Cathepsins B, D, and K have been suggested to play important roles in the metastatic potential of several types of cancer, and have been implicated in the pathology of a wide variety of diseases. Most assume cathepsins B, D, and K to be restricted to the lysosomes where they are involved in protein degradation. However, many researchers have found that under many diseased conditions cathepsins are released into the cytosol and appear in the serum. The use of several chemotherapeutic drugs have been found to cause a release of cathepsins into the cytosol, which leads to activation of caspases and apoptosis. Cathepsin K has been associated with accelerated bone degradation in patients with osteoporosis, rheumatoid arthritis, lung cancer, prostate cancer, and breast cancer. A high activated cathepsin D level in breast tumor tissue has been associated with an increased incidence of relapse and metastasis. In fact cathepsin B and D levels have been used as markers to predict the prognosis of breast cancer and uterine cancer patients. There is an urgent need for new, more effective protease inhibitors as therapeutic agents. A continuation of the design and synthesis (hydroxyethyl)amine isosteres as inhibitors as cathepsin D based on SAR data is proposed. In the initial phase of this project 96 of these (hydroxyethyl)amine isosteres have proven to be very potent inhibitors of cathepsin D activity. Also, the continuation of the design and synthesis of aldehyde and thio-semicarbazone inhibitors of cathepsin B is proposed. In the Initial phase of this project 16 new cathepsin B inhibitors have been developed. As an addition in this phase of the project, new inhibitors of cathepsin K will be synthesized and assayed. The synthetic inhibitors will be tested on whole cells and lysates of whole cells. Detailed kinetic studies and structure activity correlations will then be drawn for the synthetic inhibitors. This project is designed as a research effort which will allow undergraduate students training in many aspects of biomedical research not normally available at a rural undergraduate institution.
The design, via molecular modeling, and preparation of cathepsin B, D, and K inhibitors is proposed. Cathepsin D is an aspartyl protease associated with metastasis of several types of cancer. Cathepsin B and K are cysteinyl proteases associated with tumor progression, Alzheimer's disease, osteoporosis, and rheumatoid arthritis, as well as bone degradation associated with breast cancer, prostate cancer, and lung cancer. The synthetic compounds will be tested for their potency as protease inhibitors of cathepsin D, cathepsin B, or cathepsin K by fluorometric assay techniques. In this phase of the grant we propose to develop cathepsin K inhibitors, in addition to improving upon our previous work in the development of potent cathepsin B and D inhibitors. We propose to clone the genes for the proenzymes, express the procathepsins, activate the enzymes and assay the synthetic compounds for their inhibition of cathepsins B, D, and K in whole cells and cell lysates. Rate constants, inhibition constants, and other kinetic parameters, as well as structure-activity correlations will be documented. Considering the rationale behind this approach, some of the compounds may well be very potent inhibitors of these biologically important proteases. This work could lead to useful biochemical tools for determining the role of specific enzymes in diseased conditions, and may result in the development of new therapeutics for the treatment of these devastating diseases. Investigations of this type in undergraduate institutions will allow students to participate in research training from the inception of the idea. The computer modeling component will allow students to learn the current techniques in the rational design of chemical compounds. The students will then learn to develop and perform a research protocol in synthesizing the compounds designed by computer modeling. They will synthesize, isolate, purify, and characterize the compounds by current chemical and instrumental techniques (i.e. HPLC chromatography, NMR spectroscopy, etc.). The students will learn gene cloning techniques, protein expression and isolation, and enzyme activation techniques. In testing the biological activities of the potential inhibitors, the undergraduate students will learn tissue culture techniques and enzyme analysis. In this component the students will learn to optimize conditions and modify existing assays to fit experimental conditions. Determination of kinetic parameters will require the students to collect, analyze, and conduct statistical analysis of the data. Therefore, the project will provide students with practical experience with many aspects of biomedical research. Student participation in this type of research project should demonstrate the vital link between the chemical structures of compounds and their biological activities. Therefore, this project would greatly improve the atmosphere for and the availability of research in this rural undergraduate institution.
|McConnell, Rose M; Inapudi, Kalyani; Kadasala, Naveen et al. (2012) New cathepsin D inhibitor library utilizing hydroxyethyl isosteres with cyclic tertiary amines. Med Chem 8:1146-54|