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Мodulation of Аpoptosis in Cardio-Vascular Diseases

#3145


Modulation of Apoptosis as a New Approach for Prevention and Therapy of Acute Heart Diseases

Tech Area / Field

  • MED-DRG/Drug Discovery/Medicine
  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-OTH/Other/Medicine
  • BIO-OTH/Other/Biotechnology

Status
3 Approved without Funding

Registration date
04.11.2004

Leading Institute
Institute of Physiologically Active Substances, Russia, Moscow reg., Chernogolovka

Supporting institutes

  • State Research Institute of Organic Chemistry and Technology, Russia, Moscow

Collaborators

  • University of Michigan / School of Public Health, USA, MI, Ann Arbor\nUS Department of Health & Human Services / National Institute of Health, USA, MD, Bethesda

Project summary

Despite advances in treatment, cardio-vascular diseases are the major cause of morbidity and mortality in the Western world and Russia. That is why, the development of novel therapeutic approaches, which are based on the recent concept of molecular mechanisms of such diseases, is of great value. Acute heart diseases as myocardial infarction, ishemia-reperfusion, and atherosclerosis were shown recently to involve apoptosis as a form of myocardial cell death. Apoptosis is a well-controlled form of cell death, characterized by typical morphological changes of suicidal cells. These changes are different from necrosis and include shrinkage of cytoplasm, chromatin condensation and DNA fragmentation. These phenomena result from a series of different biochemical events in which caspases, a family of cystein proteases, play a central role. Caspase-3 is one of the key effectors of apoptosis. Intracellular Ca2+ level also plays a great role in apoptosis. Recently, increased oxidative stress has been shown to promote apoptosis.

It’s clear now that the mitochondria play the key role in cell’s calcium homeostasis and in initiating the irreversible stage of cell dearth cascade via release of proapoptotic agents and/or disruption of cellular calcium and energy metabolism. The mechanisms of mitochondrial involvement may include the activation of mitochondrial permeability pores (MPP), which tightly connected with production of reactive oxygen species and lipid peroxidation, releasing of cytochrome C, AIF, procaspases et al. As a part of a central mechanism of amplification of the apoptotic signal, mitochondrial permeability transition may be an appropriate target for therapeutic agents designed to prevent the degeneration of cells. In fact, intracellular Ca2+ level, caspase-3 activity and ROS generation may be the targets of newer therapeutical treatment for various heart diseases. It is proved by recent publications that several caspase-3 inhibitors and antioxidants are able to inhibit apoptotic pathways and reduce heart damage.

However, it should be noted that many molecular mechanisms of apoptosis are still unknown. There are very few publications about low-molecular synthetic substances that influence on apoptosis, while the most of them are concerned with apoptosis induction in tumor cells. The broad significance of apoptosis in the cardiovascular system only began to be recognized more widely recently. That is why, a direct synthesis of analogs of substances that were revealed to influence on key apoptosis events such as intracellular calcium, caspase-3 activity and ROS, seems to be very perspective. Endogenic sulfur-containing substances play a fundamental role in cell biochemical processes. For this reason organosulfur components of natural matrixes, influencing on apoptotic process, may be an attractive base for the search of new low-molecular therapeutic agents for acute cardio-vascular diseases treatment. Our own recent investigations on influence of several natural and synthetic organosulfur compounds on cell calcium homeostasis and ROS generation confirm an outlook of this approach.

During the Project realization a broad range of novel low-molecular sulfur-containing substances will be synthesized. The influence of synthesized substances on apoptosis and the apoptosis factors as intracellular calcium level, mitochondrial functions, lipid peroxidation, caspase-3 activity and ROS generation will be investigated. On the base of the obtained results a correct prognostic QSAR model will be created to correlate biological activities with the structures of developed substances. The most active substances will be proposed as potential cardio-vascular drugs of new kind of action to prevent and treat the acute cardiac diseases.

The aim of the Project is synthesis of novel low-molecular sulfur-containing substances as potential modulators of apoptosis for the development of new approaches to the prevention and treatment of various cardio-vascular diseases. Influence on apoptosis will be achieved by modulation of intracellular calcium level, inhibition of caspase-3 and scavenging of reactive oxygen species (ROS).

The Project authors have an expertise in organic synthesis, cell molecular mechanisms investigations including intracellular Ca2+ homeostasis, ROS generation, enzymes and QSAR approaches. Project participants have a long-time experience in research work on ISTC (# 870, 1055.2) and CRDF (# 2035, 2488) projects.

Expected results and their application. As a result of the Project realization a series of new low-molecular sulfur-containing substances will be synthesized as potential apoptosis modulators by influence on intracellular Ca2+ homeostasis, ROS generation, and cell caspases inhibition. Using QSAR approaches, the novel pharmacological substances will be designed for the prevention of heart injuries resulting from apoptosis. These substances may be considered as a basis for the creation of cardio-vascular drugs of new type of action.

The Project results will allow to broaden knowledge of apoptosis mechanism and to reveal molecular targets for the modulation of these processes. The effective apoptosis modulators, developed as a result of the Project realization, may be applied not only for the treatment of heart injuries, but also for the influence on apoptotic events resulting in neurodegenerative diseases.

Utility. The development of novel pharmacological substances, influencing on the earlier events of acute cardio-vascular diseases will provide the effective prevention of assisted with such injuries complications which may cause a death of patient. The pharmacological substances to be developed during the Project performing, will be low-molecular compounds accessible by organic synthesis. Due to, the technology for their probable production seems to be inexpensive and sufficiently simple. In the case of successful realization of the Project aims the novel original class of cardio-vascular pharmacological substances will be proposed for the clinical trials and following technological development.

The scope of activities of the Project meets the main Goals and Objectives of ISTC. The most of Project participants, the former weapon scientists and specialists have a great experience in the fields, related with the main Project tasks: organic synthesis, cell mechanisms studying, QSAR creation. It will give them a possibility to use their knowledge and experience for Public Health problems resolution. Moreover, the Project realization will allow to extend the possibilities for the integration of Russian scientists into world scientific cooperation, provide a support for peaceful fundamental and applied investigations in the field of prevention and treatment of cardio-vascular diseases, assist to Russian Public Health development, and promote the development of Russian pharmaceutical market.

To achieve the Project aims we are planning to perform the following tasks:


· A broad range of analogs of natural sulfur-containing substances will be synthesized, which are believed to influence on key apoptosis events. For design of target substances such structural fragments will be used as disulfide-, trisulfide, sulfoxide, cysteine-, allyl groups and others. The combination of different structural elements will provide a direct synthesis of lipido- and water-soluble substances.
· The influence of sulfur-containing substances synthesized during the previous Project stage, on cell Ca2+ homeostasis, mitochondrial functions, caspase-3 and reactive oxygen species generation (ROS) and lipid peroxidation. The influence of sulfur-containing substances on blood cells of healthy donors and cardio-vascular diseases patients will be investigated.
· The influence of sulfur-containing substances on the main apoptosis biomarkers will be studied.
· On the base of the results of the previous Project stages the relationship between anti-apoptotic activity and structure of tested substances will be evaluated and QSAR model for physiological activity prediction will be created.
· QSAR data obtained on the previous Project stage will be used for the synthesis of more effective apoptosis modulators.
· On the base of Project results (the influence of tested substances on such apoptosis events as intracellular Ca2+, mitochondrial functions, lipid peroxidation, caspase-3 and ROS generation) the most active synthetic sulfur-containing compounds will be proposes as potential cardio-vascular drugs of new type.

The following technical approaches and methodologies will be used: flash chromatography; analytical and preparative high-performance liquid chromatography; analytical and preparative gas chromatography; nuclear magnetic resonance (NMR) spectroscopy; infra-red (IR) spectroscopy; chromato-mass spectrometry (GC-MS), fluorescence method for registration of intracellular free calcium, using FURA-2 dye and standard reagents (thapsigargin, ionomycin, digitonin, 2,5-di-tert-butyl hydroquinone, and cyclopiazonic acid), “oxidative burst” model with phorbol 12-myristate 13-acetate and N-formyl-peptide as superoxide anions stimulators, colorimetric (or fluorimetry) method of caspase-3 activity determination, DNA fragmentation method for the detection of apoptosis. Activity of mitochondrial respiration chain will be studied using polarography method by measuring oxygen absorbtion with Clark electrode. Opening PTP will be indicated by mitochondria swelling. Swelling, which represent the change of mitochondrial forms, will be measured as a decrease of the optical density of the mitochondria suspension. Lipid peroxidation in mitochondrial membranes will be followed by the accumulation of substances that reacted with thiobarbituric acid, and monitored spectrophotometrically.


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