Chapter 15
Using Proteolytic Hypomorphs to Detect Small Molecule
Mechanism of Action
Eachan O. Johnson and Deborah T. Hung
Abstract
With increasing prevalence of antimicrobial resistance, a fundamental goal of antibiotic discovery is to
uncover new small molecules that prevent growth of pathogenic bacteria through diverse mechanisms of
action. This goal is particularly pertinent for tuberculosis, caused by Mycobacterium tuberculosis. In this
chapter, we describe the application of a chemical-genetic method, PROSPECT (primary screening of
strains to prioritize expanded chemistry and targets), for sensitively detecting small molecule bioactivity
using a pooled panel of hypomorphs (strains depleted in a particular essential gene) of M. tuberculosis. We
describe statistical and heuristic approaches to assign small molecule mechanism of action from the resulting
chemical-genetic interaction profiles.
Key words Mycobacterium tuberculosis, Genetics, Chemical biology, Chemical genetics, Mechanism
of action, Next generation sequencing
1 Introduction
When streptomycin was introduced into the clinic in 1944, it
provided for the first time an effective, nonsurgical treatment for
tuberculosis, caused by the pathogenic bacterium Mycobacterium
tuberculosis [1]. However, streptomycin allergies limited the general use of this otherwise life-saving aminoglycoside antibiotic,
notably leading to treatment failure for the writer George Orwell
[2]. In addition, resistance of M. tuberculosis to streptomycin
emerged by the late 1950s, heralding the beginning of the end of
its clinical utility.
The cycle of discovery of antimicrobials, introduction to the
clinic, and emergence of resistance have played out several times
since, with resistance recorded against every clinical antituberculosis drug [3], and prevalence of resistant infections continues to rise
steadily despite modest decreases in the morbidity of tuberculosis
[4]. To produce antituberculosis drugs that are better tolerated and
tackle current resistance genotypes, it is critically important to
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
323
Using Proteolytic Hypomorphs to Detect Small Molecule
Mechanism of Action
Eachan O. Johnson and Deborah T. Hung
Abstract
With increasing prevalence of antimicrobial resistance, a fundamental goal of antibiotic discovery is to
uncover new small molecules that prevent growth of pathogenic bacteria through diverse mechanisms of
action. This goal is particularly pertinent for tuberculosis, caused by Mycobacterium tuberculosis. In this
chapter, we describe the application of a chemical-genetic method, PROSPECT (primary screening of
strains to prioritize expanded chemistry and targets), for sensitively detecting small molecule bioactivity
using a pooled panel of hypomorphs (strains depleted in a particular essential gene) of M. tuberculosis. We
describe statistical and heuristic approaches to assign small molecule mechanism of action from the resulting
chemical-genetic interaction profiles.
Key words Mycobacterium tuberculosis, Genetics, Chemical biology, Chemical genetics, Mechanism
of action, Next generation sequencing
1 Introduction
When streptomycin was introduced into the clinic in 1944, it
provided for the first time an effective, nonsurgical treatment for
tuberculosis, caused by the pathogenic bacterium Mycobacterium
tuberculosis [1]. However, streptomycin allergies limited the general use of this otherwise life-saving aminoglycoside antibiotic,
notably leading to treatment failure for the writer George Orwell
[2]. In addition, resistance of M. tuberculosis to streptomycin
emerged by the late 1950s, heralding the beginning of the end of
its clinical utility.
The cycle of discovery of antimicrobials, introduction to the
clinic, and emergence of resistance have played out several times
since, with resistance recorded against every clinical antituberculosis drug [3], and prevalence of resistant infections continues to rise
steadily despite modest decreases in the morbidity of tuberculosis
[4]. To produce antituberculosis drugs that are better tolerated and
tackle current resistance genotypes, it is critically important to
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
323