Structural Analysis of Heparanase Inhibition
Creators: name: Whitefield, Cassidy
Publisher: name: The Australian National University
Year: 2023

description: Heparanase (HPSE) is the only known mammalian endo-β-glucuronidase that catalyzes the degradation of heparan sulfate, a major component of the extracellular matrix in animals. The overactivation of heparanase is associated with larger tumor size, increased metastasis, and poor prognosis, with all cancers to date showing elevated heparanase expression. Its enzymatic activity of cleaving heparan sulfate is well known, but research now shows that heparanase is a multifunctional protein with both enzymatic and non-enzymatic activities inside and outside the cell. Because of its importance in disease, heparanase has been the target of numerous therapeutic programs over the last two decades, with selected substrate mimetics currently being studied in clinical trials. However, despite tremendous efforts, no therapy with high clinical benefit and minimal side effects has been identified. The limited structural and kinetic knowledge of the various heparanase therapeutic classes has contributed to this issue. Access to easily produced protein has also contributed to this knowledge gap due to the complex processing path of heparanase in the body. In summary, this thesis focuses on the use of biophysical and structural analysis to develop and understand new heparanase therapeutics. Within this scope, our primary focus is on developing and understanding different classes of inhibitors, heparan sulfate mimetics, and small molecule inhibitors, each of which has its own therapeutic benefits and limitations. Firstly, I demonstrate the computational redesign of heparanase to allow for high yield expression in Escherichia coli. This mutated form of heparanase exhibits essentially identical kinetics and inhibition. X-ray crystallography and molecular dynamics show that the heparanase variant has the same structure and dynamics as the wild-type protein. This variant is then used to understand the interactions of the therapeutics studied in subsequent chapters. In the following chapter, I investigate pentosan polysulfate sodium (PPS), an FDAapproved inhibitor for interstitial cystitis and a known heparanase inhibitor. However, due to its heterogeneity, the mechanism of heparanase inhibition by pentosan has been limited in characterization. Three synthetic analogues of PPS showed that the mechanism of PPS inhibition is complex, with oligosaccaride length contributing to multiple inhibitor events. ITC and protein crystallography were used to determine that PPS binds at three binding domains on the surface of heparanase. Kinetic and structural studies support that this binding mechanism causes inhibitor-induced aggregation and irreversible inhibition. This binding model could also be applied to other heparanase therapeutics currently in clinical trials. Lastly, I study a new series of small molecule inhibitors. Small molecules can overcome many of the limitations of other therapeutics due to their increased bioavailability and specificity. A large structure-activity relationship for quinazoline-like compounds was completed for heparanase inhibition. Kinetics support that these inhibitors are non-competitive inhibitors, suggesting a potential binding site on heparanase. Despite unsuccessful crystallography studies, a broad screen of heparanase mutations was conducted to try to identify this binding site, but it was unsuccessful. This mutagenesis screen did demonstrate the importance of allosteric effects on heparanase. These studies highlight the importance of structural analysis of enzyme inhibition and the importance of designing and analyzing different therapeutics in order to design ideal inhibitors in the future.