especially against resistant Gram-negative bacteria. One major contribution to Gram-negative resistance is the presence of a protective outer membrane. Consequently, an appealing option for tackling resistance is to adversely affect that outer membrane. With that in mind, we define the response regulator PhoP as a target for new 2-aminoimidazole compounds and show that they affect the integrity of the outer membrane in resistant strains of Escherichia coli and Klebsiella pneumoniae. We also provide empirical evidence for the 2-aminoimidazole mechanism of action.

Identification of Staphylococcus aureus Penicillin Binding Protein 4 (PBP4) Inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low ß-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA ß-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism's ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein's function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism's PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.

Simple Dipyrrin Analogues of Prodigiosin for Use as Colistin Adjuvants.

Multidrug resistant (MDR) bacteria are an increasing public health problem. One promising alternative to the development of new antibiotics is the use of antibiotic adjuvants, which would allow the continued use of FDA-approved antibiotics that have been rendered ineffective due to resistance. Herein, we report a series of dipyrrins and pyrrole derivatives designed as analogues of prodigiosin and obatoclax, several of which potentiate the activity of colistin against Klebsiella pneumoniae, with lead compounds also potentiating colistin against Acinetobacter baumannii and Pseudomonas aeruginosa.

Identification of a Host-Targeted Compound to Control Typhoid Fever.

Typhoid fever is caused primarily by the enteric microbe Salmonella enterica serovar Typhi and remains a major global health problem with approximately 14 million new infections and 136,000 fatalities annually. While there are antibiotic options available to treat the disease, the global increase in multidrug-resistant strains necessitates alternative therapeutic options. Host-targeted therapeutics present a promising anti-infective strategy against intracellular bacterial pathogens. A cell-based assay identified a compound that inhibits Salmonella proliferation in infected cells, 2-(3-hydroxypropyl)-1-(3-phenoxyphenyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (KH-1), which is devoid of direct activity against Salmonella. The compound inhibits the growth of both antibiotic-sensitive and -resistant Salmonella strains inside macrophages and reduces lactate dehydrogenase (LDH) release from Salmonella-infected cells. Subsequent screening of KH-1 commercial analogs identified 2-(4-fluorobenzyl)-1-(3-phenoxyphenyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (KH-1-2), which is more effective in controlling Salmonella growth inside macrophages. In vitro KH-1-2 treatment of Salmonella infection resulted in an 8- to 10-fold reduction in bacterial load in infected macrophages. In combination with suboptimal ciprofloxacin treatment, KH-1-2 further reduces Salmonella growth inside macrophages. The toxicity and efficacy of KH-1-2 in controlling Salmonella infection were examined in vivo using a mouse model of typhoid fever. No significant compound-related clinical signs and histological findings of the liver, spleen, or kidney were observed from uninfected mice that were intraperitoneally treated with KH-1-2. KH-1-2 significantly protected mice from a lethal dose of infection by an antibiotic-resistant Salmonella strain. Thus, our study provides support that this is a promising lead compound for the development of a novel host-targeted therapeutic agent to control typhoid fever. IMPORTANCESalmonella spp. cause significant morbidity and mortality worldwide. Typhoidal spp. (e.g., S. Typhi) cause a systemic disease typically treated with antibiotics. However, growing antibiotic resistance is resulting in increased treatment failures. We screened a compound library for those that would reduce Salmonella-induced macrophage toxicity, identifying compound KH-1. KH-1 has no direct effects on the bacteria but limits Salmonella survival in macrophages and protects against lethal infection in a mouse model of typhoid fever. A suboptimal concentration of ciprofloxacin worked in conjunction with the compound to further decrease Salmonella survival in macrophages. An analog (KH-1-2) was identified that possessed increased activity in vitro in macrophages and in vivo against both antibiotic-sensitive and -resistant strains. Thus, we report the identification of a lead compound that may be a useful scaffold as a host-directed antimicrobial against typhoid fever.

Phenotypic screening of compound libraries as a platform for the identification of antibiotic adjuvants: Identification of colistin adjuvants from a natural product library.

The identification of antibiotic adjuvants, small molecules that potentiate the activity of conventional antibiotics, provides an orthogonal approach to the development of new antibiotics in the fight against drug resistant bacterial infections. Methods to identify novel adjuvants could potentially aid efforts to overcome the increasing prevalence of resistance and stave off the onset of a "post-antibiotic era." Phenotypic whole cell screens allow for the identification of hits with the necessary properties to access their biomolecular target, and may also facilitate the discovery of novel adjuvant targets. A phenotypic screening platform is outlined, in which a natural product library was explored for activity with antibiotics from several mechanistically distinct classes against clinically important bacterial species. General approaches to delineating the mechanism of action of hit compounds identified from phenotypic screens are described, followed by specific approaches to uncovering the mechanism of action of the colistin adjuvants identified from the natural product screen.

2-Aminoimidazoles Inhibit Mycobacterium abscessus Biofilms in a Zinc-Dependent Manner.

Biofilm growth is thought to be a significant obstacle to the successful treatment of Mycobacterium abscessus infections. A search for agents capable of inhibiting M. abscessus biofilms led to our interest in 2-aminoimidazoles and related scaffolds, which have proven to display antibiofilm properties against a number of Gram-negative and Gram-positive bacteria, including Mycobacterium tuberculosis and Mycobacterium smegmatis. The screening of a library of 30 compounds led to the identification of a compound, AB-2-29, which inhibits the formation of M. abscessus biofilms with an IC50 (the concentration required to inhibit 50% of biofilm formation) in the range of 12.5 to 25 µM. Interestingly, AB-2-29 appears to chelate zinc, and its antibiofilm activity is potentiated by the addition of zinc to the culture medium. Preliminary mechanistic studies indicate that AB-2-29 acts through a distinct mechanism from those reported to date for 2-aminoimidazole compounds.

Synthesis, Stereochemical Confirmation, and Derivatization of 12(S),16ϵ-Dihydroxycleroda-3,13-dien-15,16-olide, a Clerodane Diterpene That Sensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

Over the past decades, antibiotic resistance has grown to a point where orthogonal approaches to combating infections caused by resistant bacteria are needed. One such approach is the development of non-microbicidal small molecules that potentiate the activity of conventional antibiotics, termed adjuvants. The diterpene natural product 12(S),16ϵ-dihydroxycleroda-3,13-dien-15,16-olide, which we refer to as (-)-LZ-2112, is known to synergize with oxacillin against methicillin-resistant Staphylococcus aureus (MRSA). To explore this activity, (-)-LZ-2112 was synthesized and the structure confirmed through X-ray analysis. Preliminary structure-activity relationship studies following the synthesis of several analogs identified key structural elements responsible for activity and indicate that scaffold simplification is possible. A preliminary mode of action study suggests mecA plays a role in the adjuvant activity of (-)-LZ-2112.