Leonard Petrucelli, Ph.D.
Repeat DNA expansions are commonly found mutations in various diseases including Huntington's disease, amyotrophic lateral sclerosis (ALS), front temporal dementia (FTD) and cerebrospinal ataxia. The repeat expansions are transcribed and translated via repeat-associated non-ATG (RAN) translation and give rise to pathological features such as RNA foci formation, sequestration of RNA binding proteins and repeat peptide toxicity. Researchers have been focusing on the role of transcription factor DSIF (DRB sensitivity-inducing factor), a complex of Spt4 and Spt5 on repeat DNA transcription elongation. Knocking down of Spt4 selectively reduced transcription and translation of (CAG)81 repeats of Htt gene yeast model and also those of (GGGGCC)66/(CCCCGG)66 in C9orf72 of ALS/FTD in vivo model. Thus, Spt4 dependent elongation of repeat DNA sequences shed a light on Spt4 as a promising therapeutic target.
To prevent repeat DNA associated pathology at a fundamental level, I design peptidomimetic drugs and also use pre-selected small molecule inhibitors for in cellullo screening. My approach is to use cell-based luciferase assay system that expresses Spt4-hGluc1 and Spt5-hGluc2 and the dimerization of Spt4 and Spt5 gives the luciferase signal in cells. Unbound Spt4 is unstable in nature and the drug preventing dimerization will lead to the degradation of this dispensable protein. Once the leads identified we will use Fuchs' eye degeneration model for in vivo validation by applying as eye droplets. It is critical to assess the drug's role on global RNA expression level change and we will accomplish this with Pertea M's protocol in nature method (2016). Another area of interest is the basic biology of Spt4 and Spt5 and we have multiple approaches. I introduce point mutation and deletion mutation using site-directed mutagenesis to identify the critical interaction residues at the interface at both Spt4 and Spt5. Also, we are dissecting the phosphorylation sites of Spt4 and Spt5 together with the role of CDK9 on phosphorylation of DSIF. These efforts will contribute to the universal intervention of repeat-associated diseases aforementioned. Lastly, another intervention to prevent (GGGGCC) repeat DNA transcription is using small molecules that directly bind to DNA secondary structure and stabilize it. To achieve this goal, I conduct in sillico drug discovery of small molecule DNA binders that stabilizes repeat structure and selectively prevent repeat transcription of C9 FTD/ALS. With the evidence that the (GGGGCC) DNA exist in the form of g-quadruplex structure even in the presence of the complementary strands, I do virtual docking of drugs on DNA repeat binders of known g-quadruplex 3D structure (PDB ID: 2N2D). Further, we validate the top binders in vitro dual luciferase assay system and in C9 positive patient cell system.
Not only this will provide us a DNA binder for this specific disease, we expect this work will perform as a drug discovery platform for structural binder drug discovery.
July 26, 2017