Target-based antibiotics act on the products of essential genes but rarely result in bacterial death. A new study published in Cell suggests that this may be down to gene vulnerability. Their findings have identified high-value targets for future drug development.
As the name would suggest, essential bacterial genes are necessary for the core biological processes of a bacterium. Due to this, nearly all antibacterial drugs are designed to target these genes. However, in recent years, it has been apparent that inhibition of essential genes does not always result in detrimental effects to bacterial fitness.
Previous research has shown that only partial inhibition of some essential genes leads to bacterial death, whilst strong inhibition of other essential genes has no effect on the organism. The relationship between the magnitude of gene inhibition and bacterial fitness is known as gene vulnerability. When genes of high vulnerability are inhibited, the effect on fitness is greater. Now, in this study, researchers found that essential genes exist along a gradient of vulnerability.
Vulnerable genes in tuberculosis
The causative agent of tuberculosis (TB) is Mycobacterium tuberculosis (Mtb). In this study, researchers investigated the genomes of Mtb specifically as TB accounts for one-third of all deaths associated with antimicrobial resistance. Understanding gene vulnerability in Mtb would enable the identification of potential drug targets. Hopefully, this will help overcome the problem of antibiotic resistance.
Mtb CRISPRi library
The researchers developed a CRISPR interference-based (CRISPRi) functional genomics method that uses single guide RNAs (sgRNAs) to target all annotated genes in two strains of Mtb. In total, 96,700 unique sgRNAs that targeted 98.2% of all annotated Mtb genes made up the final CRISPRi library. Each essential gene was then ranked by the level of inhibition required in order to kill the bacteria.
“We developed a system that can be tuned, from no inhibition to nearly 100 percent inhibition,” says Barbara Bosch, a researcher in the Rockefeller University’s Laboratory of Host-Pathogen Biology. “This allowed us to determine whether the bacteria were having serious fitness costs, or whether they were still alive and kicking.”
Until now, the failures of target-based drug discovery have been blamed on compounds being unable to reach their destination. For example, scientists thought that drugs were unable to cross the bacterial envelope or were being pumped out of the organism. However, these new findings suggest that the problems may actually lie with the chosen targets.
Vulnerability was seen to vary greatly over the Mtb genome. Highly vulnerable genes, including novel targets unexplored for drug discovery, were identified. Importantly, invulnerable essential genes were also found.
Two of the most vulnerable genes found, inhA and rpoB, are the targets of two of the most potent TB drugs, showing that vulnerability level is a key factor in antibiotic success. In contrast, the genes coaA and def were found to be in the lower quartile of vulnerability. This potentially explains why previous attempts to develop drugs targeting these genes have failed. Peptide deformylase, encoded by def, is a particularly popular target in drug development. These new findings may encourage studies to look elsewhere in the genome for potential drug targets.
Future drug development
The study also found that tRNA synthetases were universally vulnerable in all the tested Mtb strains. This highlights an exciting potential drug target, as multi-targeting of conserved active sites has been known to reduce rates of antibiotic resistance.
In addition, many vulnerable genes are involved in processes that have been of low interest to drug developers. For example, two genes involved in protein folding, groES and groEL, were found to be significantly more vulnerable than those targeted by current TB therapies.
“We expected that genes involved in the central dogma would be vulnerable—to replicate, you need to be able to turn DNA into RNA into protein,” says Jeremy Rock, head of The Rockefeller University’s Laboratory of Host-Pathogen Biology. “But some of the most vulnerable genes were involved in protein folding and secretion. We wouldn’t necessarily have predicted that. These are under-explored targets that would be worth exploring in the future.”