Targeting PDGFR-b promoter vacancy G-quadruplex to suppress gene expression

PROJECT SUMMARY Targeting PDGFR-β promoter vacancy G-quadruplex to suppress gene expression Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is an important factor in human cancers and inflammatory diseases. The PDGFR-β signaling pathway is a validated therapeutic target for cancer treatment. Protein kinase inhibitors targeting PDGFR-β have been evaluated in clinical trials, however, they lack specificity and are ineffective against PDGFR-β mutants. G-quadruplex is a globular DNA secondary structure and a new class of molecular targets for anticancer drugs. The human PDGFR-β promoter has a G-quadruplex- forming region that functions as a transcriptional silencer and is targetable by small molecules. The PDGFR-β promoter G-quadruplexes differ significantly from the canonical G-quadruplexes with three complete G-tetrads, such as those formed in MYC and VEGF promoter systems. In contrast, we found that the G-quadruplex formed in the PDGFR-β promoter adopts a unique broken-strand structure which readily forms a vacancy G-quadruplex (vG4) with an incomplete G-tetrad that can be filled-in by an external guanine derivative. Furthermore, we showed that the guanine-fill-in-vG4 can be stabilized by the medicinal natural product berberine and we determined their ternary complex structure by NMR. Significantly, our structure indicates that the unique PDGFR- β vG4 present a specific recognition site for a berberine-guanine conjugate with a berberine linked with a guanine moiety. Therefore, we hypothesize that the unique vG4 formed in the PDGFR-β promoter can be selectively targeted by berberine-guanine conjugates over canonical G-quadruplexes to suppress PDGFR-β transcription in cancer cells. Excitingly, based on our determined NMR structure, we have successfully synthesized two berberine-guanine conjugates that bind the PDGFR-β vG4 with high-affinity and specificity using the G-fill-in recognition mode, and can get into cancer cells and lower PDGFR-β levels. The specific aims are: 1) Structural characterization of berberine-guanine conjugates in complex with the PDGFR-β promoter vG4. 2) Design and synthesize berberine-guanine conjugates using structure-based rational approach that specifically recognize the unique PDGFR-β promoter vG4 for effective gene suppression. 3) Determine the effects of berberine-guanine conjugates on PDGFR-β gene expression and activities in PDGFR-β–driven cancers. The expected outcome of this work is a structure-based rational design and development of novel berberine-guanine conjugates that specifically recognize the unique PDGFR-β promoter vG4 and supress PDGFR-β gene expression. The long- term goal of this proposal is to develop lead compounds of berberine-guanine conjugates for future preclinical testing. The results will have an important positive impact because they lay the groundwork to develop novel guanine conjugates for specific targeting of the PDGFR- silencer element. Further, the targeting compounds developed can serve as molecular probes to investigate protein interactions of the unique PDGFR-β promoter vG4 and provide mechanistic understanding of PDGFR-β gene transcriptional regulation.