Discovery of Novel Antibiotics as Covalent Inhibitors of Fatty Acid Synthesis.

The steady increase in the prevalence of multidrug-resistant Staphylococcus aureus has made the search for novel antibiotics to combat this clinically important pathogen an urgent matter. In an effort to discover antibacterials with new chemical structures and mechanisms, we performed a growth inhibition screen of a synthetic library against S. aureus and discovered a promising scaffold with a 1,3,5-oxadiazin-2-one core. These compounds are potent against both methicillin-sensitive and methicillin-resistant S. aureus strains. Isolation of compound-resistant strains followed by whole genome sequencing revealed its cellular target as FabH, a key enzyme in bacterial fatty acid synthesis. Detailed mechanism of action studies suggested the compounds inhibit FabH activity by covalently modifying its active site cysteine residue with high selectivity. A crystal structure of FabH protein modified by a selected compound Oxa1 further confirmed covalency and suggested a possible mechanism for reaction. Moreover, the structural snapshot provided an explanation for compound selectivity. On the basis of the structure, we designed and synthesized Oxa1 derivatives and evaluated their antibacterial activity. The structure-activity relationship supports the hypothesis that noncovalent recognition between compounds and FabH is critical for the activity of these covalent inhibitors. We believe further optimization of the current scaffold could lead to an antibacterial with potential to treat drug-resistant bacteria in the clinic.

Development and Optimization of a Higher-Throughput Bacterial Compound Accumulation Assay.

The Gram-negative bacterial permeability barrier, coupled with efflux, raises formidable challenges to antibiotic drug discovery. The absence of efficient assays to determine compound penetration into the cell and impact of efflux makes the process resource-intensive, small-scale, and lacking much success. Here, we present BacPK: a label-free, solid phase extraction-mass spectrometry (SPE-MS)-based assay that measures total cellular compound accumulation in Escherichia coli. The BacPK assay is a 96-well accumulation assay that takes advantage of 9 s/sample SPE-MS throughput. This enables the analysis of each compound in a four-point dose-response in isogenic strain pairs along with a no-cell control and 16-point external standard curve, all in triplicate. To validate the assay, differences in accumulation were examined for tetracycline (Tet) and two analogs, confirming that close analogs can differ greatly in accumulation. Tet cellular accumulation was also compared for isogenic strains exhibiting Tet resistance due to the expression of an efflux pump (TetA) or ribosomal protection protein (TetM), confirming only TetA affected cellular Tet accumulation. Finally, using a diverse set of antibacterial compounds, we confirmed the assay's ability to quantify differences in accumulation for isogenic strain pairs with efflux or permeability alterations that are consistent with differences in susceptibility seen for the compounds.

Subinhibitory concentrations of LFF571 reduce toxin production by Clostridium difficile.

LFF571 is a novel semisynthetic thiopeptide antibacterial that is undergoing investigation for safety and efficacy in patients with moderate Clostridium difficile infections. LFF571 inhibits bacterial protein synthesis by interacting with elongation factor Tu (EF-Tu) and interrupting complex formation between EF-Tu and aminoacyl-tRNA. Given this mechanism of action, we hypothesized that concentrations of LFF571 below those necessary to inhibit bacterial growth would reduce steady-state toxin levels in C. difficile cultures. We investigated C. difficile growth and toxin A and B levels in the presence of LFF571, fidaxomicin, vancomycin, and metronidazole. LFF571 led to strain-dependent effects on toxin production, including decreased toxin levels after treatment with subinhibitory concentrations, and more rapid declines in toxin production than in inhibition of colony formation. Fidaxomicin, which is an RNA synthesis inhibitor, conferred a similar pattern to LFF571 with respect to toxin levels versus viable cell counts. The incubation of two toxigenic C. difficile strains with subinhibitory concentrations of vancomycin, a cell wall synthesis inhibitor, increased toxin levels in the supernatant over those of untreated cultures. A similar phenomenon was observed with one metronidazole-treated strain of C. difficile. These studies indicate that LFF571 and fidaxomicin generally result in decreased C. difficile toxin levels in culture supernatants, whereas treatment of some strains with vancomycin or metronidazole had the potential to increase toxin levels. Although the relevance of these findings remains to be studied in patients, reducing toxin levels with sub-growth-inhibitory concentrations of an antibiotic is hypothesized to be beneficial in alleviating symptoms.

In vitro selection, via serial passage, of Clostridium difficile mutants with reduced susceptibility to fidaxomicin or vancomycin.

OBJECTIVES: Current treatments for Clostridium difficile infection include vancomycin, metronidazole and fidaxomicin. LFF571 is an experimental agent undergoing evaluation in humans for the treatment of moderate C. difficile infection. Reduced susceptibility of C. difficile to fidaxomicin or LFF571 in vitro can be mediated by single point mutations in genes encoding the targets, whereas the mechanism(s) mediating reduced susceptibility to vancomycin in vitro remains elusive. To further characterize mechanisms reducing susceptibility of C. difficile to vancomycin, fidaxomicin or LFF571 in vitro, selections via serial passage at low cell density were performed, followed by whole-genome sequencing. METHODS: C. difficile strain ATCC 43255 and three clinical isolates were subjected to 10 passages on medium containing a range of concentrations of fidaxomicin, LFF571 or vancomycin. Genomic DNA from isolates with reduced susceptibility was sequenced using Illumina Whole Genome Sequencing. RESULTS: Clones exhibiting decreased susceptibility to fidaxomicin harboured mutations in rpoB and CD22120 (marR homologue). Clones exhibiting decreased susceptibility to vancomycin harboured mutations in rpoC and also in CD2725, CD3659 and sdaB, which encode a putative N-acetylglucosamine transferase, exonuclease and l-serine deaminase, respectively. All mutations resulted in non-synonymous substitutions. No clones with reduced susceptibility to LFF571 were selected in this study. CONCLUSIONS: Reduced susceptibility to fidaxomicin and vancomycin was associated with mutations mediating target modifications (RNA polymerase and cell wall, respectively), as well as with mutations that may contribute to reduced susceptibility via other mechanisms. The MIC of LFF571 was unaffected for those mutants with reduced susceptibility to fidaxomicin or vancomycin.

Fatty acid biosynthesis in Pseudomonas aeruginosa is initiated by the FabY class of ß-ketoacyl acyl carrier protein synthases.

The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of ß-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the ß-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of ß-ketoacyl synthase KASI/II domain condensation enzymes.

Discovery of LFF571: an investigational agent for Clostridium difficile infection.

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.

Antibacterial optimization of 4-aminothiazolyl analogues of the natural product GE2270 A: identification of the cycloalkylcarboxylic acids.

4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for their activity against Gram positive bacterial infections. Optimization efforts focused on improving the physicochemical properties (e.g., aqueous solubility and chemical stability) of the 4-aminothiazolyl natural product template while improving the in vitro and in vivo antibacterial activity. Structure-activity relationships were defined, and the solubility and efficacy profiles were improved over those of previous analogues and 1. These studies identified novel, potent, soluble, and efficacious elongation factor-Tu inhibitors, which bear cycloalkylcarboxylic acid side chains, and culminated in the selection of development candidates amide 48 and urethane 58.

Benzothioxalone derivatives as novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferases (MurA and MurZ).

OBJECTIVES: We sought to identify and characterize new inhibitors of MurA and MurZ, which are enzymes involved in the early stages of bacterial peptidoglycan synthesis. METHODS: A library of ∼650 000 compounds was screened for inhibitors of Escherichia coli MurA in an endpoint assay measuring release of inorganic phosphate from phosphoenolpyruvate. Hits were validated by determining the concentrations required for 50% inhibition (IC(50)) of MurA from E. coli and MurA/MurZ from Staphylococcus aureus. The mode of action of selected inhibitors was explored by examining the reversibility of MurA inhibition, the binding of a radiolabelled inhibitor to MurA proteins and through docking studies. Inhibitors were further characterized by determining their antibacterial activity against E. coli and S. aureus. RESULTS: Benzothioxalone derivatives were identified that inhibited MurA from E. coli and MurA/MurZ from S. aureus with IC(50) values between 0.25 and 51 µM. Several inhibitors also exhibited activity against S. aureus with MICs in the range 4-128 mg/L. Inhibition of MurA was irreversible and a radiolabelled inhibitor from this compound class displayed stoichiometric binding to the enzyme, which was displaced by dithiothreitol. Binding was undetectable with a C115D mutant MurA protein. CONCLUSIONS: The results suggest a mode of action for the benzothioxalones that involves the formation of a disulfide bond with MurA/MurZ, via attack from an active site cysteine on the thioxalone ring carbonyl group, followed by ring opening to yield an S-acylated protein. The proposed covalent mode of action may prove useful in the design of new antibacterial agents.

Discovery of human antibodies against the C5aR target using phage display technology.

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.

Discovery of high-affinity peptide binders to BLyS by phage display.

B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.