Delivering therapeutic treatments to cancer, diabetes, and neurodegenerative targets
Within our bodies’ cells, a myriad of chemical reactions orchestrate life. These reactions ensure our health and are essential to all of our bodily functions, including metabolism and homeostasis within the bodily environment. But when these reactions are unable to function properly, disease can result.
One of the important chemical reactions that occur in our bodies is protein tyrosine phosphorylation, which is a modification of newly synthesized proteins. When regulation of this reaction is disturbed, diseases such as diabetes, cancer, and neurodegeneration arise. Jonathan Ellman, Professor of Chemistry and Pharmacology at Yale, and his team of researchers have developed a method for delivering therapeutic drugs to target certain proteins involved in the dysregulation of the protein tyrosine phosphorylation pathway.
Striking a Balance
During protein tyrosine phosphorylation, a phosphate molecule is added to an amino acid called tyrosine, which is a common building block of proteins. To help the reaction happen more efficiently, this addition is catalyzed by an enzyme called protein tyrosine kinase. Kinases are a class of proteins that add phosphates to other molecules. Proteins tyrosine kinases act concurrently with protein tyrosine phosphatases (PTPs), which remove phosphate groups from tyrosine. The body requires a proper balance of these kinases and phosphatases to ensure a proper balance of tyrosine phosphorylation levels on proteins within our cells.
The wide and seemingly unrelated range of diseases related to dysregulation of protein tyrosine phosphorylation pathways suggest a universal importance for these molecules within our bodies. Specifically, it highlights the significance of the enzymes that catalyze such reactions. Of particular interest are the aforementioned PTPs, a family of enzymes with the general function of removing phosphate groups from tyrosine. For example, bacterial PTPs have interestingly been found to exacerbate infections such as tuberculosis.
Medical interest in PTPs arose due to their implications in human disease. However, challenges involving PTP-based drugs have made such research and drug development difficult. “PTPs continue to be challenging targets for progressing inhibitors to the clinic because their active sites are highly conserved and charged,” Ellman said. Active sites are areas within enzymes such as PTP that specifically bind to protein targets. For PTPs, the target is the phosphate group on a tyrosine found within different kinds of proteins. Because PTP active sites are charged and conserved, meaning that they possess an electrical charge and are universal to many PTP types, designing drugs to specifically target and successfully react at the active site is tricky. “Thus, it is difficult to develop inhibitors that are potent and selective against a specific PTP while also having appropriate physicochemical properties to be effective drugs, such as level of polarity to efficiently cross cell membranes,” Ellman added.
Motivated to study PTPs, Ellman tackled the problem of PTP drug development by designing a platform to inhibit PTPs for disease treatment. The platform consists of glutathione-responsive selenosulfide prodrugs that have a specific function of inhibiting PTPs. Prodrugs are inactive precursors to drugs that, once processed by the body, can exert their biological function in a controlled manner. This selectivity in the prodrug’s mechanism is crucial for designing and understanding how the prodrug acts within the human body.
The Mechanism
Glutathione (GSH) is an antioxidant important for preventing free radical damage to our cells, which is spontaneous damage that occurs all over our body due to things like ultraviolet (UV) radiation from sunlight and even from the body’s own metabolism. GSH is synthesized in our body from food sources obtained from our diet. Because there is a large difference between GSH levels inside and outside of our cells, the research group used this natural difference in concentration to activate a specific PTP inhibitor, which comes from a novel group of chemicals called selenosulfide phosphatase inhibitors. This class is named after the key part of the inhibitor structure responsible for labeling the enzyme: the inhibitor targets a sulfur-containing group found within the phosphatase enzyme, hence the “sulfide” in the name. The researchers chose the GSH-responsive motif as a method for prodrug delivery due to these cellular properties.
The drug’s mechanism of action relies on its selenosulfide pharmacophore, the part of the drug that is responsible for its pharmacological interaction, which reacts with cysteine, an amino acid in the active site of PTP, to form a product that inhibits PTP. The inhibitor is useful because its structure contains sites available for certain molecules can be added in order to change the potency and selectivity of the inhibitor for a specific PTP.
The researchers then took their platform further by developing specific PTP inhibitors that could act against two PTP targets: the virulence factor mPTPA secreted by Mycobacterium tuberculosis and the striatal-enriched protein tyrosine phosphatase (STEP), a tyrosine phosphatase that is specific to the central nervous system. They chose to do this as a proof-of-concept experiment to demonstrate the efficacy of their prodrug platform. Both molecules were found to inhibit their respective targets in a potent and selective manner.
Drug Efficacy in the Test Tube
Tuberculosis, the lung disease that infects one-third of the world’s population and causes over one million annual deaths, is caused by the Mycobacterium tuberculosis bacterium. On top of that, over 50 million people develop multidrug resistant tuberculosis, and current treatments for this disease are limited. As such, when two PTPs secreted by the bacterium, mPTPA and mPTPB, were identified as potential drug targets, this discovery spurned new interest in developing tuberculosis treatments. “Tuberculosis drug resistance is a serious, ongoing problem and often occurs through mechanisms that limit a drug’s accessibility to its biomolecular target. Tuberculosis PTPs are intriguing because the bacteria secrete these enzymes rendering them much more accessible than the targets of most tuberculosis drugs, which reside within bacterial cells. However, additional research is needed to validate mPTPA and mPTPB as drug targets,” Ellman remarked.
This work also addressed a key problem in PTP inhibitor development. Namely, there is a high amount of structural similarity among PTPs that makes it difficult to achieve high selectivity of their developed inhibitors. The researchers evaluated the selectivity of their mPTPA inhibitor against a collection of known human PTPs, and also a generic cysteine protease, which is an enzyme that breaks down proteins using a key cysteine amino acid found within the protein of interest. Here they found that their mPTPA inhibitor had great selectivity against each enzyme in this panel, indicating that their inhibitor could act in a controlled and predictable manner.
Drug Efficacy in a Biological Setting
After testing their PTP inhibitors in a test-tube setting, the next step was to evaluate their prodrug in a cellular context. However, in animal models, it was found that both mPTPA and mPTPB inhibitors were needed for significant antibacterial activity. Because they chose only to develop an inhibitor against mPTPA at this stage of their research, they instead decided to develop selenosulfide prodrug inhibitors to another PTP target in order to do a more simple and straightforward analysis of the prodrug activity in the cell.
The second target, STEP, is a central nervous system (CNS)-specific tyrosine phosphatase that may be a therapeutic target for neurological disorders like Alzheimer’s disease. After testing a variety of potential prodrugs, they identified one that could inhibit STEP in rat cortical neurons.
After demonstrating the activity and specificity of their PTP inhibitors, they reported their success in developing a prodrug strategy to facilitate the delivery of a novel class of PTP inhibitors into cells in an efficient manner. Their development of inhibitors for two PTPs that can selectively inhibit mPTPA and STEP very potently also acted as a robust proof-of-concept demonstration, showing that their strategy for targeting PTPs is feasible and has great potential.
Future Promises of PTP-Inhibitor Drugs
In the future, Ellman hopes to expand upon this research. “We intend to investigate a number of questions to advance the approach. For example, we will evaluate proteome-wide specificity of identified inhibitors,” he said. Of the inhibitors developed in his lab so far, their group will need to see how these inhibitors act across the entire proteome, which is the collection of all proteins present in our cells. In doing so, they can determine if the inhibitor acts on a different protein or group of proteins that was not anticipated, which could have severe consequences if the inhibitor targeted a protein essential for our survival.
Furthermore, Ellman hopes to expand upon the collection of PTP inhibitors already developed in his lab. “We additionally intend to test the generality of the approach by developing potent and selective inhibitors of other PTPs as well as other enzymes,” Ellman said. If successful, this could result in a greater number of potential drugs for disease treatment involving PTP inhibition. For example, some PTPs have been implicated in cancer, and inhibitors of these enzymes have been suggested as potential drug candidates to be used in combination with immunotherapy treatments. Although such treatments would require more study and clinical tests, the future of cancer treatment using PTP inhibitors remains promising. The use of PTP inhibitors extends beyond cancer treatment, having vast implications in both neurodegenerative disorders and diabetes, two diseases with wide prevalence in society that warrant crucial further research and drug development.
About the Author
Mindy Le is a junior in Ezra Stiles College studying Molecular, Cellular, and Developmental Biology. She is an avid squirrel enthusiast who works in Professor Patrick Sung’s lab, researching DNA repair in the context of breast and ovarian cancer.
Acknowledgements
The author would like to thank both Professor Jonathan Ellman and Caroline Chandra Tjin for their time and dedication to their research.
References
[1] Chandra Tjin, C. et al. “Glutathione-responsive selenosulfide prodrugs as a platform strategy for potent and selective mechanism-based inhibition of protein tyrosine phosphatases.” ACS Cent. Sci. 3: 1322-1328.
[2] Interview with Professor Jonathan Ellman, Yale Department of Chemistry and of Pharmacology, and Caroline Chandra Tjin (graduate student), e-mail interview on 02 February, 2018.
Extra Reading
[1] Tonks, N. K. 2013. “Protein tyrosine phosphatases–from housekeeping enzymes to master regulators of signal transduction.” FEBS J. 280: 346-378.
[2] He, R. J.; Yu, Z. H.; Zhang, R. Y.; Zhang, Z. Y. 2014. “Protein tyrosine phosphatases as potential therapeutic targets.” Acta Pharmacol. Sin. 35: 1227-1246.
[3] Stanford, S. M.; Bottini, N. 2017. “Targeting tyrosine phosphatases: Time to end the stigma.” Trends Pharmacol. Sci. 38: 524-540.
[4] Zhang, Z. Y. 2017. “Drugging the undruggable: therapeutic potential of targeting protein tyrosine phosphatases.” Acc. Chem. Res. 50: 122-129.
[5] Reich, H. J.; Hondal, R. J. 2016. “Why nature chose selenium.” ACS Chem. Biol. 11: 821-841.