Investigation and Characterization of Small Molecule Modulators for Mitochondrial Processing Peptidase

Mitochondria are an extremely complex organelle comprised of four discrete compartments and are vital for the proper functioning of key cellular pathways including, energy production, growth, differentiation, and cellular signaling. The mitochondrial genome encodes for 13 proteins, however the mitochondrial proteome is estimated to contain approximately 600- 1000 proteins; most of these must be imported from the cytosol. Depending on a protein's intended location, there are host signals and import machinery that ensure the correct appropriate localization. Proteins destined for the mitochondrial matrix typically possess a mitochondrial targeting sequence (MTS) found at the N-terminus. These proteins are funneled through the translocon of the outer membrane (TOM complex), the translocon of the inner membrane (TIM23 complex) into the matrix. Here, the MTS is cleaved off by mitochondrial processing peptidase (MPP), allowing the mature protein to fold into its native confirmation.Many proteins, such as MPP, are essential for life, therefore it is difficult to study them using classical approaches or through methods such as RNAi as these may cause global side- effects and take a great deal of time for effect manifestation. As such, our lab devises ways to bypass these issues through the development of small molecule modulators for our proteins of interest. For MPP, this was accomplished through a high-throughput screen (HTS) of approximately 130,000 compounds at UCLA's CNSI screening center.We focused on 2 molecules resulting from the screen, MitoBloCK-50 and -51, which were found to be inhibitors of MPP. Both compounds possess many similarities in the effects we see via MPP inhibition including cleavage of Su9-DHFR, import of Su9-DHFR, import of Cpn10, hPink1 import, and the import of CytB2-DHFR (1-167). Conversely, we also saw differences in their inhibition of MPP and their effect on downstream pathways including, the import of CytC1, the import of CytB2-DHFR (A63P), Parkin recruitment on the surface of mitochondria, and zebrafish development. These similarities and differences between the 2 MitoBloCK compounds could signify differences in their binding and inhibition of MPP, and these differences result in slightly different downstream effects. Overall, we have discovered and initated the characterization of two MPP inhibitors and their respective analogs, potentially helping to elucidate the Pink1-Parkin mitochondrial degradation pathway and the pathways involved in copper metabolism, melanogenesis, and melanin formation