High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

Anuradha Kumar,Charles Mutai,Linyun Zhu,Meng Zhang,David R Sherman,Shittu Hafsat,Reiling P Liao,Ming-Wei Wang,Andrew J Roe

doi:10.1371/journal.pone.0039961

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a bacterial pathogen that claims roughly 1.4 million lives every year. Current drug regimens are inefficient at clearing infection, requiring at least 6 months of chemotherapy, and resistance to existing agents is rising. There is an urgent need for new drugs that are more effective and faster acting. The folate pathway has been successfully targeted in other pathogens and diseases, but has not yielded a lead drug against tuberculosis. We developed a high-throughput screening assay against Mtb dihydrofolate reductase (DHFR), a critical enzyme in the folate pathway, and screened a library consisting of 32,000 synthetic and natural product-derived compounds. One potent inhibitor containing a quinazoline ring was identified. This compound was active against the wild-type laboratory strain H37Rv (MIC99 = 207 µM). In addition, an Mtb strain with artificially lowered DHFR levels showed increased sensitivity to this compound (MIC99 = 70.7 µM), supporting that the inhibition was target-specific. Our results demonstrate the potential to identify Mtb DHFR inhibitors with activity against whole cells, and indicate the power of using a recombinant strain of Mtb expressing lower levels of DHFR to facilitate the discovery of antimycobacterial agents. With these new tools, we highlight the folate pathway as a potential target for new drugs to combat the tuberculosis epidemic.

Highlights

Tuberculosis (TB) has a long history in humans, with evidence of TB-related deaths dating back to the 3rd century BC [1]
Reduced folate is a critical nutrient in living organisms, and disruption of the folate pathway is widely exploited for chemotherapy [6,22]
The sensitivity of the wild-type Mycobacterium tuberculosis (Mtb) and dihydrofolate reductase (DHFR) kd was tested against various inhibitors and displayed as the ratio of a compound’s MIC99 when measured in wild-type Mtb over the MIC99 measured in DHFR kd in the absence of ATc. doi:10.1371/journal.pone.0039961.g004

Summary

Introduction

Tuberculosis (TB) has a long history in humans, with evidence of TB-related deaths dating back to the 3rd century BC [1]. Roughly 1.4 million people die every year from TB [2], despite the availability of chemotherapies to combat the bacterium that causes this disease (Mycobacterium tuberculosis, Mtb). Several factors have led to new strains of Mtb that are resistant to many or sometimes all of the first and second line drugs [4]. Depending on the drug susceptibility of the infecting strain, patients must endure prolonged treatment with agents that are more expensive, more toxic, and difficult to administer. There have been increased efforts to develop new and improved therapeutics for tuberculosis, and this has led to a few promising drug candidates [5].

Methods

Results

Conclusion