Final Report Summary - DOS (Synthesis of small molecules and evaluation of their biological activity: discovery of new protein-protein interaction inhibitors)
Vital cellular functions are all regulated by protein complexes. In order for a complex to work correctly, interactions among its subunits must have the right intensity. If they are too weak or too strong, the complex does not work well, causing disease. Thus, modulation of protein-protein interactions (PPIs) offers a strategy for therapy. The project goal was to discover new PPI inhibitors by the synthesis and biological evaluation of small molecules.
In the search for new protein-protein interaction (PPI) inhibitors, polo-like kinase 1 (PLK1) was selected as the biological target due to its relevance in therapy and relative stability. In order to develop PLK1 inhibitors, we followed three approaches: 1) peptidomimetic macrocycle, 2) peptide stapling, and 3) fragment-based discovery (FBD) approach. For each strategy, a set of compounds was identified by virtual screening, prepared by liquid or solid phase synthesis techniques (except for fragments which were commercially available), and tested by fluorescence polarization to evaluate the binding affinity to PLK1.
The main results are outlined below:
- Peptidomimetic macrocycle approach: a peptidomimetic macrocycle was identified by virtual screening, synthesized by liquid phase synthesis techniques, and evaluated by fluorescence polarization, but no activity was found;
- Peptide stapling approach: four stapled peptides were prepared and their biological activity evaluated by fluorescence polarization, but they showed either the same activity or a lower activity than the corresponding linear peptides;
- FBD approach: the binding affinity of more than 400 fragments for the PLK1 polo-box domain was measured by fluorescence polarization, and hits were identified;
- X-ray crystal structures were determined for complexes of the PLK1 polo-box domain with high binding affinity compounds;
- The active fragments were combined into larger molecules by a linking approach. Large molecules displayed a higher binding affinity for the PLK1 polo-box domain than the single fragments according to fluorescence polarization, but they stabilized the protein according to thermo-shift assay.
From a scientific viewpoint, the work made a significant contribution to the field of chemical biology by improving the understanding of protein-protein interactions. This topic is very important due to the large number (~100,000) of proteins in human body, yet still underexplored. From a medical perspective, this work paved the way to the development of new anticancer drugs. As PLK1 is involved in a large number of human cancers, the marketing of a PLK1 inhibitor would have a benefit for millions of people worldwide. Therefore, the results of this project are of relevance to both academia and industry.
During the fellowship, the researcher has received training in the use of molecular modelling (Schrodinger® software), biophysical techniques (fluorescence polarization and thermos-shift assay), high-performance liquid chromatography, and protein-ligand crystallization techniques (soaking and co-crystallization). He also enhanced his theoretical and practical skills in organic synthesis by learning new synthetic approaches such as solid phase synthesis, and by performing reactions in which he had little experience, such as ‘click’ reactions, cyclization reactions, and protection/deprotection reactions. The fellow regularly presented his research results at group meetings, which facilitated detailed discussion with colleagues and supervisor.
The researcher had the opportunity to attend a full range of postgraduate lectures covering all aspects of chemistry, as well as research seminars given by leading visiting lecturers, including several winners of the Nobel Prize. Furthermore, the researcher attended several workshops offered by the University of Cambridge Career Service for his personal and professional development: CV and cover letters, bibliographic research, scientific writing, peer reviewing, grant proposals, lecturing, undergraduate and graduate student supervisions, career options, leadership, and personality profile. He was selected to attend the Teaching Associate Programme offered by the University of Cambridge in preparation for a final exam to join the Higher Education Academy. He also attended three courses of Chinese language (basic 1, basic 2 and elementary 1 levels) offered by the University of Cambridge Language Centre to enhance his language skills.
In addition to training activities, the fellow took part in various integration activities. On arrival, he received full support from the university staff in the necessary administrative procedures: registration, accommodation, health insurance, and safety regulations. He also attended an induction seminar where he was given information on campus life and training facilities and was introduced to key personnel within the department. He fully integrated in the group of Prof. D. R. Spring as a postdoctoral researcher. He provided support to PhD students with both experiments and thesis proof-reading. He was also part of a small team of lab members for the creation and managing of a new compound library. He enjoyed full autonomy in experiment planning, total flexibility in working time, and maximum professional and emotional support from his supervisor.
In the search for new protein-protein interaction (PPI) inhibitors, polo-like kinase 1 (PLK1) was selected as the biological target due to its relevance in therapy and relative stability. In order to develop PLK1 inhibitors, we followed three approaches: 1) peptidomimetic macrocycle, 2) peptide stapling, and 3) fragment-based discovery (FBD) approach. For each strategy, a set of compounds was identified by virtual screening, prepared by liquid or solid phase synthesis techniques (except for fragments which were commercially available), and tested by fluorescence polarization to evaluate the binding affinity to PLK1.
The main results are outlined below:
- Peptidomimetic macrocycle approach: a peptidomimetic macrocycle was identified by virtual screening, synthesized by liquid phase synthesis techniques, and evaluated by fluorescence polarization, but no activity was found;
- Peptide stapling approach: four stapled peptides were prepared and their biological activity evaluated by fluorescence polarization, but they showed either the same activity or a lower activity than the corresponding linear peptides;
- FBD approach: the binding affinity of more than 400 fragments for the PLK1 polo-box domain was measured by fluorescence polarization, and hits were identified;
- X-ray crystal structures were determined for complexes of the PLK1 polo-box domain with high binding affinity compounds;
- The active fragments were combined into larger molecules by a linking approach. Large molecules displayed a higher binding affinity for the PLK1 polo-box domain than the single fragments according to fluorescence polarization, but they stabilized the protein according to thermo-shift assay.
From a scientific viewpoint, the work made a significant contribution to the field of chemical biology by improving the understanding of protein-protein interactions. This topic is very important due to the large number (~100,000) of proteins in human body, yet still underexplored. From a medical perspective, this work paved the way to the development of new anticancer drugs. As PLK1 is involved in a large number of human cancers, the marketing of a PLK1 inhibitor would have a benefit for millions of people worldwide. Therefore, the results of this project are of relevance to both academia and industry.
During the fellowship, the researcher has received training in the use of molecular modelling (Schrodinger® software), biophysical techniques (fluorescence polarization and thermos-shift assay), high-performance liquid chromatography, and protein-ligand crystallization techniques (soaking and co-crystallization). He also enhanced his theoretical and practical skills in organic synthesis by learning new synthetic approaches such as solid phase synthesis, and by performing reactions in which he had little experience, such as ‘click’ reactions, cyclization reactions, and protection/deprotection reactions. The fellow regularly presented his research results at group meetings, which facilitated detailed discussion with colleagues and supervisor.
The researcher had the opportunity to attend a full range of postgraduate lectures covering all aspects of chemistry, as well as research seminars given by leading visiting lecturers, including several winners of the Nobel Prize. Furthermore, the researcher attended several workshops offered by the University of Cambridge Career Service for his personal and professional development: CV and cover letters, bibliographic research, scientific writing, peer reviewing, grant proposals, lecturing, undergraduate and graduate student supervisions, career options, leadership, and personality profile. He was selected to attend the Teaching Associate Programme offered by the University of Cambridge in preparation for a final exam to join the Higher Education Academy. He also attended three courses of Chinese language (basic 1, basic 2 and elementary 1 levels) offered by the University of Cambridge Language Centre to enhance his language skills.
In addition to training activities, the fellow took part in various integration activities. On arrival, he received full support from the university staff in the necessary administrative procedures: registration, accommodation, health insurance, and safety regulations. He also attended an induction seminar where he was given information on campus life and training facilities and was introduced to key personnel within the department. He fully integrated in the group of Prof. D. R. Spring as a postdoctoral researcher. He provided support to PhD students with both experiments and thesis proof-reading. He was also part of a small team of lab members for the creation and managing of a new compound library. He enjoyed full autonomy in experiment planning, total flexibility in working time, and maximum professional and emotional support from his supervisor.