As previously reported, BAS00127538 is potently active against Gram-positive species.16 In particular, BAS00127538 showed activity against (MIC 0.5), irrespective of vancomycin- or methicillin resistance. (MRSA), ATCC (vancomycin intermediate-resistant NTS (vancomycin intermediate-resistant cancels out as we only considered the relative free energies NCTC 8325 was measured as previously explained.33 To inhibit efflux, NCTC 8325 was produced in the presence of 20 g/mL of reserpine. Each data point is the average of three replicates, and the error bars represent standard deviation. Chemical synthesis 1-Methyl-2,4-diphenyl-6-((1(atmospheric-pressure chemical ionization) 443.2 M+. 1-Isopropyl-2,4-diphenyl-6-((1(Table 1). We next compared the antibacterial activity of BAS00127538, ASN10791182, 4400-0093, LRP8 antibody and 51633428 against an extended panel of bacterial species (Table S2). As previously reported, BAS00127538 is usually potently MF63 active against Gram-positive species.16 In particular, BAS00127538 showed activity against (MIC 0.5), irrespective of vancomycin- or methicillin resistance. BAS00127538 was also active against the Gram-negative bacteria and and (Table S2). Compounds ASN10791182, 4400-0093, and 56133428 were tested further for cytotoxicity and their ability to bind to Lipid II. Compounds ASN10791182 and 4400-0093 showed a 30-fold and 70-fold reduction, respectively, in Lipid II-binding affinity compared to BAS00127538, whereas 56133428 and BAS00127537 Lipid II binding was reduced ~15-fold (Table 1). Reduction in Lipid II-binding affinities coincided with a reduction in cytotoxicity (approximately fivefold for ASN10791182, greater than tenfold for 4400-0093, approximately twofold for 56133428) as well as antibacterial activity (32-fold for ASN10791182 and 4400-0093, 16-fold for 56133428) compared to BAS00127538 (Table 1). None of the other compounds showed antibacterial activity (Table S1). Of these compounds, only Z56760026 and BAS00127537 bound Lipid II ((USA300) MRSAMIC (g/mL)0.516816Cytotoxicity (J774)CC50% (M)18.79233.2 128 Open in a separate window Notes: MIC was determined by microbroth dilution assay. Binding to immobilized 3-Lipid II was analyzed by SPR. CC50% equals compound concentration resulting in 50% J774 macrophage cell survival measured by MTT assay following incubation for 6 hours. Abbreviations: SPR, surface plasmon resonance; MIC, MF63 minimal inhibitory concentration; MRSA, methicillin-resistant (h mg/mL)26.9as computed by Equation 1. Abbreviations: LIE, linear conversation energy; MD, molecular dynamics. Subsequently, all five compounds, SF-5-219, SF-5-330, SF-5-331, SF-5-332, and SF-5-334, were synthesized according to Figure 4 to challenge the modeling and to evaluate the compounds experimentally. First, we examined the antibacterial activities, Lipid II binding, and cytotoxicity of the BAS00127538 derivatives (Table 4). The modeling results were generally predictive of the relative antibacterial activities of the pyridinium analogs (Table 4), with the exception of the isobutyl analog, a result that may indicate an alternative binding mode for the compound. All analogs were shown to bind to Lipid II in the surface plasmon resonance measurements,16 with more variability in the MICs. Based on their broad-range antibacterial activity, SF-5-330 and SF-5-331 were selected to determine their mechanism of action (Physique 5). BAS00127538 most potently inhibited cell wall synthesis (IC50 of 0.19 g/mL vs 0.42 g/mL, 0.39 g/mL, and 0.62 g/mL for DNA, protein, or lipid synthesis, respectively) in accordance with our previous findings.16 SF-5-330 and SF-5-331 inhibited all pathways at low concentrations. Open in a separate window Physique 4 Synthesis of pyridinium analogs of BAS00127538. Open in a separate windows Physique 5 The effect of BAS00127538 and analogs around the macromolecular pathways for DNA, cell wall, protein, and lipid synthesis. Notes: The dose-dependent activity of the following compounds on each pathway was measured: (A) BAS00127538 (MIC 0.5 g/mL), (B) SF-5-330 (MIC 1 g/mL), and (C) SF-5-331 (MIC 1 g/mL). Abbreviation: MIC, minimal inhibitory concentration. Table 4 Functional analysis of BAS00127538 and derivatives 1094 (MRSA)0.5111116HFH-30123 (MRSA)0.5111116EF1509 (VRE)0.160.50.50.518F118 (VRE)0.16552832NR-15410 (KPC)83210163232NR-15411 (KPC)163225.4323232BAA-16052.588820.158736832ATCC 19606488820.158736832″type”:”entrez-nucleotide”,”attrs”:”text”:”X13273″,”term_id”:”7312″,”term_text”:”X13273″X132736432483232ATCC 2785364328323232ATCC 13047323216323232ATCC 1304816328323232CC50 vs HeLa (g/mL)b0.560.250.330.260.82.25Lipid II binding (can be achieved at 1 mg/kg. In summary, these studies focused on the potentially active scaffold of BAS00127538 pointed out the functional importance of the positions of the phenyl groups, the positively charged pyrylium/pyridinium, and hydrophobicity of the indole side chain in the substitution pattern. Optimization at these positions may lead to the development of small-molecule antibiotic targeting Lipid II with broad-spectrum activity. Supplementary materials Table S1 Structure and functional analysis of BAS00127538 analogs recognized by similarity search killing(VISA)7006990.5161680.58are initial and have not been published by any journal. Some of the data were offered at the 2014 Interscience Conference of Antimicrobial Brokers and Chemotherapy getting MF63 together with, Washington, DC, USA as a poster. The accompanying poster abstract can be found at http://www.icaaconline.com/php/icaac2014abstracts/data/papers/2014/F-978.htm. MF63 A full copy of the poster can be provided.