Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.