¹Colorado State University
²Stanford Synchrotron Radiation Lightsource

Pulmonary Fibrosis is a lung disease that is characterized by injury of the alveolar epithelium. The air sacs in the lungs of PF patients are bigger because of scarring of the lungs that is caused by alveolar inflammation and proliferation of fibroblasts. It’s difficult to breathe for PF patients because it takes a longer time and it’s not easy for oxygen to reach the blood stream once it gets from the bronchioles into the alveoli. PF patients have an accumulation of NO in their blood stream and an overexpression of DDAH in their lungs. Studies show that DDAH inhibition reduces collagen production and it suppresses proliferation {1}. A lot of the DDAH inhibitors are mercury containing compounds because the inhibitors have a preference for sulfur, so when they bind to DDAH in its catalytic site, inhibition occurs since the active site of DDAH has two sulfur cysteins. These inhibitors were noted in the chemical screening publication written by Yohannes Ghebremariam and John Cooke at Stanford{2}. The screening assays for measurement of citrulline production and other compounds were developed to see how DDAH changes depending on what binds to the enzyme. Subsequently, another finding that was discovered at Stanford was that proton pump inhibitors also bind to DDAH. The PPI we looked into was omeprazole, also known as Prilosec.

Omeprazole, just like any other PPI, has sulfur. Proton pump inhibitors have benzene rings and sulfur atoms in the center that are used to reduce gastric acid. After they’re absorbed, they are able to inhibit proton pumps by binding to their cysteine residues. And it has been mentioned previously that DDAH has two cysteine residues in its active site. Essentially, this research lead to the idea of creating an inhaler that could be used to inhibit DDAH inside the lungs. This also allows us to avoid systematic intake of PPIs because they increase the risk of getting a stroke or heart attack.

At SLAC, we spent a lot of time trying to co-crystallize DDAH with omeprazole. Our findings showed that the holoform of DDAH has zinc and in order for us to bind omeprazole into the active site, we had to create crystallization conditions where we could grow crystals of DDAH, sequester the metal ions with EDTA and simultaneously add the PPI into its catalytic site. We have several refined structures with the Zn bound in the active site but at different occupancies (depending on how much EDTA was added to sequester the metal ions). One of the structures has a resolution of 1.5 Å, which is nice because there is no known publication with zinc bound to human DDAH and we've reached the highest resolution (so far) too. Unfortunately, we were not able to shoot the DDAH crystals with a higher concentration of EDTA and omeprazole because the synchrotrons were shut down for maintenance. We think that once we will find the optimum concentration of EDTA that is needed to sequester the metal ions, omeprazole will be able to bind to DDAH and we will hopefully get a crystal structure with the PPI bound to the active site. If we are successful in achieving this goal, we can take further steps in developing therapeutic strategies for treating pulmonary fibrosis.