The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

Lu Huang,Monique E Theriault,H Mike Petrassi,Case W Mcnamara,David G Russell,Amanda C Brown,Davide Pisu,Shumin Tan,Nicole L Kushner

doi:10.3389/fcimb.2018.00275

Abstract

Mycobacterium tuberculosis (Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone. The absence of these players unnecessarily limits the breadth of novel host target pathways to be interrogated. In this study, we detail a new drug screening platform based on dissociated murine TB granulomas, named the Deconstructed Granuloma (DGr), that utilizes fluorescent Mtb reporter strains screened in the host immune environment of the infection site. The platform has been used to screen a collection of known drug candidates. Data from a representative 384-well plate containing known anti-bacterial compounds are shown, illustrating the robustness of the screening platform. The novel deconstructed granuloma platform represents an accessible, sensitive and robust high-throughput screen suitable for the inclusive interrogation of immune targets for Host-Directed Therapeutics.

Highlights

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), poses an ongoing threat to human health
Complex high throughput screening (HTS) Platform for Tuberculosis new anti-microbial compounds have focused on either targetbased screening against enzymatic activities that have been shown genetically to be essential for bacterial survival (Mdluli and Spigelman, 2006; Lamichhane, 2011), or phenotypic screens exploring bacterial survival in rich broth (Collins and Franzblau, 1997; Pethe et al, 2010)
To accelerate anti-TB drugs and Host-direct therapy (HDT) discovery, we need a discovery platform with minimal assumptions that provides a more holistic representation of the complex biology of the infected host. With this aim in mind we have developed a high throughput screening (HTS) assay utilizing cells recovered from Mtbinfected mouse lungs (Deconstructed Granuloma, DGr), which we are using to screen compounds with efficacy against Mycobacterium tuberculosis (Mtb) within the context of host-derived immune cells

Summary

INTRODUCTION

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), poses an ongoing threat to human health. There is the sense that the “low-hanging fruit” have already been picked, and compounds with known modes of action continue to re-emerge from ongoing screens For these reasons we feel that it is critical to develop new, innovative approaches to drug discovery that explore and exploit the host-derived pressures on Mycobacterium tuberculosis (Mtb) to identify compounds with enhanced efficacy within the host. To accelerate anti-TB drugs and HDT discovery, we need a discovery platform with minimal assumptions that provides a more holistic representation of the complex biology of the infected host With this aim in mind we have developed a high throughput screening (HTS) assay utilizing cells recovered from Mtbinfected mouse lungs (Deconstructed Granuloma, DGr), which we are using to screen compounds with efficacy against Mtb within the context of host-derived immune cells. We believe that this novel HTS platform will reveal new avenues toward the manipulation of the host immune environment to restrict bacterial growth and survival, and to act in concert with existing drug treatments and increase their in vivo potency

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUDING REMARKS