Suicide inhibitors of the human enzyme PARP-14

Abstract

Introduction: PARP-14 is a human enzyme that use NAD+ to perform ADP-ribosylation on targets, which serves as a signalling system. The enzyme is important in for instance DNA repair and immunity, as well as regulating other PARPs in the enzyme family.
Background: There are several features of PARP-14 that needs to be further studied, such as its connection to some diseases and cancers. It is of great interest to find a selective inhibitor against the enzyme to enable further studies as well as of therapeutic relevance.
Aim: The purpose of this study is to test two inhibitors, nr. 49 and 45, against PARP-14 with the intention of them exhibiting suicide inhibition.
Methods: Firstly, the catalytic domain of PARP-14 encoded by a plasmid was transformed to competent Escherichia coli which produced the protein. The cells were lysed, and the protein was purified by IMAC and SEC FPLC, to analyze the purity SDS-PAGE was used. The PARP-14 enzymatic activity was then tested with its substrate by detecting ADPr activity by Western Blotting. Different conditions of the inhibitors incubated with PARP-14 was tested and compared to the normal activity of PARP-14. The substances were tested for suicide inhibition by trying to outcompete them by adding a great excess of NAD+.
Results: The transformation of bacteria and protein production as successful, and after purification by IMAC and SEC the protein exhibited ADPr activity on Western blot. Both substance 49 and 45 displayed inhibitory effects on PARP-14 activity, and compound 49 had a greater effect at high concentration. When testing for suicide inhibition, there was an observable decrease in PARP-14 activity, and this was the greatest for pH 9, then 8,2 and the least at pH 7,5 after an overnight incubation.
Conclusion: The substances effectively decrease the activity of the enzyme and shows great potential in being suicide inhibitors of PARP-14.

Popular Abstract

The definition of life and its boundaries is difficult to limit, but what is certain is that chemical reactions are fundamental for all life. Inside our bodies, an abundance of reactions take place, and these are catalysed by enzymes. The focus of this study is on the human enzyme PARP-14, which serves an important role in fighting illness as well as protecting our genetic code. This enzyme is also observed to be involved in some diseases and certain types of blood and liver cancer, and it has potential in being a medical target. Cancer is one malady that is of great interest in modern drug research. A close relative to PARP-14; PARP-1, is already an approved target in treatment of breast and ovarian cancer. PARP-14 is also observed to be involved in some cancers, but the full purpose of this enzyme in the cell is still obscure and need to be further studied. When using inhibitors against enzymes, their activity is blocked, and this is convenient when studying their purpose, since their inhibition result in an absence of a function. Further studies of the PARP-family are of great interest, and selective inhibitors are of importance to conduct this.

The aim of this study was to test two potential inhibitors of PARP-14. An ambition was also that these inhibitors when in contact with the enzyme, get bound to enzyme’s reactive site, not able to leave. This is called a suicide inhibition, since it results in the enzyme becoming permanently inactive, as if committing suicide.

The substances, nr 49 and 45, were synthesized and provided by prof. Dana Ferraris, which have produced inhibitors against PARP-14 before with known inhibitory effect, although no suicide inhibitors. To be able to test the new compounds, the PARP-14 protein was produced by bacteria, which were decomposed, and the protein of interest was collected by purification methods. The purified protein was then tested for enzymatic activity to verify that the protein was functional. The protein could then be tested with and without inhibitors, at different conditions, to see if the inhibitors had a desirable effect on PARP-14. If the enzymatic activity of PARP-14 is lower with the inhibitor present compared to not, this indicates on inhibition. To investigate if the inhibitors also exhibited suicide inhibition, methods were used to try to outcompete them. When this method is used, the real substrate of the enzyme is added in a great access over inhibitor. If the inhibitor is irreversible bound to the enzyme, this will block the real substrate from interacting with the enzyme.

The protein was successfully produced and after two essential purification steps it was verified that it exhibited activity. The inhibitors were then tested, and they proved to be inhibiting PARP-14 effectively. They also displayed suicide inhibitory effects, since when trying to outcompete them, a decreased PARP-14 activity persisted. This effect was the greatest after overnight incubation and at a pH well above the physiological of 7,4.

• author: Olsson, Hannah
• supervisor: Herwig Schüler
• organization: Department of Chemistry
• course: KEMK10 20241
• year: 2024
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: ADP-ribosylation, biochemistry, PARP-14, Suicide inhibition
• language: English
• id: 9155309
• date added to LUP: 2024-05-30 14:07:01
• date last changed: 2024-05-30 14:07:01