Project Abstract Traumatic brain injury (TBI) is a leading cause of morbidity and mortality world-wide and in the United States;1,2 however, there are no FDA-approved medications to ameliorate the devastating consequences of secondary injury caused by complex neuropathological cascades following the initial neuronal insult. Cathepsin B and L represent promising drug targets, as knock-out and inhibition studies of these cysteine proteases in animal models reveal improvement in behavioral and neurocognitive sequelae.3–7 There is a clear need for new therapeutic options to treat secondary injury following TBI, and cathepsin B and L inhibitors have pharmaceutical promise. Over 60% of FDA-approved drugs are derived from or inspired by natural products (NPs).8 Cyanobacteria are known to produce NPs with a range of bioactivity, including activity as protease inhibitors.9,10 NPs are biosynthesized by megaenzymes that are encoded as discrete genomic packages called biosynthetic gene clusters (BGCs).11 We hypothesize that a genomic approach can be used to enhance drug discovery efforts from cyanobacteria for novel cathepsin B and L inhibitors through the three aims outlined below. First, an innovative pharmacophore-based genome mining pipeline will be developed (Aim 1). In this aim, pharmacophores deduced from virtual docking experiments and known cathepsin inhibitors will be used to predict enzymatic domains responsible for the creation of the desired pharmacophore. This retrobiosynthetic prediction will be used to make bioinformatic models to interrogate sequenced cyanobacterial genomes to find candidate BGCs. The BGCs of highest interest will be identified, delineated, and the compounds produced through either cultivation or heterologous expression (Aim 2). Using a full retrobiosynthetic prediction for gallinamide A, a compound that demonstrates nanomolar cathepsin L inhibition, we will search for the BGC within the genomic library at Scripps or in new cyanobacterial collections. Additional BGCs from Aim 1 will be developed in this aim as well. If the BGC is not constitutively expressed or if the BGC is not associated with a cultivatable organism, heterologous expression will be pursued (Aim 3). Compound isolation and molecular networking to analyze and annotate the chemical space of the natural products produced will follow. Compounds will be tested in bioassays to evaluate cathepsin B and L activity on purified enzymes as well as in neuronal and glial cellular studies. The gap between the identified pharmaceutical need and the discovery of novel bioactive molecules can be bridged by the innovative multidisciplinary approach presented in this application. The proposed project will be carried out as part of an NIH F32 NRSA Fellowship at Scripps Institution of Oceanography, UC San Diego, under the co-sponsorship of Professors William Gerwick and Vivian Hook and a team of collaborators that will train the postdoctoral fellow in virtual docking experiments, bioinformatics, heterologous gene expression, and cathepsin-related bioassays.

Project Narrative There are currently no FDA-approved medications to treat the neuropathology that follows traumatic brain injury; however, inhibition of cathepsins B and L have been associated with improved neurocognitive and behavioral outcomes after neuronal injury in animal models. Cyanobacteria are known to produce natural product compounds with neurologic bioactivity, including cathepsin and other protease inhibitors. In this proposal, we hypothesize that novel cathepsin inhibitors can be discovered from cyanobacteria through an innovative genomic approach, which includes pharmacophore-based genome mining, delineation of biosynthetic gene clusters, cultivation, and heterologous gene expression.