In this study, combinatorial libraries were used in conjunction with ultra-high throughput sequencing to comprehensively determine the impact of each of the 19 possible amino acid substitutions at each residue position in the TEM-1β-lactamase enzyme. The libraries were introduced into E. coli and mutants were selected for ampicillin resistance. The selected colonies were pooled and subjected to ultra-high throughput sequencing to reveal the sequence preferences at each position. The depth of sequencing provided a clear, statistically significant picture of what amino acids are favored for ampicillin hydrolysis for all 263 positions of the enzyme in one experiment. Although the enzyme is generally tolerant of amino acid substitutions, several surface positions far from the active site are sensitive to substitutions suggesting a role for these residues in enzyme stability, solubility or catalysis. In addition, information on the frequency of substitutions was used to identify mutations that increase enzyme thermodynamic stability. Finally, a comparison of sequence requirements based on the mutagenesis results versus those inferred from sequence conservation in an alignment of 156 class A β-lactamases reveals significant differences in that several residues in TEM-1 do not tolerate substitutions and yet extensive variation is observed in the alignment, and vice versa. An analysis of the TEM-1 and other class A structures suggests residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes.

Inhibition of tRNA aminoacylation has proven to be an effective antimicrobial strategy, impeding an essential step of protein synthesis. Mupirocin, the well-known selective inhibitor of bacterial isoleucyl-tRNA synthetase, is one of three aminoacylation inhibitors now approved for human or animal use. However, design of novel aminoacylation inhibitors is complicated by the steadfast requirement to avoid off-target inhibition of protein synthesis in human cells. Here we review available data regarding known aminoacylation inhibitors as well as key amino-acid residues in aminoacyl-tRNA synthetases (aaRSs) and nucleotides in tRNA that determine the specificity and strength of the aaRS-tRNA interaction. Unlike most ligand-protein interactions, the aaRS-tRNA recognition interaction represents coevolution of both the tRNA and aaRS structures to conserve the specificity of aminoacylation. This property means that many determinants of tRNA recognition in pathogens have diverged from those of humans-a phenomenon that provides a valuable source of data for antimicrobial drug development.

BackgroundMany patients suffering from depressive disorders are refractory to treatment with currently available antidepressant medications, while many more exhibit only a partial response. These factors drive research to discover new pharmacological approaches to treat depression. Numerous studies demonstrate evidence of inflammation and elevated oxidative stress in major depression. Recently, major depression has been shown to be associated with elevated levels of DNA oxidation in brain cells, accompanied by increased gene expression of the nuclear base excision repair enzyme, poly(ADP-ribose) polymerase-1. Given these findings and evidence that drugs that inhibit poly(ADP-ribose) polymerase-1 activity have antiinflammatory and neuroprotective properties, the present study was undertaken to examine the potential antidepressant properties of poly(ADP-ribose) polymerase inhibitors.MethodsTwo rodent models, the Porsolt swim test and repeated exposure to psychological stressors, were used to test the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, for potential antidepressant activity. Another poly(ADP-ribose) polymerase inhibitor, 5-aminoisoquinolinone, was also tested.ResultsPoly(ADP-ribose) polymerase inhibitors produced antidepressant-like effects in the Porsolt swim test, decreasing immobility time, and increasing latency to immobility, similar to the effects of fluoxetine. In addition, 3-aminobenzamide treatment increased sucrose preference and social interaction times relative to vehicle-treated control rats following repeated exposure to combined social defeat and unpredictable stress, mediating effects similar to fluoxetine treatment.ConclusionsThe poly(ADP-ribose) polymerase inhibitors 3-aminobenzamide and 5-aminoisoquinolinone exhibit antidepressant-like activity in 2 rodent stress models and uncover poly(ADP-ribose) polymerase as a unique molecular target for the potential development of a novel class of antidepressants.