SUNNYLIFE PHARMA INC. — Department of Health and Human Services SBIR Phase I: NIAID

Description

The human papillomavirus HPV E protein is needed for viral replication Infection with high risk HPV types can progress to pre malignant lesions called dysplasias which over a period of years can eventuate in invasive and metastatic epithelial malignancies HPV E proteins bind to the ubiquitin ligase E AP and this complex targets p and other E binding proteins for proteasome mediated destruction This E dependent loss of p enables HPV to bypass host cell defenses and facilitates activation of the cell cycle E AP and other E interacting proteins utilize a charged leucine helical LxxLL peptide motif to bind to E Using molecular modeling based on the structure of this E AP motif we previously identified a series of novel flavone like molecules that inhibit HPV E binding to E AP Exposure of cervical cancer cells to these compounds led to increases inp and p Cip Waf proteins and decreased proliferation of HPV expressing cell lines We used computational modeling to predict where these compounds bind onto the recently described high resolution three dimensional co crystal structure of HPV E with an LxxLL peptide The highest scoring fits placed the flavone inhibitors in a hydrophobic pocket that forms molecular bonds with leucines in the E AP binding pocket and a series of flanking arginines of E These data support our structure based screening and compound selection based on inhibition of E association with E AP Importantly LxxLL peptide binding induces an allosteric change in the E protein needed for entry of p into the complex Our inhibitors may interfere with this conformational change in E and or its protein protein interaction with p Flavonoid small molecules inhibit HPV E but have poor aqueous solubility and unfavorable drug metabolism properties and thus are suboptimal as therapeutics Additionally development of small molecule HPV E inhibitor therapeutics will require a proprietary compound class We took advantage of the flavone based structure activity relationships and modified the core structure to establish a novel and highly druggable chemical series Pilot studies prove we can alter the scaffold and retain E inhibitory activity While less potent than the flavones this SBIR grant will allow us to rapidly increase potency using established assays of competitive inhibition of E AP binding E thermo stabilization and p degradation in vitro and in cells A focused series of mutated E proteins that disrupt the hydrophobic and charged surfaces of the E binding pocket let us explore the binding interface between E and inhibitory compounds We have observed some point mutations disrupt the E E AP interaction but do not alter compound binding to E and conversely some mutants retain E AP binding but restrict compound association These data substantiate our positioning of compounds on the E structure and instruct design of more specific and potent HPV E inhibitors We have shown we can select and synthesize inhibitors of the E E AP interaction and thus potentially a novel treatment for the millions afflicted with pre malignant and malignant HPV associated cancers There is no effective medical therapy for human papillomavirus HPV which infects millions of men and women those with persistent infections of specific subtypes are at a high risk for cancers that often metastasize and account for of cancers worldwide HPV vaccines are of no use to those already infected are out of reach financially for economically disadvantaged countries and it is unknown whether the vaccines will provide complete coverage over a long time frame We propose to make therapeutic molecules to block the activity of an HPV specific protein named E that is important for its replication thus preventing continued infection and the potential for development of cancer