RESUMO
Natural products are important precursors for antibiotic drug design. These chemical scaffolds serve as synthetic inspiration for chemists who leverage their structures to develop novel antibacterials and chemical probes. We have previously studied carolacton, a natural product macrolactone fromSorangium cellulosum, and discovered a simplified derivative, A2, that maintained apparent biofilm inhibitory activity, although the biological target was unknown. Herein, we utilize affinity-based protein profiling (AfBPP) in situ during biofilm formation to identify the protein target using a photoexcitable cross-linking derivative of A2. From these studies, we identified glucan binding protein B (GbpB), a peptidoglycan hydrolase, as the primary target of A2. Further characterization of the interaction between A2 and GbpB, as well as PcsB, a closely related homologue from the more pathogenic S. pneumoniae, revealed binding to the catalytic CHAP (cysteine, histidine, aminopeptidase) domain. To the best of our knowledge, this is the first report of a small-molecule binder of a conserved and essential bacterial CHAP hydrolase, revealing its potential as an antibiotic target. This work also highlights A2 as a useful tool compound for streptococci and as an initial scaffold for the design of more potent CHAP binders.
RESUMO
Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P.â aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.
Assuntos
Antibacterianos/farmacologia , Benzofenonas/farmacologia , Produtos Biológicos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Benzofenonas/síntese química , Benzofenonas/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Norfloxacino/antagonistas & inibidores , Norfloxacino/química , Norfloxacino/farmacologia , Pseudomonas aeruginosa/citologia , Relação Estrutura-AtividadeRESUMO
The oral microbiome is a dynamic environment inhabited by both commensals and pathogens. Among these is Streptococcus mutans, the causative agent of dental caries, the most prevalent childhood disease. Carolacton has remarkably specific activity against S. mutans, causing acid-mediated cell death during biofilm formation; however, its complex structure limits its utility. Herein, we report the diverted total synthesis and biological evaluation of a rationally designed library of simplified analogs that unveiled three unique biofilm phenotypes further validating the role of natural product synthesis in the discovery of new biological phenomena.
Assuntos
Biofilmes/efeitos dos fármacos , Produtos Biológicos/farmacologia , Macrolídeos/farmacologia , Streptococcus mutans/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Produtos Biológicos/síntese química , Produtos Biológicos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Macrolídeos/síntese química , Macrolídeos/química , Estrutura Molecular , Tamanho da Partícula , Fenótipo , Streptococcus mutans/citologia , Streptococcus mutans/metabolismo , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In Escherichia coli and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an n-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical S-adenosylmethionine (SAM) enzymes, which use an [Fe4S4] cluster to reductively cleave SAM to generate a 5'-deoxyadenosyl 5'-radical. Classical LSs also contain a second [Fe4S4] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike E. coli biotin synthase, contain two [Fe4S4] clusters. We identify the cluster ligands to both iron-sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to E. coli biotin synthase and in contrast to E. coli LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.
RESUMO
Targeting Streptococcus mutans is the primary focus in reducing dental caries, one of the most common maladies in the world. Previously, our groups discovered a potent bactericidal biaryl compound that was inspired by the natural product honokiol. Herein, a structure activity relationship (SAR) study to ascertain structural motifs key to inhibition is outlined. Furthermore, mechanism studies show that bacterial membrane disruption is central to the bacterial growth inhibition. During this process, it was discovered that analog C2 demonstrated a 4-fold better therapeutic index compared to the commercially available antimicrobial cetylpyridinium chloride (CPC) making it a viable alternative for oral care.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Cetilpiridínio/farmacologia , Lignanas/farmacologia , Streptococcus mutans/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Membrana Externa Bacteriana/efeitos dos fármacos , Compostos de Bifenilo/química , Lignanas/química , Testes de Sensibilidade Microbiana , Microbiota/efeitos dos fármacos , Fenóis/isolamento & purificação , Fenóis/farmacologia , Streptococcus mutans/crescimento & desenvolvimentoRESUMO
The prevalence of biofilm diseases, and dental caries in particular, have encouraged extensive research on S. mutans biofilms, including methods of preventing its formation. Numerous small molecules with specific anti-biofilm activity against this pathogen have been isolated and synthesized. Generally, these molecules can be characterized into three categories: sucrose-dependent anti-adhesion, sucrose-independent anti-adhesion and cellular signaling interference. This review aims to provide an overview of the current small molecule strategies used for targeting S. mutans biofilms, and a perspective of the future for the field.
RESUMO
Understanding the broader biological impact of carolacton, a macrolactone natural product, has been ongoing for the past decade. Multiple studies have shown connections to regulatory systems, acid tolerance mechanisms, biofilm formation, and recently folate dehydrogenase (FolD). Progress elucidating the cause of biofilm-specific activity in Streptococcus mutans has been limited due to low-throughput analyses of carolacton-treated cells. We disclose the discovery of a simplified carolacton-inspired analog that demonstrates inhibitory activity against S. mutans biofilm cells. This discovery permitted a proof of concept chemical genetic screen of S. mutans mutants identifying the carbon catabolite protein A signaling pathway as a putative target.
Assuntos
Biofilmes/efeitos dos fármacos , Macrolídeos/química , Macrolídeos/farmacologia , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/genética , Biofilmes/crescimento & desenvolvimento , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Estudo de Prova de Conceito , Transdução de Sinais , Proteína Estafilocócica A/metabolismo , Streptococcus mutans/químicaRESUMO
The oral microbiome is a complex ecological niche where both commensal and pathogenic bacteria coexist. Previous reports have cited that the plant isolate honokiol is a potent inhibitor of S. mutans biofilms. Herein we report a cross-coupling method that provides access to a concise library of honokiol-inspired analogs. Through this work we determined that the inhibitory activity of honokiol is highly dependent on the growth conditions. Further, we identify a series of analogs that display significant potency against oral bacteria leading to the discovery of a potent antimicrobial.