Projects / Programmes
Design and characterization of allosteric modifiers of cysteine cathepsins
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
allosteric regulation, cysteine cathepsins, drug design
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
26028 |
PhD Marko Novinec |
Biochemistry and molecular biology |
Head |
2011 - 2013 |
0 |
Organisations (1)
Abstract
Human cysteine cathepsins, members of the papain-like cysteine peptidases family, have been vastly studied for their involvement in numerous physiological and pathological processes. Aside from being responsible for non-specific protein turnover in lysosomes, cysteine cathepsins perform a number of specific physiological roles. The regulation of cysteine cathepsins by endogenous competitive inhibitors has been thoroughly investigated, there is however little known about allosteric regulation of these enzymes.
Allostery is the coupling of conformational changes between two separated sites and it is an important mechanism in the regulation of numerous proteins. Targeting allosteric sites is becoming an increasingly popular strategy in drug development because allosteric drugs can be designed to fine-tune the activities of their targets. In this project we will characterize mechanisms of allosteric communication in cysteine cathepsins and design novel allosteric modifiers of these enzymes for use in vitro and in vivo.
It has been proposed that allosteric communication in proteins is mediated by conserved networks of residues. We will use the computational algorithm statistical coupling analysis to identify such networks in cysteine cathepsins. From these results we will predict the locations of possible allosteric sites. Our primary targets will be cathepsins K and B. Both enzymes have received a lot of attention in recent decades due to their involvement in severe pathological conditions. The former has been intensely studied due to its involvement in bone turnover and a series of specific cathepsin K inhibitors have been developed for the treatment of osteoporosis, while the latter plays a role in the tissue remodeling processes associated with osteoarthritis and cancer development.
Potential allosteric modifiers will be identified by in silico screening of libraries of commercially available compounds for potential binders at allosteric sites. Identified compounds will be obtained and thoroughly tested for effects on their targets. In comparison with active site-directed inhibitors, allosteric modifiers can potentially exhibit a much wider spectrum of effects, affecting activity, stability, etc. of their targets. Identified modifiers will therefore be extensively tested to determine their specificities and activities with particular emphasis on their kinetic mechanisms of action. The most effective modifiers will also be tested for their activites in cell cultures and structurally characterized by determining the crystal structures of enzyme-modifier complexes. Our final goal is to design modifiers capable of fine-tuning the activities of cathepsins K and B. Such substances could serve as ideal therapeutics because they are able to reduce unwanted proteolysis to the level essential for normal physiological function.
Significance for science
The relevance of this project to the development of science can be evaluated from two perspectives, the novelty of methodology used and the scientific value of the project. Considering the methodology, our study is one of the first to have successfully utilized a combination of computational methods to both predict the locations of allosteric sites in enzymes and design modifiers targeted at these sites. Regarding its scientific value, our work represents a major step forward in our understanding of the mechanisms involved in the regulation of cysteine cathepsin activity at the molecular level. The importance of allosteric regulation is widely documented for numerous enzymes involved in diverse biological processes, it had been, however, largely overlooked or dismissed as a strategy in the regulation of cysteine cathepsins. This project was the first to directly address and demonstrate the existence and importance of allosteric regulation in cysteine cathepsins. The identified cathepsin K inhibitor NSC13345 is the first known low-molecular-weight allosteric inhibitor of cysteine cathepsins. The compound has all the properties theoretically expected from allosteric inhibitors, but rarely found in practice. It has high specificity for cathepsin K and it has the ability to specifically inhibit the degradation of collagen, the most important natural substrate without abolishing the hydrolysis of other substrates. These properties qualify NSC13345 as an excellent candidate for drug development as well as for experimental use. The same conclusions can be reached for several other compounds identified in this study. Taken as a whole, this project presented a novel approach to studying the mechanisms of protein function and will as such undoubtedly serve as inspiration for numerous studies on other proteins in the future.
Significance for the country
The results of our project are of direct interest to the pharmaceutical industry. Cysteine cathepsins, like many other peptidases, are very appealing drug targets. Several cathepsin K inhibitors are currently undergoing clinical trials for the treatment of osteoporosis. Design of allosteric inhibitors offers an attractive alternative to active site-directed probes currently in development. Morevoer, the rational approach to allosteric drug design introduced in this project is much more economical and error-free than random screening of compound libraries in vitro. Diseases associated with excessive proteolytic activity,such as osteoporosis, arthritis, atherosclerosis, etc. are becoming an increasingly pressing health problem in modern society. Because of its potential application in the treatment of these diseases, the results of our project may also contribute to improved health and quality of life of the residents of Slovenia. From the academic perpsective the project intensified our collaboration with the research group of prof. Antonio Baici (University of Zürich, Switzerland) who is one of the best experts in enzyme kinetics today and sprouted novel collaborations with the groups of prof. Rama Ranganathan (University of Texas, Dallas, USA) and prof. Amedeo Caflisch (University of Zürich, Switzerland) who are both world-leading experts in the fields of bioinformatics and computational biochemistry. In these collaborations we have acquired knowledge of novel experimental and computational methods that have not been previously established in Slovenian laboratories. Thereby, this project also contributed to the development of the Chair of Baiochemistry at UL FCCT as an independent research group performing state-of-the-art research. This is in turn reflected in the quality of education passed on to the students of our faculty by both being able to present them firsthand with quality research as well as offer them the opportunity to participate in ongoing research activities.
Most important scientific results
Final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2012,
final report,
complete report on dLib.si
