Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases.

Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.

Discovery and development of inhibitors of acetyltransferase Eis to combat Mycobacterium tuberculosis.

Aminoglycosides are bactericidal antibiotics with a broad spectrum of activity, used to treat infections caused mostly by Gram-negative pathogens and as a second-line therapy against tuberculosis. A common resistance mechanism to aminoglycosides is bacterial aminoglycoside acetyltransferase enzymes (AACs), which render aminoglycosides inactive by acetylating their amino groups. In Mycobacterium tuberculosis, an AAC called Eis (enhanced intracellular survival) acetylates kanamycin and amikacin. When upregulated as a result of mutations, Eis causes clinically important aminoglycoside resistance; therefore, Eis inhibitors are attractive as potential aminoglycoside adjuvants for treatment of aminoglycoside-resistant tuberculosis. For over a decade, we have studied Eis and discovered several series of Eis inhibitors. Here, we provide a detailed protocol for a colorimetric assay used for high-throughput discovery of Eis inhibitors, their characterization, and testing their selectivity. We describe protocols for in vitro cell culture assays for testing aminoglycoside adjuvant properties of the inhibitors. A procedure for obtaining crystals of Eis-inhibitor complexes and determining their structures is also presented. Finally, we discuss applicability of these methods to discovery and testing of inhibitors of other AACs.

Introduction to the themed collection on antimicrobial resistance.

Guest editors Jayanta Haldar, Sylvie Garneau-Tsodikova and Micha Fridman introduce the RSC Medicinal Chemistry themed collection on 'Antibiotic microbial resistance'.

Small molecule inhibitors of the fosfomycin resistance enzyme FosM from Mycobacterium abscessus.

Fosfomycin is a safe broad-spectrum antibiotic that has not achieved widespread use because of the emergence of fosfomycin-modifying enzymes. Inhibition of fosfomycin-modifying enzymes could be used to help combat pathogens like Mycobacterium abscessus. Our previous work identified several inhibitors for the enzyme FosB from Staphylococcus aureus. We have tested those same compounds for inhibition of FosM, the fosfomycin-modifying enzyme from M. abscessus. The work described here will be used as the basis for more detailed studies into the inhibition of FosM.

Droplet Tn-Seq identifies the primary secretion mechanism for yersiniabactin in Yersinia pestis.

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.

Discovery and development of novel substituted monohydrazides as potent antifungal agents.

Novel substituted monohydrazides synthesized for this study displayed broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains. The activity of these compounds was either comparable or superior to amphotericin B against most of the fungal strains tested. These compounds possessed fungistatic activity in a time-kill assay and exhibited no mammalian cell toxicity. In addition, they prevented the formation of fungal biofilms. Even after repeated exposures, the Candida albicans ATCC 10231 (strain A) fungal strain did not develop resistance to these monohydrazides.

Identification and analysis of small molecule inhibitors of FosB from Staphylococcus aureus.

Antimicrobial resistance (AMR) poses a significant threat to human health around the world. Though bacterial pathogens can develop resistance through a variety of mechanisms, one of the most prevalent is the production of antibiotic-modifying enzymes like FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. FosB enzymes are found in pathogens such as Staphylococcus aureus, one of the leading pathogens in deaths associated with AMR. fosB gene knockout experiments establish FosB as an attractive drug target, showing that the minimum inhibitory concentration (MIC) of fosfomycin is greatly reduced upon removal of the enzyme. Herein, we have identified eight potential inhibitors of the FosB enzyme from S. aureus by applying high-throughput in silico screening of the ZINC15 database with structural similarity to phosphonoformate, a known FosB inhibitor. In addition, we have obtained crystal structures of FosB complexes to each compound. Furthermore, we have kinetically characterized the compounds with respect to inhibition of FosB. Finally, we have performed synergy assays to determine if any of the new compounds lower the MIC of fosfomycin in S. aureus. Our results will inform future studies on inhibitor design for the FosB enzymes.

Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Aromatic hydrazides: A potential solution for Acinetobacter baumannii infections.

The emergence of multidrug-resistant bacteria and the poor efficacy of available antibiotics against these infections have led to the urgent need for novel antibiotics. Acinetobacter baumannii is one of high-priority pathogens due to its ability to mount resistance to different classes of antibiotics. In an effort to provide novel agents in the fight against infections caused by A. baumannii, we synthesized a series of 46 aromatic hydrazides as potential treatments. In this series, 34 compounds were found to be low- to sub-µM inhibitors of A. baumannii growth, with MIC values in the range of 8 µg/mL to ≤0.125 µg/mL against a broad set of multidrug-resistant clinical isolates. These compounds were not highly active against other bacteria. We showed that one of the most potent compounds, 3e, was bacteriostatic and inhibitory to biofilm formation, although it did not disrupt the preformed biofilm. Additionally, we found that these compounds lacked mammalian cytotoxicity. The high antibacterial potency and the lack of mammalian cytotoxicity make these compounds a promising lead series for development of a novel selective anti-A. baumannii antibiotic.

Discovery and Mechanistic Analysis of Structurally Diverse Inhibitors of Acetyltransferase Eis among FDA-Approved Drugs.

Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis-proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.

Inhibition of Fosfomycin Resistance Protein FosB from Gram-Positive Pathogens by Phosphonoformate.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico-based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosBSa) and Bacillus cereus (FosBBc). In addition, we have determined a crystal structure of FosBBc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.

Selective Inhibition of the Periodontal Pathogen Porphyromonas gingivalis by Third-Generation Zafirlukast Derivatives.

Periodontal diseases are inflammatory diseases triggered by pathogenic oral bacterial species, such as Porphyromonas gingivalis. Zafirlukast (ZAF) has displayed antibacterial activity against P. gingivalis. Herein, we report the design, synthesis, and selective antibacterial activity of 14 novel third-generation ZAF derivatives. Two second-generation ZAF derivatives were tested as they were not previously tested against P. gingivalis ATCC 33277. Most third-generation derivatives displayed superior/selective antibacterial activity against P. gingivalis compared to ZAF and its second-generation derivatives. The compounds displayed bactericidal activity against P. gingivalis, inhibited biofilm growth, displayed no hemolytic activity, and displayed less cytotoxicity against mammalian cells than ZAF. The superior/selective antibacterial activity of ZAF derivatives against P. gingivalis, increased safety profile, and inhibition of biofilm growth compared to ZAF indicate that the compounds, especially 21a, 21b, and 24g, show promise as antibacterial agents for future studies to test their potential for preventing and treating P. gingivalis-induced periodontal diseases.

Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity.

A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 µM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

Discovery and Optimization of 6-(1-Substituted pyrrole-2-yl)-s-triazine Containing Compounds as Antibacterial Agents.

Antimicrobial drug resistance is a major health issue plaguing healthcare worldwide and leading to hundreds of thousands of deaths globally each year. Tackling this problem requires discovery and development of new antibacterial agents. In this study, we discovered novel 6-(1-substituted pyrrole-2-yl)-s-triazine containing compounds that potently inhibited the growth of Staphylococcus aureus regardless of its methicillin-resistant status, displaying minimum inhibitory concentration (MIC) values as low as 1 µM. The presence of a single imidazole substituent was critical to the antibacterial activity of these compounds. Some of the compounds also inhibited several nontubercular mycobacteria. We have shown that these molecules are potent bacteriostatic agents and that they are nontoxic to mammalian cells at relevant concentrations. Further development of these compounds as novel antimicrobial agents will be aimed at expanding our armamentarium of antibiotics.

Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut.

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

Structure-based design of haloperidol analogues as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis to overcome kanamycin resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.

Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Porphyromonas gingivalis: where do we stand in our battle against this oral pathogen?

Periodontal diseases, such as gingivitis and periodontitis, are inflammatory diseases triggered by pathogenic bacteria that lead to damage of the soft tissue and bone supporting the teeth. Amongst the identified oral periodontopathogenic bacteria, Porphyromonas gingivalis is able to enhance oral dysbiosis, which is an imbalance in the beneficial commensal and periodontal pathogenic bacteria that induces chronic inflammation. Given the critical role of oral pathogenic bacteria like P. gingivalis in the pathogenesis of periodontitis, local and/or systemic antibacterial therapy has been suggested to treat this disease, especially in its severe or refractory forms. Nevertheless, the majority of the antibacterial agents currently used for the treatment of periodontal diseases are broad-spectrum, which harms beneficial bacterial species that are critical in health, inhibit the growth of pathogenic bacteria, contribute in protecting the periodontal tissues to damage and aid in its healing. Thus, the development of more effective and specific antibacterial agents is needed to control oral pathogens in a polymicrobial environment. The strategies for the development of novel antibacterial agents include natural product isolation as well as synthetic and semi-synthetic methodologies. This review presents an overview of the periodontal diseases gingivitis and periodontitis along with current antibacterial treatment options (i.e., classes of antibacterial agents and the mechanism(s) of resistance that hinder their usage) used in periodontal diseases that specifically target oral pathogens such as P. gingivalis. In addition, to help medicinal chemists gain a better understanding of potentially promising scaffolds, this review provides an in-depth coverage of the various families of small molecules that have been investigated as potential anti-P. gingivalis agents, including novel families of compounds, repositioned drugs, as well as natural products.

Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen Oxide and Its Analogues.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently found in the airways of cystic fibrosis (CF) patients due to the dehydrated mucus that collapses the underlying cilia and prevents mucociliary clearance. During this life-long chronic infection, P. aeruginosa cell accumulates mutations that lead to inactivation of the mucA gene that results in the constitutive expression of algD-algA operon and the production of alginate exopolysaccharide. The viscous alginate polysaccharide further occludes the airways of CF patients and serves as a protective matrix to shield P. aeruginosa from host immune cells and antibiotic therapy. Development of inhibitors of alginate production by P. aeruginosa would reduce the negative impact from this viscous polysaccharide. In addition to transcriptional regulation, alginate biosynthesis requires allosteric activation by bis (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO) inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind c-di-GMP. However, P. aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P. aeruginosa. Future development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P. aeruginosa.

Development of Single-Stranded DNA Bisintercalating Inhibitors of Primase DnaG as Antibiotics.

Many essential enzymes in bacteria remain promising potential targets of antibacterial agents. In this study, we discovered that dequalinium, a topical antibacterial agent, is an inhibitor of Staphylococcus aureus primase DnaG (SaDnaG) with low-micromolar minimum inhibitory concentrations against several S. aureus strains, including methicillin-resistant bacteria. Mechanistic studies of dequalinium and a series of nine of its synthesized analogues revealed that these compounds are single-stranded DNA bisintercalators that penetrate a bacterium by compromising its membrane. The best compound of this series likely interacts with DnaG directly, inhibits both staphylococcal cell growth and biofilm formation, and displays no significant hemolytic activity or toxicity to mammalian cells. This compound is an excellent lead for further development of a novel anti-staphylococcal therapeutic.