Development of novel bisphenol derivatives with a membrane-targeting mechanism as potent gram-positive antibacterial agents.

Antibiotic resistance is becoming increasingly severe. The development of small molecular antimicrobial peptides is regarded as a promising design strategy for antibiotics. Here, a series of bisphenol derivatives with amphiphilic structures were designed and synthesized as antibacterial agents by imitating the design strategy of antimicrobial peptides. After a series of structural optimizations, lead compound 43 was identified, which exhibited excellent antibacterial activity against Gram-positive bacterial strains (MICs = 0.78-1.56 µg/mL), poor hemolytic activity (HC50 > 200 µg/mL), and low cytotoxicity (CC50 > 100 µg/mL). Further biological evaluation results indicated that 43 exerted antibacterial effects by directly destroying bacterial cell membranes and displayed rapid bactericidal properties (within 0.5-1 h), leading to a very low probability of drug resistance. Moreover, in a murine model of corneal infection, 43 exhibited a strong in vivo antibacterial efficacy. These findings indicate that 43 is a promising candidate compound for the treatment of bacterial infections.

Development of cannabidiol derivatives as potent broad-spectrum antibacterial agents with membrane-disruptive mechanism.

The emergence of antibiotic resistance has brought a significant burden to public health. Here, we designed and synthesized a series of cannabidiol derivatives by biomimicking the structure and function of cationic antibacterial peptides. This is the first report on the design of cannabidiol derivatives as broad-spectrum antibacterial agents. Through the structure-activity relationship (SAR) study, we found a lead compound 23 that killed both Gram-negative and Gram-positive bacteria via a membrane-targeting mechanism of action with low resistance frequencies. Compound 23 also exhibited very weak hemolytic activity, low toxicity toward mammalian cells, and rapid bactericidal properties. To further validate the membrane action mechanism of compound 23, we performed transcriptomic analysis using RNA-seq, which revealed that treatment with compound 23 altered many cell wall/membrane/envelope biogenesis-related genes in Gram-positive and Gram-negative bacteria. More importantly, compound 23 showed potent in vivo antibacterial efficacy in murine corneal infection models caused by Staphylococcus aureus or Pseudomonas aeruginosa. These findings would provide a new design idea for the discovery of novel broad-spectrum antibacterial agents to overcome the antibiotic resistance crisis.

Design of phenothiazine-based cationic amphiphilic derivatives incorporating arginine residues: Potential membrane-active broad-spectrum antimicrobials combating pathogenic bacteria in vitro and in vivo.

Multidrug-resistant bacteria infections pose an increasingly serious threat to human health, and the development of antimicrobials is far from meeting the clinical demand. It is urgent to discover and develop novel antibiotics to combat bacterial resistance. Currently, the development of membrane active antimicrobial agents is an attractive strategy to cope with antimicrobial resistance issues. In this study, the synthesis and biological evaluation of cationic amphiphilic phenothiazine-based derivatives were reported. Among them, the most promising compound 30 bearing a n-heptyl group and two arginine residues displayed potent bactericidal activity against both Gram-positive (MICs = 1.56 µg/mL) and Gram-negative bacteria (MICs = 3.125-6.25 µg/mL). Compound 30 showed low hemolysis activity (HC50 = 281.4 ± 1.6 µg/mL) and low cytotoxicity (CC50 ＞ 50 µg/mL) toward mammalian cells, as well as excellent salt resistance. Compound 30 rapidly killed bacteria by acting on the bacterial cell membrane and appeared less prone to resistance. Importantly, compound 30 showed potent in vivo efficacy in a murine model of bacterial keratitis. Hence, the results suggested compound 30 has a promising prospect as a broad-spectrum antibacterial agent for the treatment of drug-resistant bacterial infections.

Electrochemical Selenylation of Sulfoxonium Ylides for the Synthesis of gem-Diselenides as Antimicrobials against Fungi.

Organoselenium compounds are important scaffolds in pharmaceutical molecules. Herein, we report metal-free, electrochemical, highly chemo- and regioselective synthesis of gem-diselenides through the coupling of α-keto sulfoxonium ylides with diselenides. The versatility of the electrochemical manifold enabled the selenylation with ample scope and broad functional group tolerance, as well as setting the stage for modification of complex bioactive molecules. Detailed mechanistic studies revealed that the key C-Se bond was constructed using n-Bu4NI as an electrolyte and catalyst through the electrosynthetic protocol. Finally, the desired α-keto gem-diselenides showed excellent antimicrobial activity against Candida albicans, which can be identified as the lead compounds for further exploration.

Membrane-Active Nonivamide Derivatives as Effective Broad-Spectrum Antimicrobials: Rational Design, Synthesis, and Biological Evaluation.

Antibiotic resistance is emerging as a "global public health concern". To address the growing epidemic of multidrug-resistant pathogens, the development of novel antimicrobials is urgently needed. In this study, by biomimicking cationic antibacterial peptides, we designed and synthesized a series of new membrane-active nonivamide and capsaicin derivatives as peptidomimetic antimicrobials. Through modulating charge/hydrophobicity balance and rationalizing structure-activity relationships of these peptidomimetics, compound 51 was identified as the lead compound. Compound 51 exhibited potent antibacterial activity against both Gram-positive bacteria (MICs = 0.39-0.78 µg/mL) and Gram-negative bacteria (MICs = 1.56-6.25 µg/mL), with low hemolytic activity and low cytotoxicity. Compound 51 displayed a faster bactericidal action through a membrane-disruptive mechanism and avoided bacterial resistance development. Furthermore, compound 51 significantly reduced the microbial burden in a murine model of keratitis infected by Staphylococcus aureus or Pseudomonas aeruginosa. Hence, this design strategy can provide a promising and effective solution to overcome antibiotic resistance.

Design and Synthesis of Phenyl Sulfide-Based Cationic Amphiphiles as Membrane-Targeting Antimicrobial Agents against Gram-Positive Pathogens.

Due to the emergence of antimicrobial resistance and the lack of new antibacterial agents, it has become urgent to discover and develop new antibacterial agents against multidrug-resistant pathogens. Antimicrobial peptides (AMPs) serve as the first line of defense for the host. In this work, we have designed, synthesized, and biologically evaluated a series of phenyl sulfide derivatives by biomimicking the structural features and biological functions of AMPs. Among these derivatives, the most promising compound 17 exhibited potent antibacterial activity against Gram-positive bacteria (minimum inhibitory concentrations = 0.39-1.56 µg/mL), low hemolytic activity (HC50 > 200 µg/mL), and high membrane selectivity. In addition, 17 can rapidly kill Gram-positive bacteria within 0.5 h through membrane-targeting action and avoid antibiotic resistance. More importantly, 17 showed high in vivo efficacy against Staphylococcus aureus in a murine corneal infection model. Therefore, 17 has great potential as a lead compound for the treatment of Gram-positive bacterial infections.

Design, synthesis, and biological evaluation of tetrahydroquinoline amphiphiles as membrane-targeting antimicrobials against pathogenic bacteria and fungi.

The rising prevalence of drug-resistant pathogens is one of the biggest threats to human health. The development of new antibiotics that can overcome drug resistance is in urgent need. Herein, we designed and synthesized a series of amphiphilic tetrahydroquinoline derivatives as small-molecule-based antimicrobial peptidomimetics. Two lead compounds 36 and 52 which contained the tetrahydroquinoline core, hydrophobic alkyl chains (n-nonyl or isoprenyl group), different spacer lengths (n = 4 or 8), and cationic guanidine moiety, showed poor hemolytic activity, low cytotoxicity, and potent broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi. The further biological evaluation revealed that compounds 36 and 52 can kill bacteria and fungi rapidly via membrane-targeting action and avoid drug resistance development. More importantly, compounds 36 and 52 exhibited similarly potent in vivo antimicrobial activities in a murine corneal infection caused by Staphylococcus aureus ATCC29213 or Pseudomonas aeruginosa ATCC9027, as compared to vancomycin or gatifloxacin. These results suggest that compounds 36 and 52 have great potential as new broad-spectrum antimicrobial agents to combat microbial resistance.

Membrane-Active Antibacterial Agents Based on Calix[4]arene Derivatives: Synthesis and Biological Evaluation.

Bacteria have developed increasing resistance to currently used antimicrobial agents. New classes of antimicrobial drugs are urgently required to fight drug-resistant pathogens. Here, we designed and synthesized a series of calix[4]arene derivatives as antibacterial agents by biomimicking the structural properties and biological functions of antibacterial peptides. After introducing cationic hydrophilic moieties and preliminary structural optimization, we obtained a lead compound (16) that exhibited excellent antibacterial activity against Gram-positive bacteria, low toxicity toward mammalian cells and poor hemolytic activity. The antibacterial mechanism studies showed that compound 16 can destroy bacterial cell membrane directly, leading to bacterial death and a low tendency to develop bacterial resistance.

Curcumin prevents As3+-induced carcinogenesis through regulation of GSK3ß/Nrf2.

BACKGROUND: Arsenic (As3+) is a carcinogen with considerable environmental and occupational relevancy. Its mechanism of action and methods of prevention remain to be investigated. Previous studies have demonstrated that ROS is responsible for As3+-induced cell transformation, which is considered as the first stage of As3+ carcinogenesis. The NF-E2 p45-related factor-2 (Nrf2) signaling pathway regulates the cellular antioxidant response, and activation of Nrf2 has recently been shown to limit oxidative damage following exposure to As3+ METHODS AND RESULTS: In this study, molecular docking was used to virtually screen natural antioxidant chemical databases and identify molecules that interact with the ligand-binding site of Keap1 (PDB code 4L7B). The cell-based assays and molecular docking findings revealed that curcumin has the best inhibitory activity against Keap1-4L7B. Co-immunoprecipitation (Co-IP) results indicated that curcumin is a potent Keap1 Kelch domain-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. The increased activation of Nrf2 and its target antioxidant genes by curcumin could significantly decrease As3+-generated ROS. Moreover, curcumin induced autophagy in As3+-treated BEAS-2B via inducing autophagy by the formation of a p62/LC-3 complex and increasing autophagic flux by promoting transcription factor EB (TFEB) and lysosome-associated membrane protein 1 (LAMP1) expression. Knockdown of Nrf2 abolished curcumin-induced autophagy and downregulated ROS. Further studies showed that inhibition of autophagosome and lysosome fusion with bafilomycin a1 (BafA1) could block curcumin and prevented As3+-induced cell transformation. These results demonstrated that curcumin prevents As3+-induced cell transformation by inducing autophagy via the activation of the Nrf2 signaling pathway in BEAS-2B cells. However, overexpression of Keap-1 showed a constitutively high level of Nrf2 in As3+-transformed BEAS-2B cells (AsT) is Keap1-independent regulation. Overexpression of Nrf2 in AsT demonstrated that curcumin increased ROS levels and induced cell apoptosis via the downregulation of Nrf2. Further studies showed that curcumin decreased the Nrf2 level in AsT by activating GSK-3ß to inhibit the activation of PI3K/AKT. Co-IP assay results showed that curcumin promoted the interaction of Nrf2 with the GSK-3ß/ß-TrCP axis and ubiquitin. Moreover, the inhibition of GSK-3ß reversed Nrf2 expression in curcumin-treated AsT, indicating that the decrease in Nrf2 is due to activation of the GSK-3ß/ß-TrCP ubiquitination pathway. Furthermore, in vitro and in vivo results showed that curcumin induced cell apoptosis, and had anti-angiogenesis and anti-tumorigenesis effects as a result of activating the GSK-3ß/ß-TrCP ubiquitination pathway and subsequent decrease in Nrf2. CONCLUSIONS: Taken together, in the first stage, curcumin activated Nrf2, decreased ROS, and induced autophagy in normal cells to prevent As3+-induced cell transformation. In the second stage, curcumin promoted ROS and apoptosis and inhibited angiogenesis via inhibition of constitutive expression of Nrf2 in AsT to prevent tumorigenesis. Our results suggest that antioxidant natural compounds such as curcumin can be evaluated as potential candidates for complementary therapies in the treatment of As3+-induced carcinogenesis.

Development of Amphiphilic Coumarin Derivatives as Membrane-Active Antimicrobial Agents with Potent In Vivo Efficacy against Gram-Positive Pathogenic Bacteria.

Increases in drug-resistant pathogens are becoming a serious detriment to human health. To combat pathogen infections, a new series of amphiphilic coumarin derivatives were designed and synthesized as antimicrobial agents with membrane-targeting action. We herein report a lead compound, 25, that displayed potent antibacterial activity against Gram-positive bacteria, including MRSA. Compound 25 exhibited weak hemolytic activity and low toxicity to mammalian cells and can kill Gram-positive bacteria quickly (within 0.5 h) by directly disrupting the bacterial cell membranes. Additionally, compound 25 demonstrated excellent efficacy in a murine corneal infection caused by Staphylococcus aureus. These results suggest that 25 has great potential to be a potent antimicrobial agent for treating drug-resistant Gram-positive bacterial infections.

Synthesis and biological evaluation of indole-based peptidomimetics as antibacterial agents against Gram-positive bacteria.

The emergence of bacterial multidrug resistance and the lack of new antimicrobial agents urgently demand the discovery and development of novel antibacterials that avoid bacterial resistance. Antimicrobial peptidomimetics represent a promising approach for overcoming antibiotic resistance. Herein we report the synthesis and evaluation of indole-based amphiphilic antimicrobial peptidomimetics, bearing hydrophobic side chains and hydrophilic cationic moieties. Among these derivatives, compound 28 demonstrated potent antimicrobial activity against Gram-positive bacteria, low hemolytic activity and low toxicity towards mammalian cells, as well as good stability in salt conditions. Moreover, compound 28 showed the rapid killing of bacteria via membrane-targeting action without developing bacterial resistance. More importantly, compound 28 displayed high antimicrobial potency against Gram-positive bacteria in a murine model of bacterial keratitis, and was found to be more efficient than vancomycin. Thus, compound 28 had great potential as a promising lead compound for the treatment of Gram-positive bacterial infection.

Rational Design of Dipicolylamine-Containing Carbazole Amphiphiles Combined with Zn2+ as Potent Broad-Spectrum Antibacterial Agents with a Membrane-Disruptive Mechanism.

Antibiotic resistance has become one of the most urgently important problems facing healthcare providers. A novel series of dipicolylamine-containing carbazole amphiphiles with strong Zn2+ chelating ability were synthesized, biomimicking cationic antimicrobial peptides. Effective broad-spectrum 16 combined with 12.5 µg/mL Zn2+ was identified as the most promising antimicrobial candidate. 16 combined with 12.5 µg/mL Zn2+ exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria (MICs = 0.78-3.125 µg/mL), weak hemolytic activity, and low cytotoxicity. Time-kill kinetics and mechanism studies revealed 16 combined with 12.5 µg/mL Zn2+ had rapid bacterial killing properties, as evidenced by disruption of the integrity of bacterial cell membranes, effectively preventing bacterial resistance development. Importantly, 16 combined with 12.5 µg/mL Zn2+ showed excellent in vivo efficacy in a murine keratitis model caused by Staphylococcus aureus ATCC29213 or Pseudomonas aeruginosa ATCC9027. Therefore, 16 combined with 12.5 µg/mL Zn2+ could be a promising candidate for treating bacterial infections.

Design, Synthesis, and Biological Evaluation of Membrane-Active Bakuchiol Derivatives as Effective Broad-Spectrum Antibacterial Agents.

Infections caused by drug-resistant bacteria seriously endanger human health and global public health. Therefore, it is urgent to discover and develop novel antimicrobial agents to combat multidrug-resistant bacteria. In this study, we designed and synthesized a series of new membrane-active bakuchiol derivatives by biomimicking the structure and function of cationic antibacterial peptides. The most promising compound 28 displayed potent antibacterial activity against both Gram-positive bacteria (minimum inhibitory concentration, MIC = 1.56-3.125 µg/mL) and Gram-negative bacteria (MIC = 3.125 µg/mL), very weak hemolytic activity, and low cytotoxicity. Compound 28 had rapid bactericidal properties and avoided bacterial resistance. More importantly, compound 28 showed strong in vivo antibacterial efficacy against Staphylococcus aureus and Pseudomonas aeruginosa in murine corneal infection models. This design strategy is expected to provide an effective solution to the antibiotic crisis.

De Novo Design of Flavonoid-Based Mimetics of Cationic Antimicrobial Peptides: Discovery, Development, and Applications.

The alarming rate at which pathogens are developing resistance to conventional antibiotics represents one of the major global challenges to public health care. The prevalence of multidrug-resistant microorganisms is a major impetus for the discovery and development of new antimicrobials. Nature has, to date, been the source of most of the antibiotics discovered and used, including cationic antimicrobial peptides (CAMPs). CAMPs are key components of the innate immune system that are widely found in humans, animals, plants, and microorganisms and that serve as a first line of defense for the host. The attractive features of CAMPs have led to their recognition as potential new antimicrobials. However, they possess several inherent flaws that limit their clinical application including low stability, poor oral bioavailability, poor in vivo efficacy, and a high production cost. To address these issues, small molecule-based peptidomimetic antimicrobials have been designed to biomimic the structural features and biological function of CAMPs. Plant-derived flavonoids (e.g., xanthones and flavones) are active components in traditional herbal medicines and have been reported to contain a variety of significant pharmacological actions including antibacterial, antiviral, antioxidant, and anticancer activities. Over the past decade, we have developed a new chemical strategy to design, discover, and develop xanthone- or flavone-based peptidomimetics and have designed, synthesized, and biologically evaluated a library of approximately 450 new xanthone or flavone derivatives. The designed, structurally diverse compounds can be generally classified into two subfamilies, namely, peptidic and nonpeptidic amphiphilic xanthone or flavone derivatives. In this Account, we describe our efforts on the design, synthesis, biological property evaluation, and mechanism of action model studies of synthetic mimics of CAMPs. The flavonoid compounds are an important component of these rationally designed mimics because they function as hydrophobic aromatic moieties conjugated with different length lipid moieties, behave like an unnatural hydrophobic residue, and provide a rigid scaffold, with the reduced conformational flexibility more likely to provide an active conformation. The mimics can effectively disrupt the integrity of the bacterial membranes. Our endeavors encompass design principles, chemical synthesis, in vitro screening, structural optimization, extensive structural-activity relationship analysis, and a mechanism of action study through biophysical technologies including NMR spectroscopy techniques and computer dynamics simulations, drug resistance assays, in vivo pharmaceutical kinetics (PK) analyses, and in vivo efficacy evaluations of selected promising compounds against drug-resistant bacteria and fungi. Our major contributions to the discovery and development of flavonoid-based mimics as antimicrobials include effectively addressing several limitations associated with CAMPs and have led to promising compounds with a notable potential for further development as new therapeutic antimicrobial agents for the treatment of drug-resistant bacteria- or fungi-induced infections.

Development of Highly Potent Carbazole Amphiphiles as Membrane-Targeting Antimicrobials for Treating Gram-Positive Bacterial Infections.

The development of new antimicrobial agents capable of curing drug-resistant bacteria-induced infections is becoming a major challenge to the global healthcare system. To develop antimicrobials with new molecular entities, a series of novel carbazole-based compounds were designed and synthesized by biomimicking the structural properties and biological function of antimicrobial peptides. Compound 29 was selected as a lead compound from the structure-activity relationship analyses and biological activity evaluation. Compound 29 showed excellent antimicrobial activity against Gram-positive bacteria (MICs = 0.78-1.56 µg/mL), poor hemolytic activity (HC50 > 200 µg/mL), and low cytotoxicity to mammalian cells. Compound 29 had fast bactericidal properties and effectively prevented bacterial resistance in laboratory simulations. Antibacterial mechanism studies revealed that compound 29 directly destroyed bacterial cell membranes, leading to bacterial deaths. Importantly, compound 29 displayed an excellent efficacy in a murine bacterial keratitis model caused by Staphylococcus aureus ATCC29213.

Design, synthesis, and evaluation of amphiphilic sofalcone derivatives as potent Gram-positive antibacterial agents.

New antimicrobial agents are urgently needed to overcome drug-resistant bacterial infections. Here we describe the design, synthesis and evaluation of a new class of amphiphilic sofalcone compounds as antimicrobial peptidomimetics. The most promising compound 14, bearing two arginine residues, showed poor hemolytic activity, low cytotoxicity, and excellent antimicrobial activity against Gram-positive bacteria, including MRSA. Compound 14, had good stability in various salt conditions, killed bacteria rapidly by directly disrupting bacterial cell membranes and was slow at developing bacterial resistance. Additionally, compound 14 exhibited effective in vivo efficacy in the murine model of bacterial keratitis caused by Staphylococcus aureus ATCC29213. Our studies suggested that compound 14 possessed promising potential to be used as a novel antimicrobial agent to combat drug-resistant Gram-positive bacteria.

In Vitro and in Vivo Evaluation of Membrane-Active Flavone Amphiphiles: Semisynthetic Kaempferol-Derived Antimicrobials against Drug-Resistant Gram-Positive Bacteria.

Because of the rapid increase in bacterial resistance, there is an urgent need for developing new antimicrobial agents to combat multidrug-resistant pathogens. In this study, we designed and synthesized a series of kaempferol derivatives as antimicrobial agents biomimicking the structural properties and biological functions of host defense peptides. After fine-tuning of hydrophobic and cationic hydrophilic moieties linked to the flavone scaffold of kaempferol, we obtained a lead compound (52) that displayed high membrane selectivity (>128), poor hemolytic activity, low cytotoxicity to mammalian cells, and excellent activity against Gram-positive bacteria (minimum inhibitory concentrations = 1.56 µg/mL), including methicillin-resistant Staphylococcus aureus. Compound 52 can kill bacteria quickly by destroying the bacterial membranes and avoid developing bacterial resistance. Moreover, compound 52 exhibited potent in vivo antibacterial activity against S. aureus in a murine corneal infection model. These results indicated that compound 52 had the therapeutic potential as a novel membrane-active antimicrobial to combat Gram-positive bacterial infections.

Potent in vitro and in vivo antimicrobial activity of semisynthetic amphiphilic γ-mangostin derivative LS02 against Gram-positive bacteria with destructive effect on bacterial membrane.

Semisynthetic γ-mangostin derivative LS02 is a novel cationic amphiphilic peptidomimetic antimicrobial agent containing a hydrophobic scaffold and three hydrophilic and positively charged residues of arginine. LS02 showed low in vitro toxicity, potent activities against Gram-positive bacteria including MRSA (MIC = 1.56-6.25 µg/mL) and avoidance of drug resistance. The mode of action studies indicated that LS02 killed bacteria by disrupting bacterial cell membranes. LS02 not only exhibited good water solubility, low hemolytic activity and cell cytotoxicity, but also displayed excellent in vitro and in vivo antibacterial activity, indicating its great potential of being a lead compound as a novel membrane-active antibacterial agent capable of combating bacterial resistance.

Design and synthesis of oligo-lipidated arginyl peptide (OLAP) dimers with enhanced physicochemical activity, peptide stability and their antimicrobial actions against MRSA infections.

Multi-drug resistant pathogens have been of increasing concern today. There is an urgent need for the discovery of more potent antibiotics. Cationic antimicrobial peptides (CAMPs) are known to be effective antimicrobial agents against resistant pathogens. However, poor activity under physiological conditions is one of the major limitations of CAMPS in clinical applications. In this study, a series of oligo-lipidated arginyl peptide OLAP dimers comprised of a saturated fatty acid chain (with m number of carbon units) and p repeating units of arginyl fatty acid chains (with n number of carbon units) were designed and studied for their antimicrobial activities as well as their physico-chemical property in various physiological conditions, such as in human serum albumin and high salt conditions. Our results showed that OLAP-11 exhibits potent antimicrobial activity against Gram-positive bacteria with improved physico-chemical activity in various physiological conditions. OLAP-11 is also less susceptible to human serum and trypsin degradation. The HPLC-MS analysis showed that the lipid-arginine bond is very stable. SYTOX Green assay and scanning electron microscopy both show that the OLAP-11 killed bacteria via inner membrane disruption. In addition, OLAP-11 is inner membrane targeting, making it difficult for bacteria to develop resistance. Overall, the design of the OLAP dimers provides an alternative approach to improve the physicochemical activity, peptide stability of CAMPs with potent inner membrane disruption and low in vitro toxicity to increase their potential for clinical applications in the future.

Antimicrobial activity profiles of Amphiphilic Xanthone derivatives are a function of their molecular Oligomerization.

Currently, membrane-targeting small antimicrobial peptidomimetics (SAP) are important in antibiotic development because bacteria appear to develop resistance to these surface-active compounds less readily. However, the molecular membrane-targeting action of SAPs has received little attention. In this study, we investigated the effect of oligomerization of amphiphilic xanthone, a model SAP, on its antimicrobial properties against both Gram-positive and Gram-negative bacteria. First, oligomer formation by an amphiphilic xanthone, compound 2 (also coded as AM052), was investigated via solution-state nuclear magnetic resonance (NMR) spectroscopy. Then, the effects of oligomerization on membrane disruption were further studied via biophysical approaches. The results showed that the antimicrobial activities of SAPs develop in several stages: oligomer formation in aqueous solution, initial binding of oligomers to the membrane-water interface followed by insertion into the membrane bilayer, aggregation of antimicrobial oligomers in the membrane, and induced membrane leakage. Ultimately, the presence of the oligomers in the bacterial membrane leads to decreased membrane fluidity and bacterial cell death. Interestingly, the early formation of large oligomers leads to stronger membrane disruption and more rapid bacterial killing. However, reduced antimicrobial activities against Gram-negative bacteria were observed for compounds that formed larger oligomers because the LPS layer acts as a barrier to large complexes. Taken together, our results suggest that oligomerization of SAPs has a strong impact on their antimicrobial properties.