The main goal of the proposed project is understanding the interaction between the onco-suppressor kinase HIPK2 and its target p53 by an integrated structural biology approach.
I will combine crystallography and single particle cryo-electron microscopy (SP Cryo-EM) to achieve structural determination at different resolutions and attempt providing a picture of the molecular interactions that govern recognition and catalysis.
HIPK2 is a nuclear Ser/Thr kinase that co-regulates specific transcription factors during two fundamental biological processes: embryonic differentiation and development, and cellular response to DNA-damaging agents. A well-understood function of HIPK2 is its role in response to high DNA damage. A severe DNA damage elicits the HIPK2 kinase activity on p53, promoting changes in its affinity towards pro-apoptotic genes, and sending irreversible apoptosis signals. Dysregulation of HIPK2 that impairs p53 pro-apoptotic functions has been described in several tumor types, thus making HIPK2, and its activity on p53, an attractive antitumoral drug target to rescue and potentiate to counteract onco-transformation due to DNA damage. Despite of being a well-recognized anticancer target, molecular effectors selectively modulating the activity of HIPK2 and/or of its functional complex with p53 are currently not known, possibly because of the absence of structural information on this system.
With the aim to achieve a molecular strategy to selectively modulate the HIPK2 activity on p53, we plan to study the structure of the HIPK2-p53 complex by integrating SP Cryo-EM and X-ray crystallography. This information will allow the identification or the rational design of compounds able to selectively bind the complex, promoting an allosteric regulation of the interaction. In parallel, we will sieve existing drugs/compounds and natural products to examine their effect on the HIPK2 activity on p53. The most promising compounds will be tested in cellular systems.
In this project, we pursue the structural characterization of the crucial complex between p53 and the nuclear Ser/Thr kinase HIPK2 that influences cellular fate in case of severe DNA-damage. Dysfunctions of both partners alter the specific phosphorylation activity of HIPK2 toward p53 that regulates its pro-apoptotic function, thus compromising the apoptotic response of a damaged cell in favor of tumor development.
Playing a key role in virtually all the human cancers, p53 has been the object of numerous studies focused on its biological and clinical aspects, given its potential for therapeutic applications. Since 2002, when HIPK2 was found to physically and functionally interacting with p53 in response to DNA damage, the role of this kinase as oncosuppressor has been investigated in cellular and living systems. Different aspects of its pro-apoptotic activity have been clarified, but the molecular basis of HIPK2 activation and p53 recognition and phosphorylation still escapes a detailed description, given the absence of structural and functional characterizations in vitro. Elucidating the biophysical aspects of this process would increase our understanding on their function, making a substantial contribution to human physiology, and opening the possibility to translate this knowledge into future therapeutic tools (e.g., the structural data will provide a platform for in silico screening of existing drugs to increase HIPK2 activity on p53 or other physiological partners).
The driving idea to gain structural information on molecular targets crucial for rescuing damaged tissue from tumorigenesis will be used for the development of feasible innovative strategy in treating cancer. The reactivation of the pro-apoptotic pathways stimulated by the kinase activity of HIPK2 toward p53 in damaged cells might be a novel possible therapeutic approach in blocking tumor progression. The determination of structure of HIPK2 in complex with p53 (with/without Axin) will provide information to design, synthetize and characterize small ad hoc molecules that specifically recognize and interact on sites different from the ATP binding task or from the HIPK2-p53 interacting region promoting conformational changes of the kinase that stabilise the active forms.
To this purpose, we plane to sieve existing drugs/compounds and natural products from the secondary metabolism of actinomycetes using biochemical methodologies. To our knowledge, molecular effectors performing such activation on HIPK2 are still unknown.
Achieving the goals of this project will bring new relevant knowledge to the field of programmed cell death and speed up the identification of molecular effectors directed towards that complex.
For this reason, the most innovative task of this research is the identification of systems or molecules able to target functional HIPK2 complexes with p53 and molecular partners to hamper or enhance functional complex formation.
The objectives we propose are very ambitious and endowed with challenges. The strength of the proposed research resides in the multidisciplinary approach and collaborations with international, highly qualified groups, joining the expertise in molecular biology, biochemistry, X-ray crystallography, single particle cryo-electron microscopy, bioinformatics and combinatorial biosynthesis.
Overall, the long-term outcome of the proposed studies is to provide an alternative/innovative strategy to expand the current available portfolio of anticancer treatments and improve their effectiveness targeting a specific molecular interaction that activate physiological apoptotic response in malignant cells.