Enzymes are proteins that catalyze a majority of the chemical transformation required by living organisms and as such they play central roles in virtually all biological processes. Enzyme inhibition also provides a powerful approach for the treatment of disease. Unfortunately, the development of potent and selective small molecule inhibitors with acceptable physicochemical properties has proven to be very challenging for a number of enzyme targets. To discover novel, small molecule enzyme inhibitors the Ellman lab has therefore developed the Substrate Activity Screening (SAS) method, which is the first substrate-based fragment discovery and optimization approach.The key desirable aspects of the approach are the elimination of false positives that plague many HTS efforts and signal amplification in substrate detection as a result of substrate turnover.
We first developed and applied the SAS approach to the highly abundant serine and cysteine proteases for which there are a large number of important human and pathogen disease targets. The Ellman group has used the approach to develop highly potent and selective inhibitors of cathepsin S, which is implicated in autoimmune disorders and cancer. Indeed, due to the potency and selectivity of inhibitor 1 (Figure 1A), it served as the starting point for further drug development by Boehringer Ingelheim, and in collaboration with Matt Bogyo at Stanford,provided the key binding motif for near-infrared activity-based-probes for imaging cathepsin S activity in vivo. Potent, selective and orally available inhibitors such as 2 have also been developed against cruzain (Figure 1B), which is an essential protease of Trypanosoma cruzi and is the causative agent of Chagas’ disease. The McKerrow lab at UCSF has found that inhibitor 2 eliminated all symptoms of disease in infected mice and produced a parasitological cure in a significant percentage of the animals. Other targets for which potent small molecule inhibitors have been identified by the SAS method include inhibitors of the malaria proteases DPAP1 and DPAP3 and of the caspases.
Figure 1. Potent and selective protease inhibitors identified by the SAS method
We have also applied the SAS method to other protein classes. For example, we have targeted the large family of protein tyrosine phosphatases (PTPs), which have been implicated as important chemotherapeutic targets for many diseases but have proven to be refractory to traditional drug discovery approaches. Using the SAS method we have identified completely non-peptidic, selective inhibitors of several PTPs. For example, inhibitor 3 was the first reported non-peptidic inhibitor of Mycobacterium tuberculosis PTPB, which is an established virulence factor for tuberculosis. In ongoing studies with Paul Lombroso and Angus Nairn at Yale Medical School we have also identified a series of selective inhibitors such as 4 against STriatal Enriched protein tyrosine Phosphatase (STEP), which is implicated in a number of neurodegenerative diseases. Indeed, we have synthesized another class of unpublished STEP inhibitors that result in considerable cognitive improvement in multiple animal models for Alzheimer’s disease.
Figure 2. Potent and selective protein tyrosine phosphatase inhibitors
The depth and breadth of cutting edge research at Yale School of Medicine provides many exciting opportunities for collaboration. As one example, we collaborate with Prof. Ha Ya (Yale Pharmacology), who is a leading expert on membrane proteases, to design and synthesized mechanism-based protease inhibitors of the rhomboid protease GlpG. Crystal structure analysis of the enzyme-inhibitor complex enhances our general understanding of the mechanisms and mode of binding of physiologically important membrane proteases (Figure 3).
Figure 3. X-ray crystal structure of the rhomboid membrane protease GlpG in complex with a substrate-based fluorophosphonate inhibitor