The prevalence of drug-resistant bacteria in the clinic has propelled a concerted effort to find new classes of antibiotics that will circumvent current modes of resistance. We have previously described a set of bisamidine antibiotics that contains a core composed of two indoles and a central linker. The first compounds of the series, MBX 1066 and MBX 1090, have potent antibacterial properties against a wide range of Gram-positive and Gram-negative bacteria. We have conducted a systematic exploration of the amidine functionalities, the central linker, and substituents at the indole 3-position to determine the factors involved in potent antibacterial activity. Some of the newly synthesized compounds have even more potent and broad-spectrum activity than MBX 1066 and MBX 1090.
antibiotic; antibacterial; broad-spectrum; indole; Cadogan-Sundberg reaction; Reissert indole synthesis; McMurry reductive homocoupling reaction; amidine; imidazoline; tetrahydropyrimidine
The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.
Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC50] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM2 % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.
Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screening against purified S. aureus (Sa) and B. anthracis (Ba) replicative helicases. Chemical optimization has produced compound 59 with nanomolar potency against the DNA duplex strand unwinding activities of both B. anthracis and S. aureus helicases. Selectivity index (SI = CC50/IC50) values for 59 were greater than 500. Kinetic studies demonstrated that the benzobisthiazole-based bacterial helicase inhibitors act competitively with the DNA substrate. Therefore, benzobisthiazole helicase inhibitors represent a promising new scaffold for evaluation as antibacterial agents.
Optimization; DNA helicase; DNA replication; Inhibitor
The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated ϕE264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.
The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described coumarin-based inhibitors of an underexploited bacterial target, namely, the replicative helicase. Here we report the synthesis and evaluation of optimized coumarin-based inhibitors with 9–18-fold increased potency against S. aureus (Sa) and B. anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC50 values of 3 and 1 µM, respectively, against the DNA duplex strand-unwinding activities of both B. anthracis and S. aureus helicases without affecting the single strand DNA-stimulated ATPase activity. Selectivity index (SI = CC50/MIC) values against S. aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were 20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent antibacterial activity against multiple ciprofloxacin-resistant MRSA strains with MIC values ranging between 0.5–4.2 µg/mL.
The type II secretion (T2S) system in Gram-negative bacteria is comprised of the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, we used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive up-regulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the IPTG concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73,000) were detected as primary hits due to stimulation of luminescence with a z-score ≥5. Direct secretion assays of the 9 most potent hits, representing 5 chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm, but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 – 25 μM. In addition, 7 of the 9 compounds also inhibited the T2S-mediated extracellular secretion of phospholipases C by P. aeruginosa and of protease activity by Burkholderia pseudomallei.
P. aeruginosa; type II secretion; high throughput screening; inhibitors
Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC50s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.
Botulinum neurotoxins (BoNTs), and in particular serotype A, are the most poisonous of known biological substances, and are responsible for the flaccid paralysis of the disease state botulism. Because of the extreme toxicity of these enzymes, BoNTs are considered highest priority biothreat agents. To counter BoNT serotype A (BoNT/A) poisoning, the discovery and development of small molecule, drug-like inhibitors as post-intoxication therapeutic agents has been/is being pursued. Specifically, we are focusing on inhibitors of the BoNT/A light chain (LC) (ie, a metalloprotease) subunit, since such compounds can enter neurons and provide post-intoxication protection of the enzyme target substrate. To aid/facilitate this drug development effort, a pharmacophore for inhibition of the BoNT/A LC subunit was previously developed, and is continually being refined via the incorporation of novel and diverse inhibitor chemotypes. Here, we describe several analogs of a promising therapeutic chemotype in the context of the pharmacophore for BoNT/A LC inhibition. Specifically, we describe: 1) the pharmacophoric ‘fits’ of the analogs and how these ‘fits’ rationalize the in vitro inhibitory potencies of the analogs and 2) pharmacophore refinement via the inclusion of new components from the most potent of the presented analogs.
botulinum; neurotoxin; inhibitor; pharmacophore; metalloprotease; biothreat
NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC50 = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity.
Botulinum neurotoxin A; small molecule inhibitor; metalloprotease; lethal factor; indole; benzothiophene
Antimicrobial susceptibilities of 233 Gram-positive and 180 Gram-negative strains to two novel bis-indoles were evaluated. Both compounds were potent inhibitors of Gram-positive bacteria, with MIC90 values of 0.004 to 0.5 μg/ml. One bis-indole, MBX 1162, exhibited potent activity against all Gram-negative strains, with MIC90 values of 0.12 to 4 μg/ml, even against high-level-resistant pathogens, and compared favorably to all comparator antibiotics. The bis-indole compounds show promise for the treatment of multidrug-resistant clinical pathogens.
The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates up to four protein toxin effectors into human cells, facilitating the establishment and dissemination of infections. To discover inhibitors of this important virulence mechanism, we developed two cellular reporter assays and applied them to a library of 80,000 compounds. The primary screen was based on the dependence of the transcription of T3SS operons on the T3SS-mediated secretion of a negative regulator and consisted of a transcriptional fusion of the Photorhabdus luminescens luxCDABE operon to the P. aeruginosa exoT effector gene. Secondary assays included direct measurements of the T3SS-mediated secretion of a P. aeruginosa ExoS effector-β-lactamase fusion protein as well as the detection of the secretion of native ExoS by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture supernatants. Five inhibitors in three chemical classes were demonstrated to inhibit type III secretion selectively with minimal cytotoxicity and with no effects on bacterial growth or on the type II-mediated secretion of elastase. These inhibitors also block the T3SS-mediated secretion of a YopE effector-β-lactamase fusion protein from an attenuated Yersinia pestis strain. The most promising of the inhibitors is a phenoxyacetamide that also blocks the T3SS-mediated translocation of effectors into mammalian cells in culture. Preliminary studies of structure-activity relationships in this phenoxyacetamide series demonstrated a strict requirement for the R-enantiomer at its stereocenter and indicated tolerance for a variety of substituents on one of its two aromatic rings.
Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC50 ≤ 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ∼5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial vs. mammalian cells (CC50/MIC >16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (Ki = 8 μM) and is rapidly bactericidal at 4× MIC.
S. aureus; B. anthracis; helicase; high throughput screen; aminocoumarin
Staphylococcus epidermidis and Staphylococcus aureus are the leading causative agents of indwelling medical device infections because of their ability to form biofilms on artificial surfaces. Here we describe the antibiofilm activity of a class of small molecules, the aryl rhodanines, which specifically inhibit biofilm formation of S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and E. gallinarum but not the gram-negative species Pseudomonas aeruginosa or Escherichia coli. The aryl rhodanines do not exhibit antibacterial activity against any of the bacterial strains tested and are not cytotoxic against HeLa cells. Preliminary mechanism-of-action studies revealed that the aryl rhodanines specifically inhibit the early stages of biofilm development by preventing attachment of the bacteria to surfaces.
Given the limited number of structural classes of clinically available antimicrobial drugs, the discovery of antibacterials with novel chemical scaffolds is an important strategy in the development of effective therapeutics for both naturally occurring and engineered resistant strains of pathogenic bacteria. In this study, several diarylamidine derivatives were evaluated for their ability to protect macrophages from cell death following infection with Bacillus anthracis, a gram-positive spore-forming bacterium. Four bis-(imidazolinylindole) compounds were identified with potent antibacterial activity as measured by the protection of macrophages and by the inhibition of bacterial growth in vitro. These compounds were effective against a broad range of gram-positive and gram-negative bacterial species, including several antibiotic-resistant strains. Minor structural variations among the four compounds correlated with differences in their effects on bacterial macromolecular synthesis and mechanisms of resistance. In vivo studies revealed protection by two of the compounds of mice lethally infected with B. anthracis, Staphylococcus aureus, or Yersinia pestis. Taken together, these results indicate that the bis-(imidazolinylindole) compounds represent a new chemotype for the development of therapeutics for both gram-positive and gram-negative bacterial species as well as against antibiotic-resistant infections.
We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaBBA displayed distinct enzymatic and kinetic properties. DnaBBA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaBBA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaBBA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaBBA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaBBA is highly efficient in DNA unwinding and its coupling to ATP consumption.
A homogeneous, sensitive, cellular bioluminescent high throughput screen was developed for inhibitors of gyrase and other DNA damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as ¼-MIC with Z’ scores above 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2,000 known bioactive compounds, 13 compounds generated reproducible signals ≥50% of that of the control (ciprofloxacin at ¼-MIC) using bioluminescence readings after 7h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other three are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis, but by a variety of different DNA damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA damaging agents, which may lead to potent new anti-bacterials.
P. aeruginosa; gyrase; high throughput screen; luciferase
Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles (“thiopyridines”), inhibited both purified ENR (50% inhibitory concentration [IC50] = 3 to 25 μM) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 μg/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [14C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift). Synthesis of a variety of compounds in this chemical series revealed a correlation between IC50 and MIC, and the results provided initial structure-activity relationships. Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development.
Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outocme of H. pylori-related disease.