DNA GyrB docking with thiazolesProject finaced by
UEFISCDI

SYNTHIAZO

Financing contract no.: 210/2014

Project no.: PN-II-PT-PCCA-2013-4-2075

Synthesis, screening and controlled release of some novel thiazole, bithiazole and thiazolidin-4-one compounds with antioxidant, antiproliferative and antimicrobial activity

 

Summary

The project is a multidisciplinary approach with some important interdisciplinary valences aiming to bring its contribution in the solving of generic problems identified within the framework of Research Domain No. 6. BIOTECHNOLOGIES, with specific fit in the Research direction 6.1 Biotechnologies: 6.1.1 Design and development of new drugs with maximal efficiency and minimal secondary effects.

The final aim of the SYNTHIAZO project is to develop new thiazole, bithiazole and thiazolidin-4-one compounds with controlled delivery and antioxidant/antiproliferative effects and/or specific antitumoral activity, and antimicrobial properties in order to respond at two recurring problems in clinical medicine:

  • multiple drug resistance (MDR) of infectious agents;
  • the proliferation and metastasis of drug-resistant tumor cell lines, combined with an insufficient specificity of current anticancer drugs.

In the challenging process of new drug discovery, the partners tasked themselves not only with solving a whole range of difficult scientific and technical problems but even dissemination of project’s without affecting the allocation of intellectual property rights. Therefore, partners will work together and the outcomes of the project will be promoted at national and EU level.

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Consortium

The Consortium consists of four participating entities: three from the academic area (a national R&D institute and two universities) and one from industry (a SME).

 

Grant holder (Coordinator - CO): The National Research and Development Institute for Cryogenics and Isotopic Technologies – ICSI Rm. Valcea

Department of Research and Development and Technology Transfer: http://www.icsi.ro

  • project manager: PhD. Radu Tamaian

orcid.org/0000-0001-6380-1460

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Partner 1 (P1): University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj-Napoca

Pharmaceutical chemistry: http://www.umfcluj.ro

  • P1 project responsible: Prof. Brînduşa Tiperciuc

orcid.org/0000-0002-1333-5649

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Partner 2 (P2): University of Bucharest

3Nano-SAE Research Center: http://www.3nanosae.org

  • P2 project responsible: PhD. Cornelia Nichita

orcid.org/0000-0002-4928-4564

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Partner 3 (P3): SC BioLumiMedica SRL Bucharest: http://biolumimedica.ro

  • P3 project responsible: Biophys. Ioan Daniel Stamatin

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General objectives

  • To apply predictive and computational methods used in drug development, by combining ligand-based metods and a structure-based approach in order to comply with the principle of the 3Rs of animal use in alternative test method development (Replacement, Reduction and Refinement), according to the DIRECTIVE 2010/63/EU on the protection of animals used for scientific purposes.
  • To synthesize novel drug candidates (thiazole, bithiazole and thiazolidin-4-one compounds) as a clinical alternative to fight against MDR infectious agents and against of the current chemotherapy resistant tumor cell lines.
  • To design and develop controlled delivery systems for the novel pharmacoactive agents with the help of functionalised nanomaterials and by specific targeting technology (bioconjugates technology) using monoclonal antibodies (mAb) or fragments antigen-binding (Fab fragments).
  • To confirm the structure of novel compounds and of their assembling with the carriers and to apply in the preclinical pharmaceutical tests the principles of the 3Rs to perform more biochemical assays and cell-based assays to evaluate the potential toxicity and the enzymatic/biological activity of newly synthesised compounds.
  • To train the younger generations for a better understanding of the scientific bases of dug discovery process and for upgrading of their technical skills (use of the advanced analytical instruments).

SMART objectives

Undertaking the aim of the project, we have developed a set of clearly defined "SMART" (Specific, Measurable, Attainable, Realistic, Time-bound)  objectives

  • Building a database with the three-dimensional structures of desired molecular targets based on experimentally-determined structures from RCSB Protein Data Bank and homology models of missing crystal structures, prior to molecular docking;
  • Generation of a large combinatorial library with the help of a dedicated software (e.g., SmiLib v2.0 – Rapid Assembly of Combinatorial Libraries in SMILES Notation) through virtual reactions between the so-called building blocks (small Markush molecules – functional groups yielding newly synthesized reaction virtual compounds) and scaffold molecules (Markush structures based on thiazole, bithiazole and thiazolidin-4-one fragments that contain the R-groups – sites of variability) to serve as hits' selection pool for computational testing (in silico experiments).
  • Identification of the most promising leading structures (thiazole derivatives) using a "hit-to-lead" approach combining two different drug design strategies:
    •  a ligand-based drug design approach (exploration of literature data for pharmacofore fragments with antimicrobial, antioxidant, antitumoral activity to establish the quantitative relations between the chemical structure, the biological activity and the retention parameters – QSAR, QSSR, QRAR);
    •  a structure-based drug design approach – the virtual screening (VS) consisting in:
      •  ADME-Tox properties filtering (to eliminate the promiscuous binders and compounds with high toxicity, metabolic stability issues or other undesirable side effects, etc) to select the lead-like thiazole derivative;
      • molecular docking to predict their binding affinity/potency to the active site(s) of molecular targets (the key enzymes previously included in our database).
  •  Selection and synthesis of best drug candidates (new molecules with thiazole, bithiazole, triazole and thiazolidin-4-one rings in their structures) from the most high-quality leads for preclinical pharmaceutical testing.
  •  Experimental determination of the lipophilicity (using thin layer reversed phase chromatography) combined with principal component analysis for some of the synthesized compounds in order to validate the predicted values of this key-feature parameter used for ADMET properties filtering and improve the reliability of software for further calculations.
  •  To design and develop controlled delivery systems for the novel pharmacoactive agents with the help of functionalised nanomaterials (i.e. carboxylated/hydroxy-lated carbon nanotubes, chitosan, sodium alginate) and specific targeting technology (bioconjugates technology) using monoclonal antibodies (mAb) or fragments antigen-binding (Fab fragments).
  •  Confirmation of the structure of newly synthesised compounds (e.g., using 1H and 13C-Nuclear Magnetic Resonance Spectroscopy, etc) and of their assembling with carriers (e.g., using Scanning Electron Microscopy – SEM, Atomic Force Microscopy – AFM, Surface-Enhanced Raman Spectroscopy – SERS, sodium dodecyl sulfate polyacrylamide gel electrophoresis – SDS-PAGE and/or Western blot).
  •  Preclinical pharmaceutical testing of synthesised drug candidates: toxicity (acute toxicity and genotoxicity assays, etc) and pharmacoactive effects:
    • a. antimicrobial activity: antimicrobial susceptibility testing (AST) on clinical samples and ATCC strains – the usual strains and the resistant strains (BLA);
    • b. antioxidant effects (chemiluminescence assays);
    • c. antiproliferative/antitumoral activity (effects against experimental and drug-resistant tumor cell lines).
  •  Training activities (twinning and tripling schemes) for the benefit of the younger generation (M.Sci students, Ph.D students, young researchers) to upgrade their academic background and their professional skills for a better understanding of drugs development process – including here:
    • an in silico tests module (PCA, QSAR, QSSR, QRAR, VS);
    • a laboratory experiments module (e.g. organic synthesis, biochemical assays, cell-based assays, biohybrids synthesis, immunoconjugates technology, AST, spectroscopy).

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Expected results

  • Development of a database with the three-dimensional structures of desired molecular targets prior to molecular docking.
  • Generation of a large combinatorial library with the help of a dedicated software to serve as hits' selection pool for computational testing (in silico experiments).
  • Identification of the most promising leading structures (thiazole derivatives) using a "hit-to-lead" approach.
  • Identification and synthesis of best drug candidates (new molecules with thiazole, bithiazole, triazole and thiazolidin-4-one rings in their structures) from the most high-quality leads for preclinical pharmaceutical testing.
  • Development of controlled delivery systems for the novel pharmacoactive agents with the help of functionalised nanomaterials (i.e. carboxylated and/or hydroxy-lated carbon nanotubes, chitosan, sodium alginate) and specific targeting technology (bioconjugates technology) using monoclonal antibodies (mAb) and/or fragments antigen-binding (Fab fragments).
  • Preclinical pharmaceutical testing of synthesised drug candidates: toxicity (acute toxicity and genotoxicity assays, etc) and pharmacoactive effects.
  • Training activities (twinning and tripling schemes) for the benefit of the younger generation (M.Sci students, Ph.D students, young researchers) to upgrade their academic background and their professional skills for a better understanding of drug discovery process.

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Budget

Requested budget: 2.760.000,00 RON

Approved budget: 1.380.000,00 RON

  • Public budget: 1.200.000,00 RON

  • Own budget: 180.000,00 RON

 

Budget allocation by stages (in RON):

  • Stage 1: Designing of new virtual derivatives 
    Total budget Public budget Own budget
    112.646,00 112.646,00 0,00

  • Stage 2: Computational simulations, synthesys of new thiazole derivatives (1st series), tests for structure verification
    Total budget Public budget Own budget
    263.854,00 245.754,00 18.100,00

  • Stage 3: Synthesys of 2nd and 3rd thiazole derivatives series. Development of biohibrids/immunoconjugates. Laboratory tests
    Total budget Public budget Own budget
    540.000,00 473.550,00 66.450,00
     

  • Stage 4: Identification of drug candidates. Identification of intellectual property rights. Development of prototypes
    Total budget Public budget Own budget
    463.500,00 4368.050,00 95.450,00
     

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Project progress: public results & dissemination

One of the main objectives of organic and medicinal chemistry is the design, synthesis and production of molecules having value as human therapeutic agents.

Computer-aided drug design (CADD) techniques were used to start the researches in the process of drug discovery:

  • rational selection of biological targets and building of a comprehensive database (the first milestone of the project);

  • building of large combinatorial library with virtual derivatives (the second milestone of the project);

  • ADME properties pre-filtering for physical and chemical properties (e.g., aqueous solubility, boiling point, LogD, LogP, etc) and identifications of PAINS, promiscuous binders, undesirable and /or flagged moieties, and toxicity screening;

  • design of drug delivery systems (biohybrids).

SmiLib v2.0

Generation of combinatorial library

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Lipophilicity study on thiazolyl-carbonyl-thiosemicarbazides and thiazolyl-azoles:

Lipophilicity, as one of the most important physicochemical parameters of bioactive molecules, was investigated for twenty-two thiazolyl-carbonyl-thiosemicarbazides and thiazolyl-azoles. The determination was carried out by reversed-phase thin-layer chromatography, using a binary isopropanol-water mobile phase. Chromatographically obtained lipophilicity parameters were correlated with calculated log P and log D and with some biological parameters, determined in order to evaluate the anti-inflammatory and antioxidant potential of the investigated compounds, by using principal component analysis (PCA). The PCA grouped the compounds based on the nature of their substituents (X, R and Y), indicating that their nature, electronic effects and molar volumes influence the lipophilicity parameters and their anti-inflammatory and antioxidant effects. Also, the results of the PCA analysis applied on all the experimental and computed parameters show that the best anti-inflammatory and antioxidant compounds were correlated with medium values of the lipophilicity parameters. On the other hand, the knowledge of the grouping patterns of the tested variables allows the reduction of the number of parameters, determined in order to establish the biological activity.

For references, please check our paper: R. Tamaian, A. Mot, R. Silaghi-Dumitrescu, I. Ionut, A. Stana, O. Oniga, C. Nastasa, D. Benedec, B. Tiperciuc, 2015, Study of the Relationships between the Structure, Lipophilicity and Biological Activity of Some Thiazolyl-carbonyl-thiosemicarbazides and Thiazolyl-azoles. Molecules, 20:22188–22201, doi:10.3390/molecules201219841

Profiles of RM values

Profiles of RM values at all mobile binary phase composition (% of organic modifier) for thiazolyl-carbonyl-thiosemicarbazides and thiazolyl-azoles

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Biological evaluation and docking of chromenyl-derivatives:

Various thiosemicarbazones (TSCs) and their heterocyclic thiadiazolines (TDZ) possess important biological effects. In addition, chromenyl derivatives exhibit a wide range of pharmacological activities. Based on these findings and as a continuation of our research on nitrogen and sulfur containing compounds, we investigated a series of previously reported chromenyl-TSCs (1a-j) and chromenyl-TDZs (2a-j) for their in vitro antimicrobial activities against two bacterial and four fungal strains. MIC and MBC/MFC (µg/mL) values of these compounds were evaluated and compared to those of Spectinomycin, Moxifloxacin and Fluconazole, used as reference drugs. For a better understanding of the drug-receptor interactions, all the compounds were further subjected to molecular docking against four targets that were chosen based on the specific mechanism of action of the reference drugs used in the antimicrobial screening. All compounds tested showed equal or higher antibacterial/antifungal activities relative to the used reference drugs. In silico studies (docking) revealed that all the investigated compounds showed good binding energies towards four receptor protein targets and supported their antimicrobial properties.

For references, please check our paper:  I. Ionuţ, D. C. Vodnar, I. Oniga, O. Oniga, B. Tiperciuc, R. Tamaian, 2016, Biological evaluation and molecular docking of some chromenyl-derivatives as potential antimicrobial agents. Pakistan Journal of Pharmaceutical Sciences, 29(1-Suppl):261–272, http://www.pjps.pk/?page_id=276&id=2181

Docking poses

Docking poses of compounds 1a-j and 2a-j against 30S ribosomal protein S12

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Design, synthesis and antifungal activity evaluation of novel thiazolin-4-ones:

Twenty-three thiazolin-4-ones were synthesized starting from phenylthioamide or thiourea derivatives by condensation with alpha-monochloroacetic acid or ethyl alpha-bromoacetate, followed by substitution in position 5 with various arylidene moieties. All the synthesized compounds were physico-chemically characterized and the IR, 1H NMR, 13C NMR and MS data were consistent with the assigned structures. The synthesized thiazolin-4-one derivatives were tested for antifungal properties against several strains of Candida and all compounds exhibited efficient anti-Candida activity, two of them (9b and 10) being over 500-fold more active than fluconazole. Furthermore, the compounds’ lipophilicity was assessed and the compounds were subjected to in silico screening for prediction of their ADME-Tox properties. Molecular docking studies were performed to investigate the mode of action towards the fungal lanosterol 14α-demethylase, a cytochrome P450-dependent enzyme. The results of the in vitro antifungal activity screening, docking study and ADME-Tox prediction revealed that the synthesized compounds are potential anti-Candida agents that might act by inhibiting the fungal lanosterol 14α-demethylase and can be further optimized and developed as lead compounds.

For references, please check our paper: A. Stana, D. C. Vodnar, R. Tamaian, A. Pîrnău, L. Vlase, I. Ionuţ, O. Oniga, B. Tiperciuc, 2017, Design, Synthesis and Antifungal Activity Evaluation of New Thiazolin-4-ones as Potential Lanosterol 14 α-Demethylase Inhibitors. International Journal of Molecular Sciences, 18(1):177, doi:10.3390/ijms18010177

Docking poses

Docking poses of thiazolin-4-ones in the active site of lanosterol-14α-demethylase

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Patent application A/00766 from September 28th, 2017: Chitosan nanoparticles based bio-support for transport of active principles and process for obtaining thereof (authors: Cornelia Nichita, Ioan Daniel Stamatin, Radu Tamaian)

SEM images of chitosan nanoparticles

SEM images of chitosan nanoparticles

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Dissemination via scientific manifestations:

 

19th International Union for Pure and Applied Biophysics Congress & 11th European Biophysics Congress – IUPAB & EBSA 2017, 16 – 20 July 2017, Edinburgh, Scotland, UK: http://www.iupab2017.org

  • R. Tamaian, C. Nastasă, O. Oniga, B. G. Tiperciuc. In silico evaluation of new 5-arylidene(chromenyl)-thiazolidinediones as safer K-Ras inhibitors, European Biophysics Journal with Biophysics Letters, 2017, 46(Suppl 1):S319 (poster P-784)

 

10th Joint Meeting of Medicinal Chemistry, 25 – 28 June 2017, Dubrovnik (Srebreno), Croatia: http://www.jmmc2017.hr

  • A. Stana, R. Tamaian, D. C. Vodnar, O. Oniga, B. Tiperciuc, Antibacterial Thiazolin-4-Ones as Potential DNA Gyrase and Topoisomerase IV Inhibitors, Book of Abstracts, p.219 (poster P-143)

 

Bucharest University Faculty of Physics 2017 Meeting, 23 – 24 June 2017 Bucharest, Romania:

  •  T. Axenie, S. Voinea, F. Al-Behadili, C. Nichita, Design and Synthesis of Chitosan Nanoparticles as a Drug Delivery System, Poster

 

The 21th ICIT International Conference “Progress in Cryogenics and Isotopes Separation”, 19 – 21 October 2016, Călimăneşti-Căciulata, Romania: http://www.icsi.ro/conference/index.php

  • R. Tamaian, I. A. Ionuţ, I. E. Oniga, O. Oniga, B. G. Tiperciuc – In silico Predictions on ADME-TOX Profile of Some Novel Chromenyl Derivatives, Book of Abstracts 2016, p.84-85 (poster P.28)

 

​The 16th Edition of the National Congress of Pharmacy in Romania – CNFR 2016 “Pharmacy – Interdisciplinary Center for Life Sciences”, 28 September – 1 October 2016, Bucharest, Romania: http://cnfr2016.ro

  • A. Stana, R. Tamaian, D. Vodnar, B. Tiperciuc, O. Oniga – New antifungal thiazolin-4-ones targeting lanosterol 14α-demethylase, Abstract Book 2016, p.73 (poster ID: 280)

 

Physics Boat 2016: Atomic structure of nanosystems from first-principles simulations and microscopy experiments – AS-SIMEX 2016, 31 May – 2 June 2016, Helsinki, Finland – Stockholm, Sweden: http://viesti.physics.aalto.fi/pub/boat/

  • R. Tamaian – Simulated scanning tunneling microscopy study of one-dimensional graphene nanoribbons for further development of drug delivery systems, Abstracts, p. 60 (poster)

 

The 2016 E-MRS Spring Meeting and Exhibit, Lille, France, 2-6 May 2016: https://www.european-mrs.com/meetings/2016-spring-meeting

  • C. Nichita,  G. Neagu, A. Balan , I. Stamatin,  Self crosslinking chitosan in high density ultrasonic field, support for drug delivery systems, Poster

 

The European Nanomedicine Meeting – ENM2015, 7 – 9 December 2015, Grenoble, France: http://www.sfnano.fr/2015

  • R. Tamaian, V. Niculescu – Biocomputational Approach for Modelling of Nano-polymeric Particles / T Lymphocytes Diffusive Transfer, Abstract book of The European Nanomedicine Meeting – ENM2015, p. 42 (poster A.37)

 

Annual Meeting of the "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, 23 – 27 November 2015, Cluj-Napoca, Romania:

  • I. Ionuţ, D. C. Vodnar, I. Oniga, O. Oniga, B. Tiperciuc, R. Tamaian – Biological evaluation and molecular docking of some novel chromenyl-derivatives as potential antimicrobial agents, Clujul Medical – Journal of Medicine and Pharmacy: Abstracts of the Annual Meeting of the "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca (ISSN 1222-2119), Supplement No. 3, Vol. 88, (poster S125)

 

Drug Discovery 2015 – ELRIGDD15, 2 – 3 September, Telford, England, United Kingdom of Great Britain and Northern Ireland: https://elrig.org/portfolio/drug-discovery-2015/

  • R. Tamaian, C. Nichita, I. D. Stamatin, D. Enciu - In silico modelling of the biodistribution of therapeutic immunoconjugates with thiazolic payload (on-line poster #11)

 

On Line Event "Drug Discovery & Development" – DDD 2015, 23 June 2015: http://selectbiosciences.com/DDD2015

  • R. Tamaian, A. Stana, I. Ionuţ, D.C. Vodnar, O. Oniga, B. Tiperciuc – Evaluation of new thiazole derivatives as antifungal agents targeting lanosterol 14-alpha demethylase (on-line poster #1)

 

The 8th International Conference on Advanced Materials – ROCAM 2015, 7 – 10 July 2015, Bucharest, Romania: http://rocam.unibuc.ro/rocam2015/

  • C. Nichita, R. Tamaian, G. Neagu, I. D. Stamatin – Nanocarrier Delivery Systems Based On Chitosan And Antioxidants. Abstract Book of The 8th International Conference on Advanced Materials - ROCAM 2015 (ISSN 1842-3574), p. 98 (poster)

 

The 2015 E-MRS Spring Meeting, 11 – 15 May, 2015, Lille, France: https://www.european-mrs.com/meetings/2015-spring

  • C. Nichita, A. Taqi, A. Bălan, R. Tamaian, S. Voinea, A.M. Soare, M. Zăuleţ, G. Neagu, I. Stamatin – Nanostructured biofilms designed with polysaccharides and bioactive compounds. Mechanical and antioxidant properties (on-line poster W-P 5)

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