Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.
The bacterial primase—an essential component in the replisome—is a promising but underexploited target for novel antibiotic drugs. Bacterial primases have a markedly different structure than the human primase. Inhibition of primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.
Fragment-based drug discovery (FBDD) using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a therapeutically relevant agent. In this review, we aim to list the considerations that make NMR fragment screening a successful process for yielding potent inhibitors. Factors that may govern the competence of NMR in fragment based drug discovery are discussed, as well as later steps that involve optimization of hits obtained by NMR-FBDD.
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