RESUMEN
Background: Plasmodium falciparum possesses a cobalamin-dependent methionine synthase (MS). MS is putatively encoded by the PF3D7_1233700 gene, which is orthologous and syntenic in Plasmodium. However, its vulnerability as an antimalarial target has not been assessed. Methods: We edited the PF3D7_1233700 and PF3D7_0417200 (dihydrofolate reductase-thymidylate synthase, DHFR-TS) genes and obtained transgenic P. falciparum parasites expressing epitope-tagged target proteins under the control of the glmS ribozyme. Conditional loss-of-function mutants were obtained by treating transgenic parasites with glucosamine. Results: DHFR-TS, but not MS mutants showed a significant proliferation defect over 96 h, suggesting that P. falciparum MS is not a vulnerable antimalarial target.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Antimaláricos/farmacología , Plasmodium falciparum/genética , 5-Metiltetrahidrofolato-Homocisteína S-MetiltransferasaRESUMEN
The translation of time-dependent drug-target occupancy to extended pharmacological activity at low drug concentration depends on factors such as target vulnerability and the rate of target turnover. Previously, we demonstrated that the postantibiotic effect (PAE) caused by inhibitors of bacterial drug targets could be used to assess target vulnerability, and that high levels of target vulnerability coupled with relatively low rates of target resynthesis resulted in a strong correlation between drug-target residence time and the PAE following compound washout. Although the residence time of inhibitors on UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in Pseudomonas aeruginosa (paLpxC) results in significant PAE, inhibitors of the equivalent enzyme in Escherichia coli (ecLpxC) do not cause a PAE. Hyperactivity of the fatty acid biosynthesis enzyme FabZ or the inclusion of sub-MIC levels of azithromycin lead to the observation of a PAE for three inhibitors of ecLpxC. FabZ hyperactivity has been shown to stabilize ecLpxC, and using mass spectrometry, we demonstrate that the appearance of a PAE can be directly linked to a 3-fold increase in the stability of ecLpxC. These studies substantiate the importance of target turnover in time-dependent drug activity.
Asunto(s)
Antibacterianos , Preparaciones Farmacéuticas , Antibacterianos/farmacología , Bacterias , Escherichia coli/genética , Pseudomonas aeruginosaRESUMEN
Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 µM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Animales , IMP Deshidrogenasa/genética , Inosina , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
Target vulnerability correlates the level of drug-target engagement required to generate a pharmacological response. High vulnerability targets are those that require only a relatively small fraction of occupancy to achieve the desired pharmacological outcome, whereas low vulnerability targets require high levels of engagement. Here, we demonstrate that the slope of the correlation between drug-target residence time and the post-antibiotic effect (PAE) can be used to define the vulnerability of bacterial targets. For macrolides, a steep slope is observed between residence time on the E. coli ribosome and the PAE, indicating that the ribosome is a highly vulnerable drug target. The analysis of the residence time-PAE data for erythromycin, azithromycin, spiramycin, and telithromycin using a mechanistic pharmacokinetic-pharmacodynamic model that integrates drug-target kinetics into predictions of drug activity lead to the successful prediction of the cellular PAE for tylosin, which has the longest residence time (7.1 h) and PAE (5.8 h). Although the macrolide data support a connection between residence time, PAE, and bactericidality, many bactericidal ß-lactam antibiotics do not give a PAE, illustrating the role of factors such as protein resynthesis in the expression of target vulnerability.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Desarrollo de Medicamentos , Pruebas de Sensibilidad MicrobianaRESUMEN
The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.