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.