Structural and Functional Studies of Three Human Hydrolases: DUF89, HtrA1, and Uba2
Structural biology has offered deepened understanding of the biological world at the molecular level. An elucidated structure can be used to understand enzymatic mechanisms and design new, novel inhibitory molecules. Our studies have examined the structures of three proteins and paired them with in vitro assays to probe the activity and small-molecule inhibition of two therapeutically intriguing enzymes.
First, we have solved the three-dimensional structure of the gene product of C6orf211 using X-ray crystallographic techniques. The architecture revealed a DUF89 fold with a coordinate magnesium in the deepest surface pocket. In vitro functional studies identified a metal-dependent phosphatase activity against a host of phosphometabolites, and strongest against fructose 1-phosphate. The enzymatic promiscuity, low Km, and ubiquitous low-level expression suggest the hDUF89 protein may play an in vivo role in metabolite repair.
Next, the extracellular protease HtrA1 was pursued as a possible therapeutic target for age-related macular degeneration (AMD). An AMD predisposing haplotype has been linked to HTRA1 where elevated expression levels were previously noted. In vitro assays were developed to probe the efficacy of AI-CADD predicted binders. Our inhibition studies identified several low micromolar hits, and efforts to characterize the nature of the interaction between enzyme and ligand via macromolecular crystallographic approaches are ongoing.
Last, we pursued many avenues for inhibitor development against the Aos1/Uba2 heterodimer. The SUMOylation system has been widely identified as a suitable drug candidate due to its roles in many untreatable cancers. Of note, the most potent natural product inhibitor for Aos1/Uba2 is anacardic acid, a fat-soluble natural product with known off-target effects. Avenues for inhibitor identification therefore included natural product chemistry to produce anacardic acid derivatives, CADD, AI-CADD, and FBDD. Our in vitro binding studies have identified strong candidates for subsequent lead development via chemical modifications following ongoing crystallographic efforts.