Michael S. Wolfe
Mathias P. Mertes Professor, Department of Medicinal Chemistry
University of Kansas

Targeting the Malarial Presenilin Homolog (2017-18)

A malarial protein-cutting enzyme, or protease, found in cell membranes is essential for parasite growth, but no compounds inhibit the enzyme without affecting related human enzymes. The goal of this pilot project is to purify the malarial protease in active form and develop assays suitable for compound screening and discovery of selective inhibitors with the potential to treat malaria.

Presenilin and presenilin-like proteins are membrane-embedded aspartyl proteases that use water in the lipid bilayer to cleave the transmembrane domains (TMDs) of their substrates. While the presenilins assemble with three other membrane proteins to become the protease complex known as gammasecretase, presenilin homologs (PSHs) are proteolytically active as single polypeptides and do not require any other protein cofactors. The malaria parasite, Plasmodium falciparum, encodes a single PSH that is apparently essential to parasite growth. Specific inhibitors of mPSH should be useful chemical biological tools to address the functions of the protease in the parasite life cycle and have the potential to become a new class of antimalarial agents. Published assays for mPSH are limited to cell-based reporter systems, and these have been used to test known inhibitors of the presenilin and presenilin-like proteases for inhibition of mPSH. To date, however, there are no mPSH-selective inhibitors and no isolated enzyme assays. The development of such assays would be ideal for broad screening of diverse compound libraries in search of potent and selective inhibitors of mPSH. In this pilot project we will:

  1.  Clone, express, isolate and purify proteolytically active malarial presenilin homolog (mPSH). This will be accomplished using both E. coli and mammalian cells for expression. Designed epitopetagged substrates based on known cellular chimeric substrates of mPSH will be used to determine if the isolated protease is active.
  2.  Develop an assay suitable for inhibitor screening. We will modify our mPSH substrate, developed in Aim 1, by incorporating a fluorescent tag on one of the termini. From this one-year pilot project, we intend to utilize the Protein Production Group of the KU COBRE PSF, to scale up production and purification of mPSH, for the immediate purpose of assay development for screening.