RP3: Targeting ATG gene-dependent immunity for novel anti-infective therapeutics

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PROJECT SUMMARY – RP3: The goal of RP3 is to define and pharmacologically target intracellular immune mechanisms utilizing autophagy (Atg) genes (but not degradative autophagy) to control infection and inflammation. We discovered these processes in experiments designed to understand the anti-infective mechanisms of IFNγ, a cytokine essential in both humans and mice for macrophage-mediated resistance to viruses, bacteria, and parasites. IFNγ is used to treat chronic granulomatous disease and osteopetrosis, but other uses of IFNγ have been limited by toxicity, a fact generally attributed to IFNγ inducing the expression of a very large number of pro-inflammatory genes. We theorized that new therapeutics might be generated by defining the effector mechanisms responsible for the potent effects of IFNγ, and then selectively stimulating these mechanisms to generate Atg gene-directed broad-spectrum anti-infectives. This led to the discovery that IFNγ triggers Atg gene-dependent immunity against T. gondii and norovirus (NoV) infection using the same set of Atg genes (Atg5, Atg7, Atg16L1, Atg12), but not Atg14 or degradative autophagy. The fact that Atg gene- dependent mechanism(s) controlled two phylogenetically distinct pathogens suggested to us that we might be able to make compounds that are effective against a broad range of pathogens. During the first CETR funding period we confirmed this prediction by identifying Atg gene-directed broad-spectrum anti-infective compounds with activity against norovirus, salmonella, Mycobacterium tuberculosis and Toxoplasma gondii. This discovery was complemented by discoveries of the role of Atg genes in the regulation of inflammation. RP3 focuses on the Caliciviridae (most importantly NoVs) which are priority pathogens, causing >90% of the epidemic non-bacterial gastroenteritis in the world. We discovered murine NoV (MNoV) and cultured NoVs for the first time, providing a preclinical model in laboratory mice and allowing discovery of Atg gene-dependent immunity. Studies of human NoV (HNoV) infection had been limited until it was recently found that HNoV grows in cells differentiated from biopsy-derived human colonic stem cells. This, together with the discovery that MNoV grows robustly in human cells expressing the proper receptor, and that Atg gene-dependent immunity operates against both T. gondii and MNoV in human cells strongly argues that these mechanisms are evolutionarily conserved and provide the tools to identify compounds relevant to a major human disease via the following aims: Aim 1: Develop Atg gene- directed broad spectrum anti-infective compounds; Aim 2: Define the molecular basis of Atg gene- dependent IFNγ-induced control of norovirus replication, and identify targets and lead compounds that trigger this form of intracellular immunity; Aim 3: Define the molecular basis for the regulation of IFNγ responses by Atg genes and identify lead compounds that up-regulate specific IFNγ effector mechanisms; Aim 4. Collaborate with other CETR projects to identify autophagy- and Atg gene-directed broad-spectrum anti-infective compounds.