Whole Cell Luminescence-Based Screen for Inhibitors of the Bacterial Sec Machinery

TL;DR

Researchers developed a new screening method to find compounds that inhibit the bacterial Sec system, a potential target for new antibiotics.

Contribution

A scalable, whole-cell luminescence-based assay for identifying inhibitors of the bacterial Sec machinery is introduced.

Findings

A split NanoLuc luciferase-based assay was adapted for high-throughput screening of Sec inhibitors in living cells.

A counterscreen distinguishes Sec-specific inhibitors from compounds with general cellular effects.

A library of 5000 compounds yielded several moderate in vivo Sec inhibitors, validating the screening method.

Abstract

There is a pressing need for new antibiotics to combat
rising resistance
to those already in use. The bacterial general secretion (Sec) system
has long been considered a good target for novel antimicrobials thanks
to its irreplacable role in maintaining cell envelope integrity, yet
the lack of a robust, high-throughput method to screen for Sec inhibition
has so far hampered efforts to realize this potential. Here, we have
adapted our recently developed in vitro assay for
Sec activity—based on the split NanoLuc luciferase—to
work at scale and in living cells. A simple counterscreen allows compounds
that specifically target Sec to be distinguished from those with other
effects on cellular function. As proof of principle, we have applied
this assay to a library of 5000 compounds and identified a handful
of moderately effective in vivo inhibitors of Sec.
Although these hits are unlikely to be potent enough to use as a basis
for drug development, they demonstrate the efficacy of the screen.
We therefore anticipate that the methods presented here will be scalable
to larger compound libraries, in the ultimate quest for Sec inhibitors
with clinically relevant properties.

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