Optimization of a novel potent and selective bacterial DNA helicase inhibitor scaffold from a high throughput screening hit.

Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screening against purified Staphylococcus aureus (Sa) and Bacillus 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.

Coumarin-based inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicase: chemical optimization, biological evaluation, and antibacterial activities.

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 Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC(50) 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 = CC(50)/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 and 4.2 µg/mL.

Antimicrobials targeted to the replication-specific DNA polymerases of gram-positive bacteria: target potential of dnaE.

DNA polymerases pol IIIC and dnaE [i.e. pol IIIE] are essential for replicative DNA synthesis in low G:C Gram-positive eubacteria. Therefore, they have strong potential as targets for development of Gram-positive-selective antibacterial agents. This work has sought to extend to dnaE the recent discovery of antimicrobial agents based on pol IIIC-specific dGTP analogs. Compound 324C, a member of the same dGTP analog family, was found to be a potent and selective inhibitor of isolated dnaE in vitro. Surprisingly, 324C had no inhibitory effect in either intact Bacillus subtilis cells or in permeabilized cell preparations used to assess replicative DNA synthesis directly. It is proposed that the failure of 324C in the intact cell is a consequence of two major factors: (i) its template-dependent base pairing mechanism, and (ii) a specific subordinate role which dnaE apparently plays to pol IIIC. To generate an effective dnaE-selective inhibitor of replicative DNA synthesis in Gram-positive bacteria, it will likely be necessary to develop a molecule that attacks the enzyme's active site directly, without binding to template DNA.

7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity.

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N(2)-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N(2)-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram- bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10µg/ml), and were inactive against the Gram- organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.

Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases.

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 (IC(50)25 microM). 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 approximately 5 microg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC(50)/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 (K(i)=8 microM) and is rapidly bactericidal at 4 x MIC.

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action.

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 (DnaB(BA)) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB(BA) displayed distinct enzymatic and kinetic properties. DnaB(BA) 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 DnaB(BA) was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB(BA) demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB(BA) has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB(BA) is highly efficient in DNA unwinding and its coupling to ATP consumption.

Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound.

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ "hybrids" offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, "251D," were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of Staphylococcus aureus resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens.

Hybrid antibacterials. DNA polymerase-topoisomerase inhibitors.

Novel Gram-positive (Gram+) antibacterial compounds consisting of a DNA polymerase IIIC (pol IIIC) inhibitor covalently connected to a topoisomerase/gyrase inhibitor are described. Specifically, 3-substituted 6-(3-ethyl-4-methylanilino)uracils (EMAUs) in which the 3-substituent is a fluoroquinolone moiety (FQ) connected by various linkers were synthesized. The resulting "AU-FQ" hybrid compounds were significantly more potent than the parent EMAU compounds as inhibitors of pol IIIC and were up to 64-fold more potent as antibacterials in vitro against Gram+ bacteria. The hybrids inhibited the FQ targets, topoisomerase IV and gyrase, with potencies similar to norfloxacin but 10-fold lower than newer agents, for example, ciprofloxacin and sparfloxacin. Representative hybrids protected mice from lethal Staphylococcus aureus infection after intravenous dosing, and one compound showed protective effect against several antibiotic-sensitive and -resistant Gram+ infections in mice. The AU-FQ hybrids are a promising new family of antibacterials for treatment of antibiotic-resistant Gram+ infections.

Active site directed inhibitors of replication-specific bacterial DNA polymerases.

7-Substituted-N(2)-(3,4-dichlorobenzyl)guanines potently and competitively inhibit DNA polymerases IIIC and IIIE from Gram(+) bacteria. Certain derivatives are also competitive inhibitors of DNA polymerase IIIE from Gram(-) bacteria.

DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA, gyrB and parC, parE genes.

Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.

DNA polymerase III of Enterococcus faecalis: expression and characterization of recombinant enzymes encoded by the polC and dnaE genes.

Enterococcus faecalis (Ef) dnaE and polC, the respective genes encoding the DNA replication-specific DNA polymerase III E and DNA polymerase III C, were cloned and engineered for expression in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Each gene expressed a catalytically active DNA polymerase of the expected molecular weight. The recombinant polymerases were purified and each was characterized with respect to catalytic properties, inhibitor sensitivity, and recognition by specific antibody raised against the corresponding DNA polymerase III of the model Gram-positive (Gr(+)) organism, Bacillus subtilis (Bs). In conclusion, the properties of each Enterococcus polymerase enzymes were similar to those of the respective B. subtilis enzymes.

DNA polymerases of low-GC gram-positive eubacteria: identification of the replication-specific enzyme encoded by dnaE.

dnaE, the gene encoding one of the two replication-specific DNA polymerases (Pols) of low-GC-content gram-positive bacteria (E. Dervyn et al., Science 294:1716-1719, 2001; R. Inoue et al., Mol. Genet. Genomics 266:564-571, 2001), was cloned from Bacillus subtilis, a model low-GC gram-positive organism. The gene was overexpressed in Escherichia coli. The purified recombinant product displayed inhibitor responses and physical, catalytic, and antigenic properties indistinguishable from those of the low-GC gram-positive-organism-specific enzyme previously named DNA Pol II after the polB-encoded DNA Pol II of E. coli. Whereas a polB-like gene is absent from low-GC gram-positive genomes and whereas the low-GC gram-positive DNA Pol II strongly conserves a dnaE-like, Pol III primary structure, it is proposed that it be renamed DNA polymerase III E (Pol III E) to accurately reflect its replicative function and its origin from dnaE. It is also proposed that DNA Pol III, the other replication-specific Pol of low-GC gram-positive organisms, be renamed DNA polymerase III C (Pol III C) to denote its origin from polC. By this revised nomenclature, the DNA Pols that are expressed constitutively in low-GC gram-positive bacteria would include DNA Pol I, the dispensable repair enzyme encoded by polA, and the two essential, replication-specific enzymes Pol III C and Pol III E, encoded, respectively, by polC and dnaE.