Structural and Antibacterial Characterization of a New Benzamide FtsZ Inhibitor with Superior Bactericidal Activity and In Vivo Efficacy Against Multidrug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) bacterial pathogen of acute clinical significance. Resistance to current standard-of-care antibiotics, such as vancomycin and linezolid, among nosocomial and community-acquired MRSA clinical isolates is on the rise. This threat to global public health highlights the need to develop new antibiotics for the treatment of MRSA infections. Here, we describe a new benzamide FtsZ inhibitor (TXH9179) with superior antistaphylococcal activity relative to earlier-generation benzamides like PC190723 and TXA707. TXH9179 was found to be 4-fold more potent than TXA707 against a library of 55 methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, including MRSA isolates resistant to vancomycin and linezolid. TXH9179 was also associated with a lower frequency of resistance relative to TXA707 in all but one of the MSSA and MRSA isolates examined, with the observed resistance being due to mutations in the ftsZ gene. TXH9179 induced changes in MRSA cell morphology, cell division, and FtsZ localization are fully consistent with its actions as a FtsZ inhibitor. Crystallographic studies demonstrate the direct interaction of TXH9179 with S. aureus FtsZ (SaFtsZ), while delineating the key molecular contacts that drive complex formation. TXH9179 was not associated with any mammalian cytotoxicity, even at a concentration 10-fold greater than that producing antistaphylococcal activity. In serum, the carboxamide prodrug of TXH9179 (TXH1033) is rapidly hydrolyzed to TXH9179 by serum acetylcholinesterases. Significantly, both intravenously and orally administered TXH1033 exhibited enhanced in vivo efficacy relative to the carboxamide prodrug of TXA707 (TXA709) in treating a mouse model of systemic (peritonitis) MRSA infection. Viewed as a whole, our results highlight TXH9179 as a promising new benzamide FtsZ inhibitor worthy of further development.

TXH11106: A Third-Generation MreB Inhibitor with Enhanced Activity against a Broad Range of Gram-Negative Bacterial Pathogens.

The emergence of multi-drug-resistant Gram-negative pathogens highlights an urgent clinical need to explore and develop new antibiotics with novel antibacterial targets. MreB is a promising antibacterial target that functions as an essential elongasome protein in most Gram-negative bacterial rods. Here, we describe a third-generation MreB inhibitor (TXH11106) with enhanced bactericidal activity versus the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa compared to the first- and second-generation compounds A22 and CBR-4830, respectively. Large inocula of these four pathogens are associated with a low frequency of resistance (FOR) to TXH11106. The enhanced bactericidal activity of TXH11106 relative to A22 and CBR-4830 correlates with a correspondingly enhanced capacity to inhibit E. coli MreB ATPase activity via a noncompetitive mechanism. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa provide further evidence supporting MreB as the bactericidal target of the compound. Taken together, our results highlight the potential of TXH11106 as an MreB inhibitor with activity against a broad spectrum of Gram-negative bacterial pathogens of acute clinical importance.

Novel MreB inhibitors with antibacterial activity against Gram (-) bacteria.

MreB is a cytoskeleton protein present in rod-shaped bacteria that is both essential for bacterial cell division and highly conserved. Because most Gram (-) bacteria require MreB for cell division, chromosome segregation, cell wall morphogenesis, and cell polarity, it is an attractive target for antibacterial drug discovery. As MreB modulation is not associated with the activity of antibiotics in clinical use, acquired resistance to MreB inhibitors is also unlikely. Compounds, such as A22 and CBR-4830, are known to disrupt MreB function by inhibition of ATPase activity. However, the toxicity of these compounds has hindered efforts to assess the in vivo efficacy of these MreB inhibitors. The present study further examines the structure-activity of analogs related to CBR-4830 as it relates to relative antibiotic activity and improved drug properties. These data reveal that certain analogs have enhanced antibiotic activity. In addition, we evaluated several representative analogs (9, 10, 14, 26, and 31) for their abilities to target purified E. coli MreB (EcMreB) and inhibit its ATPase activity. Except for 14, all these analogs were more potent than CBR-4830 as inhibitors of the ATPase activity of EcMreB with corresponding IC50 values ranging from 6 ± 2 to 29 ± 9 µM.

Aryl and Arylalkyl Substituted 3-Hydroxypyridin-2(1H)-ones: Synthesis and Evaluation as Inhibitors of Influenza†A Endonuclease.

Seasonal influenza infections are associated with an estimated 250-500 000 deaths annually. Resistance to the antiviral M2 ion-channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap-snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap-snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X-ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating agent at the active site. We have reported the structure-activity relationships for 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5- and 6-positions of 3-hydroxypyridin-2(1H)-ones. Herein we report the structure-activity relationships associated with various para-substituted 5-phenyl derivatives of 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p-fluorophenyl substituent on their activity as endonuclease inhibitors.

Phenyl substituted 4-hydroxypyridazin-3(2H)-ones and 5-hydroxypyrimidin-4(3H)-ones: inhibitors of influenza A endonuclease.

Seasonal and pandemic influenza outbreaks remain a major human health problem. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is attractive for the development of new agents for the treatment of influenza infection. Our earlier studies identified a series of 5- and 6-phenyl substituted 3-hydroxypyridin-2(1H)-ones that were effective inhibitors of influenza endonuclease. These agents identified as bimetal chelating ligands binding to the active site of the enzyme. In the present study, several aza analogues of these phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity. In contrast to the 4-aza analogue of 6-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one, the 5-aza analogue (5-hydroxy-2-(4-fluorophenyl)pyrimidin-4(3H)-one) did exhibit significant activity as an endonuclease inhibitor. The 6-aza analogue of 5-(4-fluorophenyl)-3-hydroxypyridin-2(1H)-one (6-(4-fluorophenyl)-4-hydroxypyridazin-3(2H)-one) also retained modest activity as an inhibitor. Several varied 6-phenyl-4-hydroxypyridazin-3(2H)-ones and 2-phenyl-5-hydroxypyrimidin-4(3H)-ones were synthesized and evaluated as endonuclease inhibitors. The SAR observed for these aza analogues are consistent with those previously observed with various phenyl substituted 3-hydroxypyridin-2(1H)-ones.

3-Hydroxyquinolin-2(1H)-ones As Inhibitors of Influenza A Endonuclease.

Several 3-hydroxyquinolin-2(1H)-ones derivatives were synthesized and evaluated as inhibitors of 2009 pandemic H1N1 influenza A endonuclease. All five of the monobrominated 3-hydroxyquinolin(1H)-2-ones derivatives were synthesized. Suzuki-coupling of p-fluorophenylboronic acid with each of these brominated derivatives provided the respective p-fluorophenyl 3-hydroxyquinolin(1H)-2-ones. In addition to 3-hydroxyquinolin-2(1H)-one, its 4-methyl, 4-phenyl, 4-methyl-7-(p-fluorophenyl), and 4-phenyl-7-(p-fluorophenyl) derivatives were also synthesized. Comparative studies on their relative activity revealed that both 6- and 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one are among the more potent inhibitors of H1N1 influenza A endonuclease. An X-ray crystal structure of 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one complexed to the influenza endonuclease revealed that this molecule chelates to two metal ions at the active site of the enzyme.