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Synthesis of novel inhibitors of proteases and kinases

#A-2169


Design and synthesis of novel inhibitors of proteases and kinases based on non-protein amino acids

Tech Area / Field

  • BIO-CHM/Biochemistry/Biotechnology

Status
3 Approved without Funding

Registration date
05.11.2014

Leading Institute
SPC "Armbiotechnology" NAS RA, Armenia, Yerevan

Supporting institutes

  • Ilia State University, Georgia, Tbilisi

Collaborators

  • Institut fur Pharmakologie und Toxikologie, Universitat Wurzburg, Germany, Würzburg\nUniversity of Saskatchewan, Canada, SK, Saskatoon\nRostok University, Germany, Rostock\nUniversity of Maryland / Department of Chemistry and Biochemistry, USA, MD, College Park

Project summary

The goal of the project is modeling and synthesis of novel peptides able to affect the activity of target enzymes.
Currently non-protein amino acids and peptides based thereon are widely used in biotechnology and pharmacology. Peptides composed of non-protein α-amino acids occupy a special place among active compounds. The biological activity of synthetic peptides is stipulated by their ability to interact with enzymes. Design of a number of modern antibacterial, antiviral, antitumor and other drugs is based on the property of non protein amino acids and peptides either to inhibit or enhance the activity of target enzymes. Short peptides synthesized from non-protein amino acids are promising alternatives to small molecule drugs. These compounds have several advantages: high activity, high specificity, targeting capabilities, minimal drug-drug interactions, low toxicity etc. There are more than 60 peptides in pharmacy, approximately 270 in clinical testing, and about 400 in preclinical phase. Nevertheless there are few disadvantages that should be overcome: low stability in organism, low bioavailability, difficulties in membrane penetration, rapid excretion and risks of immunological response.
The proposed project is continuation of the research fulfilled in the framework of ISTC projects #A-1247 and #A-1677. Seventy novel aliphatic and heterocycle substituted nonprotein amino acids and peptides based thereon have been synthesized in the frame of these projects. Synthesized compounds were screened on microbiological, biochemical and cytological models aimed at revealing biologically active ones. The potential of nonprotein amino acids and peptides to interact with proteases and kinases was studied by docking. According to the obtained results as the most prospective compounds that may be recommended for drug design appeared the following nonprotein amino acids, characteristics of which are given below:
R-, S-stereoisomers of allylglycine inhibit proteinase K; in dipeptides they inhibit bovine trypsin; suppress proliferation of human lymphocytes; inhibit the growth of Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella typhimurium, Erwinia sp.
(2R,3S)-hydroxyleucine and (2S, 3R)-hydroxyleucine inhibit proteinase K and trypsin; in dipeptides inhibit trypsin. Stereosiomers of hydroxyleucine either free or in dipeptides inhibit the growth of some representatives of gram- and gram+ bacteria.
(S)-β-(N-benzylamino)alanine inhibits activity of bacterial aminotransferases.
(S)-β-[4-allyl-3-(3’-hydroxypropyl)-5-thioxo-1,2,4-triazol-1-yl]α-alanine suppresses proliferation of rats’ bone marrow cells. At the same time this compound decreases frequency of spontaneous and NG-induced mutagenesis.
(S)-β-[4-allyl-3-propyl-5-thioxo-1,2,4-triazol-1-yl]α-alanine suppresses proliferation of rats’ bone marrow cells. Tripeptide N-formyl-methionyl-glycyl-(S)-β-[4-allyl-3-propyl-5-thioxo-1,2,4-triazol-1-yl]α-alanine inhibits the growth of some representatives of gram- and gram+ bacteria.
(S)-β-[4-phenyl-3-propyl-5-thioxo-1,2,4-triazol-1-yl]α-lanine suppresses proliferation of rats’ bone marrow cells. Dipeptide N-formyl-methionyl-(S)-β-[4-phenyl-3-propyl-5-thioxo-1,2,4-triazol-1-yl]α-alanine and tripeptide N-formyl-methionyl-glycyl-(S)-β-[4-phenyl-3-propyl-5-thioxo-1,2,4-triazol-1-yl]α-alanine inhibits the growth of some representatives of gram- and gram+ bacteria and increases the frequency of spontaneous and NG-induced mutagenesis.
(S)-β-[4-allyl-3-(pyridin-4’-yl)-5-thioxo-1,2,4-triazol-1-yl]α-alanine suppresses proliferation of human lymphocytes and that of rats’ bone marrow cells. This compound increases frequency of spontaneous and NG-induced muragenesis.
(S)-β-[4-allyl-3-(pyridin-3’-yl)-5-thioxo-1,2,4-triazol-1-yl]α-alanine suppresses proliferation of human lymphocytes and that of rats’ bone marrow cells.
The proposed project assumes to use the above-described compounds for modeling and synthesis of peptides interacting with target proteins. The peptides with low inhibition constants and high specificity will be selected. And as the target for novel synthetic peptides proteases (trypsin, proteinase K, matrix metalloprotеases ММРs, angiotensin converting enzyme ACE) and kinases (protein kinase p38 and pyruvate kinase M2) will be employed.
Proteases play an essential role in vital processes of human organism. Involvement of proteases in the development of various diseases made them suitable targets for drugs. Proteases inhibitors used as antiviral drugs are peptides, which are composed of nonprotein amino acids as well.
ACE, or kinase II, is a dipeptidyl carboxy peptidase found in various tissues in the body and is integral to the moderation of blood pressure and normal heart function. At present a great number of natural and synthetic ACE inhibitory peptides are known. The most common of synthetic peptides is Captopril. Captopril and other synthetic ACE inhibitors exert various side-effects. Besides, these side effects, coupled with the fact that hypertension affects a great part of world’s population and is a risk factor for stroke and cardiovascular disease, has contributed to the ongoing search for food derived and synthetic antihypertensive peptides.
Matrix metalloproteases (ММРs) are a major group of enzymes that regulates cell-matrix composition. Ample evidence exists on the role of MMPs in normal and pathogenic processes including embryogenesis, wound healing, inflammation and cancer. The association of MMPs with cancer metastasis makes them an attractive target for development of novel antimetastatic drugs aimed at inhibiting MMP activity.
Currently intensive investigations of anti-inflammatory and antitumor characteristics of protein kinases inhibitors are in progress. Protein kinases (particularly MAP kinases) activated by mitogen belong to a large family of serine/threonine kinases. The role of kinase p38 is very essential in inflammatory processes and therefore it is considered to be a target for anti-inflammatory and antitumor preparations. Particularly, the anti-inflammatory agent pyridineimidazole and its derivatives suppress cytokines through inhibition of protein kinases p38a and p38b. The synthesis and study of protein kinases inhibitors is one of principal tendencies in contemporary pharmacological industry.
Pyruvate kinase catalyzes irreversible conversion of phosphoenolpyruvate into pyruvate and formation of ATF from ADF in the process of glycolysis. Isoform of pyruvate kinase PKM2 is expressed in tumor cells and is considered as a target for antitumor drugs. It is assumed that the low activity of PKM2 results in enhancement of anabolitic cell processes. Increase of PKM2 activity suppresses cell proliferation.
It is well established that that the metabolic changes that accompany transformation are intimately related to the growth abnormalities of malignant cells. The c-Ras, c-Myc and Oct-4 are major oncogenes, which controls many aspects of intracellular regulation and metabolism. Overexpression of these oncogenes are necessary to support the increased need for macromolecules in transformed cells. Through down-stream protein kinases, Ras and Myc oncoproteins coordinate changes in level of expression of gene families which results in increased cellular proliferation. Among these protein kinases most important are tyrosine protein kinases and serine / threonine kinases, which catalytic activities strictly regulate metabolism in proliferated and differentiated cells. Pim-1 and Pyk2 kinase regulate the expression of oncogenic transcription factors: c-Myc, Oct-4 and other. Ras and Myc driven protein kinases are: Akt / PkB, JNK and ERK, Met-kinase and Aurora kinase, Src-kinases and others. Overexpression or mutation of these protein kinases is associated with abnormal metabolism of cancer cells and progression of malignancy.
In the frame of proposed project we plan to design novel nonprotein amino acids and peptides based thereon which are able to affect these target enzymes.
One of the main endpoints in preclinical drug development process is a risk assessment of new potential drugs, i.e. estimation of their possible negative effects on cells, tissues, and whole organism. A total risk characterization is performed using all relevant toxicity data and a comprehensive exposure evaluation in association with the genotoxicity data. The result of this characterization is ultimately used to generate a final level of concern for genotoxic risk to humans.
To achieve the goal of the project the following scope of activity is suggested.
The following scope of activity is suggested:
- modeling of peptides based on nonprotein amino acids with high specificity of binding and low constants of inhibition of trypsin, matrix metalloproteinases ММРs, angiotensin converting enzyme ACE, protein kinase p38 and pyruvate kinase M2;
- synthesis of nonprotein amino acids and designed peptides;
- study of the effect of synthesized peptides on the activity of matrix metalloproteinases ММРs, angiotensin converting enzyme ACE, protein kinase p38 and pyruvate kinase M2;
- screening of novel non-protein amino acids based peptides on the expression of Ras- oncoprotein; on the activity of Oct-4 and c-Myc protooncogenic transcription factors; and on the activities of tumor promoting protein kinases.
- evaluation of cytotoxicity and genotoxicity of synthesized compounds;
- evaluation of mutagenicity of synthesized compounds;
- evaluation of antimutagenic effects of peptides with highest activity in rat bone marrow cells in vivo and in human peripheral whole blood leukocytes in vitroafter treatment with methyl methanesulfonate (MMS) with application of micronuclei test.


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