Complex Inhibitory Mechanism of Glycomimetics with Heparanase.

Heparanase (HPSE) is the only mammalian endo-ß-glucuronidase known to catalyze the degradation of heparan sulfate. Dysfunction of HPSE activity has been linked to several disease states, resulting in HPSE becoming the target of numerous therapeutic programs, yet no drug has passed clinical trials to date. Pentosan polysulfate sodium (PPS) is a heterogeneous, FDA-approved drug for the treatment of interstitial cystitis and a known HPSE inhibitor. However, due to its heterogeneity, characterization of its mechanism of HPSE inhibition is challenging. Here, we show that inhibition of HPSE by PPS is complex, involving multiple overlapping binding events, each influenced by factors such as oligosaccharide length and inhibitor-induced changes in the protein secondary structure. The present work advances our molecular understanding of the inhibition of HPSE and will aid in the development of therapeutics for the treatment of a broad range of pathologies associated with enzyme dysfunction, including cancer, inflammatory disease, and viral infections.

The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors-A Spin-Off Discovery from Fragment Screening.

The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen-deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices.

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.