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In the effort to combat antibiotic resistance, inhibitors of the essential bacterial protein FtsZ have emerged as a promising new class of compounds with clinical potential. One such FtsZ inhibitor (TXA707) is associated with potent activity against clinical isolates of methicillin-resistant Staphylococcus aureus(MRSA) that are resistant to current standard-of-care antibiotics. However, mutations in S. aureusFtsZ (SaFtsZ) that confer resistance to TXA707 have been observed, with mutations in the Gly196 and Gly193 residues being among the most prevalent. Here, we describe structural studies of an FtsZ inhibitor, TXA6101, which retains activity against MRSA isolates that express either G196S or G193D mutant FtsZ. We present the crystal structures of TXA6101 in complex with both wild-type SaFtsZ and G196S mutant SaFtsZ, as well the crystal structure of TXA707 in complex with wild-type SaFtsZ. Comparison of the three structures reveals a molecular basis for the differential targeting abilities of TXA6101 and TXA707. The greater structural flexibility of TXA6101 relative to TXA707 enables TXA6101 to avoid steric clashes with Ser196 and Asp193. Our structures also demonstrate that the binding of TXA6101 induces previously unobserved conformational rearrangements of SaFtsZ residues in the binding pocket. In aggregate, the structures reported in this work reveal key factors for overcoming drug resistance mutations in SaFtsZ and offer a structural basis for the design of FtsZ inhibitors with enhanced antibacterial potency and reduced susceptibility to mutational resistance.

ABSTRACTCombination therapy of bacterial infections with synergistic drug partners offers distinct advantages over monotherapy. Among these advantages are (i) a reduction of the drug dose required for efficacy, (ii) a reduced potential for drug-induced toxicity, and (iii) a reduced potential for the emergence of resistance. Here, we describe the synergistic actions of the third-generation oral cephalosporin cefdinir and TXA709, a new, FtsZ-targeting prodrug that we have developed with improved pharmacokinetics and enhanced in vivoefficacy against methicillin-resistant Staphylococcus aureus(MRSA) relative to earlier agents. We show that the active product of TXA709 (TXA707) acts synergistically with cefdinir in vitroagainst clinical isolates of MRSA, vancomycin-intermediate S. aureus(VISA), vancomycin-resistant S. aureus(VRSA), and linezolid-resistant S. aureus(LRSA). In addition, relative to TXA707 alone, the combination of TXA707 and cefdinir significantly reduces or eliminates the detectable emergence of resistance. We also demonstrate synergy in vivowith oral administration of the prodrug TXA709 and cefdinir in mouse models of both systemic and tissue (thigh) infections with MRSA. This synergy reduces the dose of TXA709 required for efficacy 3-fold. Viewed as a whole, our results highlight the potential of TXA709 and cefdinir as a promising combination for the treatment of drug-resistant staphylococcal infections.

ABSTRACTAntibiotics with novel mechanisms of action are urgently needed. Processes of cellular division are attractive targets for new drug development. FtsZ, an integral protein involved in cell cytokinesis, is a representative example. In the present study, the pharmacodynamic (PD) activity of an FtsZ inhibitor, TXA-709, and its active metabolite, TXA-707, was evaluated in the neutropenic murine thigh infection model against 5 Staphylococcus aureusisolates, including both methicillin-susceptible and methicillin-resistant isolates. The pharmacokinetics (PK) of the TXA-707 active metabolite were examined after oral administration of the TXA-709 prodrug at 10, 40, and 160 mg/kg of body weight. The half-life ranged from 3.2 to 4.4 h, and the area under the concentration-time curve (AUC) and maximum concentration of drug in serum (Cmax) were relatively linear over the doses studied. All organisms exhibited an MIC of 1 mg/liter. Dose fractionation demonstrated the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) to be the PD index most closely linked to efficacy (R2= 0.72). Dose-dependent activity was demonstrated against all 5 isolates, and the methicillin-resistance phenotype did not alter the pharmacokinetic/pharmacodynamic (PK/PD) targets. Net stasis was achieved against all isolates and a 1-log10kill level against 4 isolates. PD targets included total drug 24-h AUC/MIC values of 122 for net stasis and 243 for 1-log10killing. TXA-709 and TXA-707 are a promising novel antibacterial class and compound for S. aureusinfections. These results should prove useful for design of clinical dosing regimen trials.

ABSTRACTThe clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivoagainst both methicillin-sensitive Staphylococcus aureus(MSSA) and methicillin-resistant S. aureus(MRSA). Despite being efficacious in vivo, TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF3functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivoantistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureusstrains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PANactive site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PANin the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.

ABSTRACTThe bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide PC190723 was among the first FtsZ-targeting compounds to exhibit in vivoefficacy in a murine infection model system. Despite its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We describe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivoefficacy. The N-Mannich base derivative (TXY436) is ∼100-fold more soluble than PC190723 in an acidic aqueous vehicle (10 mM citrate, pH 2.6) suitable for oral in vivoadministration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to PC190723 with a conversion half-life of 18.2 ± 1.6 min. Pharmacokinetic analysis following intravenous administration of TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respectively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureusor methicillin-resistant S. aureus, we show that TXY436 is efficacious in vivoupon oral administration. In contrast, the oral administration of PC190723 was not efficacious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentrations at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.

The kinetics of base hydrolysis of cis-[(salicylato)(RNH2) (en)2COIII]2+ (R = H, Me or Et) have been studied in MeOH-H2O medium at 35.0°C, I = 0.1 (ClO4-) and 0.01 ? [OH-]Tmol dm-3 ? 0.10. The phenoxide species [(RNH2)(en)2CoO2C6H4O]+, underwent both OH--independent (SNICB) and OH--catalysed (SN1CB) hydrolysis with Kobs=k1+k2[OH-]. Both k1 and k2 are appreciably enhanced by the medium for MeNH2 and EtNH2 complexes. In contrast, for the NH3 complex, k1 decreases with increasing MeOH content and k2 was non-existent beyond 60% (v/v) MeOH. These rate variations illustrate the importance of the solute-solvent interaction, presumably partly of hydrophobic origin, and solvent structure in existing stabilizing/destabilizing effects on the initial and transition states.

The base hydrolysis of (αβS)(p-hydroxybenzoato)-(tetraethylenepentamine)cobalt(III) has been investigated in aqueous–organic solvent media using i-PrOH, t-BuOH and dimethyl sulfoxide (DMSO) as cosolvents at 20.0 ≤ T (°C) ≤ 40.0 (I=0.02 mol dm -3) with 80% (v/v) of cosolvents. Only the base-catalysed path (kobs=kOH[OH-]) is observed. The relative second order rate constant kOHos/kOHowat I=0 increases nonlinearly with increasing mol fraction (xO.S.) of the cosolvents, the rate acceleration in alcoholic cosolvents being greater than in DMSO. The destabilization of -OH in mixed solvent media alone does not explain the observed rate acceleration. The solvent composition dependence, log kOHos= log kOHow+ ∑aixosi[i=1,2,kOH0denotes kOHat I=0 in mixed solvent(s) and water (w)] indicates specific solute–solvent interactions. The values of the relative transfer free-energy data [ΔTG(t.s.) - ΔTGo(i.s.)](s←w)(25 °C)(G′), where t.s. and i.s. denote the transition state and initial state of the substrates respectively, are positive for all substrates at all compositions, indicating a greater destabilizing effect of the mixed solvent on the transition state than on the initial state. The G′ values also correlate with GE(G′ = axO.S.+ cGE) for all solvents, supporting the fact that solvent structural effects mediate the rates and energetics of the reaction. However, the solvent effects on the solvation components of ΔH‡ and ΔS‡ are mutually compensating, thus indicating that there is no change in the mechanism.

ABSTRACTMethicillin-resistant Staphylococcus aureus(MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureususing a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections.

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