Periodic Reporting for period 2 - CLATHROPROBES (Chiroptical, optical and magnetic probes for protein sensing based on cage metal complexes)
Reporting period: 2020-01-01 to 2023-12-31
The CLATHROPROBES project has been devoted to the design of novel and highly efficient chiroptical, luminescent, and other probes for sensing of proteins and their structural changes, build on the basis of cage metal complexes (clathrochelates and lacunar spatial complexes with encapsulated 3d metal ions) as molecular reporters.
The majority of known probes for protein sensing or inhibitors of protein activity is based on organic compounds built on a carbon-based scaffold. The highest number of substituents on a carbon atom is 4. In contrast, metal-based scaffolds offer higher number of substituents that significantly expand the available structural space and allow efficient targeting of a variety of proteins. However, this potentially powerful approach has been rarely used up to now, which is arguably caused by challenges in synthesis of metal-based protein binders stable in the presence of natural metal binders like amino acids, peptides, metallothioneins or others.
The aim of CLATHROPROBES has been to develop metal cage complexes as molecular reporters able to sense target proteins and structural changes that they undergo. Since the “reporting properties” of cage complexes allow direct studies of their interactions with a target, this research opens new opportunities for the design of “smart” cage molecules - imaging probes and therapeutic agents.
This project included the design and synthesis of new compounds, their complete structural and spectroscopic characterization, studies of their interactions with proteins, properties as reporters, and evaluation of their stability, bioactivity and cytotoxicity in model systems.
The majority of known probes for protein sensing or inhibitors of protein activity is based on organic compounds built on a carbon-based scaffold. The highest number of substituents on a carbon atom is 4. In contrast, metal-based scaffolds offer higher number of substituents that significantly expand the available structural space and allow efficient targeting of a variety of proteins. However, this potentially powerful approach has been rarely used up to now, which is arguably caused by challenges in synthesis of metal-based protein binders stable in the presence of natural metal binders like amino acids, peptides, metallothioneins or others.
The aim of CLATHROPROBES has been to develop metal cage complexes as molecular reporters able to sense target proteins and structural changes that they undergo. Since the “reporting properties” of cage complexes allow direct studies of their interactions with a target, this research opens new opportunities for the design of “smart” cage molecules - imaging probes and therapeutic agents.
This project included the design and synthesis of new compounds, their complete structural and spectroscopic characterization, studies of their interactions with proteins, properties as reporters, and evaluation of their stability, bioactivity and cytotoxicity in model systems.
A wide set of novel, functionalized cage complexes was synthesized, and corresponding synthetic methodologies were developed.
The following several types of novel cage complexes were obtained:
• Hexahydrazide clathrochelate complexes with high-valent first row transitional metals (Fe(IV), Mn(IV)) and Co(III) as ions incorporated into macrobicyclic cage;
• Heterofunctionalized Fe(II) clathrochelate complexes to facilitate targeting of protein macromolecules and connection of reporter units;
• Fe(II) dihalogenoclathrochelates with inherent halogen substituents for performing of Suzuki–Miyaura and Sonogashira reactions;
• Fe(I) cage complex with high stability towards strong H-acids;
• Co(II) pseudoclathrochelates with a rigid cholesteryl substituent , and a very large magnetic anisotropy;
• Hybrid metallo(IV)phthalocyaninate-capped Co(III) complexes.
Novel cage complexes were characterized by appropriate set of physico-chemical and analytical methods, including elemental analysis, mass spectrometry (ESI-MS and MALDI-TOF), UV-VIS, fluorescent, CD, IR, ATR-FTIR, NMR, EPR, spectroelectrochemistry and X-ray analysis. Single crystals of several M(II)/M(III)/M(IV) clathrochelate complexes have been successfully obtained and studied by means of X-ray diffraction structure analysis.
Original experimental data resulted from the redox reactivity characterization of hexahydrazide clathrochelate complexes with high-valent first row transitional metals (Fe(IV), Mn(IV)). The mentioned cage complexes on action of strong oxidants (cerium ammonium nitrate, lead(IV) oxide) can be oxidized to pentavalent state, while in the presence of strong and moderate reductants (sodium dithionite, ascorbic acid), a reduction to trivalent species occurs.
Of importance, the phenomena of chirality induction upon binding with proteins was discovered for inherently achiral iron(II) clathrochelates, due to this clathrochelates are considered as prospective CD reporters for protein studies. The interactions of cage metal complexes with proteins were characterized by a wide range of spectral methods (CD, UV-VIS, fluorescence), ESI-MS and ITC.
It was shown that carboxyphenyl ribbed-functionalized iron(II) clathrochelates are able to induce a pronounced CD (ICD) response upon supramolecular interactions with a macromolecular chiral inductor, such as a protein. The induced CD signals are observed in the spectral range characteristic to the clathrochelates MLCT absorption bands. The binding to a protein was found to be determined by the nature of clathrochelate terminal groups. Further, we have shown that iron(II) clathrochelates able to discriminate between serum albumins of relative structure (human and bovine albumins) by giving distinct ICD spectra. Besides, by the variation of the shape and intensity of CD bands, these cage metal complexes reflect alterations of the tertiary structure of albumins. The interactions of constitutional isomers of iron(II) clathrochelates with different globular proteins (serum albumins, lysozyme, -lactoglobulin (BLG), trypsin, insulin) had shown that a highly-intensive ICD output of the clathrochelates was observed upon their association with albumins and BLG. Based on the docking simulations, the binding of the clathrochelate molecule to the main BLG binding site is suggested.
For clathrochelates, which carry a single carboxylic acid, the anticancer activity was observed on a series of cancer cells, whereas normal cells were found to be unaffected. The studies of the mechanism of cytotoxicity have revealed that the compounds are efficiently uptaken by cancer cells via an active mechanism. In cells, they first induce the ER stress due to the accumulation of unfolded proteins as evidenced by the activation of the protein ubiquitination pathway and the increase of the amount of intracellular ubiquitinated proteins. The initial effect on the ER leads later on to the decrease of mitochondrial membrane potential, induction of oxidative stress including formation of mitochondrial ROS, upregulation of nitric oxide as well as growth arrest in the G1 phase and inhibition of DNA and RNA synthesis. All these factors cause cell apoptosis and partially necrosis.
The following several types of novel cage complexes were obtained:
• Hexahydrazide clathrochelate complexes with high-valent first row transitional metals (Fe(IV), Mn(IV)) and Co(III) as ions incorporated into macrobicyclic cage;
• Heterofunctionalized Fe(II) clathrochelate complexes to facilitate targeting of protein macromolecules and connection of reporter units;
• Fe(II) dihalogenoclathrochelates with inherent halogen substituents for performing of Suzuki–Miyaura and Sonogashira reactions;
• Fe(I) cage complex with high stability towards strong H-acids;
• Co(II) pseudoclathrochelates with a rigid cholesteryl substituent , and a very large magnetic anisotropy;
• Hybrid metallo(IV)phthalocyaninate-capped Co(III) complexes.
Novel cage complexes were characterized by appropriate set of physico-chemical and analytical methods, including elemental analysis, mass spectrometry (ESI-MS and MALDI-TOF), UV-VIS, fluorescent, CD, IR, ATR-FTIR, NMR, EPR, spectroelectrochemistry and X-ray analysis. Single crystals of several M(II)/M(III)/M(IV) clathrochelate complexes have been successfully obtained and studied by means of X-ray diffraction structure analysis.
Original experimental data resulted from the redox reactivity characterization of hexahydrazide clathrochelate complexes with high-valent first row transitional metals (Fe(IV), Mn(IV)). The mentioned cage complexes on action of strong oxidants (cerium ammonium nitrate, lead(IV) oxide) can be oxidized to pentavalent state, while in the presence of strong and moderate reductants (sodium dithionite, ascorbic acid), a reduction to trivalent species occurs.
Of importance, the phenomena of chirality induction upon binding with proteins was discovered for inherently achiral iron(II) clathrochelates, due to this clathrochelates are considered as prospective CD reporters for protein studies. The interactions of cage metal complexes with proteins were characterized by a wide range of spectral methods (CD, UV-VIS, fluorescence), ESI-MS and ITC.
It was shown that carboxyphenyl ribbed-functionalized iron(II) clathrochelates are able to induce a pronounced CD (ICD) response upon supramolecular interactions with a macromolecular chiral inductor, such as a protein. The induced CD signals are observed in the spectral range characteristic to the clathrochelates MLCT absorption bands. The binding to a protein was found to be determined by the nature of clathrochelate terminal groups. Further, we have shown that iron(II) clathrochelates able to discriminate between serum albumins of relative structure (human and bovine albumins) by giving distinct ICD spectra. Besides, by the variation of the shape and intensity of CD bands, these cage metal complexes reflect alterations of the tertiary structure of albumins. The interactions of constitutional isomers of iron(II) clathrochelates with different globular proteins (serum albumins, lysozyme, -lactoglobulin (BLG), trypsin, insulin) had shown that a highly-intensive ICD output of the clathrochelates was observed upon their association with albumins and BLG. Based on the docking simulations, the binding of the clathrochelate molecule to the main BLG binding site is suggested.
For clathrochelates, which carry a single carboxylic acid, the anticancer activity was observed on a series of cancer cells, whereas normal cells were found to be unaffected. The studies of the mechanism of cytotoxicity have revealed that the compounds are efficiently uptaken by cancer cells via an active mechanism. In cells, they first induce the ER stress due to the accumulation of unfolded proteins as evidenced by the activation of the protein ubiquitination pathway and the increase of the amount of intracellular ubiquitinated proteins. The initial effect on the ER leads later on to the decrease of mitochondrial membrane potential, induction of oxidative stress including formation of mitochondrial ROS, upregulation of nitric oxide as well as growth arrest in the G1 phase and inhibition of DNA and RNA synthesis. All these factors cause cell apoptosis and partially necrosis.
The multidisciplinary character of the project has ensured the transfer of knowledge between scientists of different research areas, sectors and countries - over 6 years of project duration, 48 researchers spent more than 170 months in laboratories of collaborating partners. Among them there were 25 Early Stage and 23 Experienced Researchers (27 females and 21 males). This exchange has enhanced the level of their professional scientific and technical expertise, extensive training of early stage researchers in different, yet complementary research areas (which is crucial for the development of their further scientific or industrial career), enhancement of research activity and network of participating organizations, new long-term collaborations, increase of academia-industry interactions.
The success of this project is the result of a close cooperation on research and technological development among both academic and industrial partners. New clathrochelate-based probes elaborated are of their specific interest. The application of cage metal complexes in a new generation of probes, markers and reporters for the purposes of detection of specific proteins, their labelling and tracing as well as imaging and staining of cells and cell components is therefore original and innovative.
The results of the project have been described in 25 original articles published in renown journals and widely disseminated & communicated to various audiences. The project has been awarded Polish Smart Development Award 2021 in the category Innovative technologies and future research.
The success of this project is the result of a close cooperation on research and technological development among both academic and industrial partners. New clathrochelate-based probes elaborated are of their specific interest. The application of cage metal complexes in a new generation of probes, markers and reporters for the purposes of detection of specific proteins, their labelling and tracing as well as imaging and staining of cells and cell components is therefore original and innovative.
The results of the project have been described in 25 original articles published in renown journals and widely disseminated & communicated to various audiences. The project has been awarded Polish Smart Development Award 2021 in the category Innovative technologies and future research.