The Istituto di Chimica del Riconoscimento Molecolare (Institute of Chemistry of Molecular Recognition) carries out research, technological development and training activities in the following areas:
1) Biomolecules (natural bioactive substances and synthesis of compounds of biological interest);
2) Chemical biotechnologies (bioconversions, enzyme technology and analytical methodologies);
3) Mechanisms of bioregulation (molecular basis of biological regulation and experimental and theoretical studies of molecular recognition).
The unifying theme of Institute activities is the chemistry of molecular recognition, which can be defined as a multidisciplinary area which studies the principles and strengths that regulate and determine biospecificity and biorecognition at the molecular level. The area involves expertise in organic, bioorganic and computational chemistry, biochemistry and biotechnology. Advances in understanding thus gained will help to discover innovative compounds, materials and biotechnological methods in the pharmaceutical, diagnostic and food fields, as well as to develop biocatalysis, bioseparation and bioregulation. All of these fields are and, even more so, will be strategic to improve the quality of life.
1) Biomolecules (natural bioactive substances and synthesis of compounds of biological interest)
Synthesis of flavors, fragrances and odorous molecules
Claudio Fuganti and Stefano Serra
The research consists in two different lines related to each other:
The group has a well established expertise in synthetic organic chemistry. The main part of the researches carried out in our laboratory is devoted to the preparation of specific bioactive substances (see point b). In order to face these synthetic problems the group has developed and exploited a number of new synthetic methods.
The combining of the skills described above with the extensive use of enzymatic methodologies allowed us to prepare a large number of chiral bioactive substances in enantiopure form. In this context, the group has involved in the study of a number of natural compounds (flavours, fragrances, terpenes) as well as in the synthesis of pharmaceutical substances.
Asymmetric and solid-phases/combinatorial synthesis of biomolecules with antitumoral activities
Monica Sani, Alessandro Volonterio and Matteo Zanda
The group has a strong experience in the stereoselective synthesis of molecules having biological interest. Over the last few years, the group has undertaken an intensive research activity in the synthesis of cytotoxic compounds. Among the recent results, it should be mentioned the total synthesis of tubulysins, a family of natural tetrapeptides that has been shown to depolymerize microtubules and induce mitotic arrest. Further developments and work in progress are directed toward the synthesis of antibody-tubulysin conjugates and peptides-tubulysin conjugates in order to deliver the cytotoxic agent to tumor cells in a selective way.
The activities of the peptide synthesis lab encompass many aspects of peptide and protein chemistry. In addition to our 25 years expertise in the peptide field, we take advantage of cutting edge technologies, such as microwave-assisted synthetic protocols and modern ligation techniques, to provide synthetic solutions to our partner groups and collaborators.
Our interests focus in particular on the synthesis and development of peptides displaying biological potential in the pharmaceutical, pharmacological and immunological field. Our tasks, among others, include the synthesis of small peptide libraries, the optimization of peptide leads, the synthesis of peptide-based immunogens, the synthesis of naturally occurring toxins and derivatives, the conjugation of peptides to fluorescent probes.
We are currently involved in different projects in collaboration with several Italian and international partners such as: other groups at CNR, University of Milan, San Raffaele Hospital (Milan), IRCCS C. Besta Neurological Institute (Milan), IRCCS Don Carlo Gnocchi Foundation (Milan), Gaslini Hospital (Genova), University of Aberdeen (Scotland), University of Queensland (Australia), University of Exeter (England), Khan Khoen University (Thailand) and others.
Chemistry and biology of dental materials
Giuseppina Nocca and Alessandro Lupi
The restoration of the dental structure, for aesthetic and functional purposes, can be performed through the use of composites, based on a three-dimensional combination of at least two chemically different materials with a distinct interface separating the components. Dental resin composites comprise a blend of hard inorganic particles bound together by a soft resin matrix (comprising a photopolymerizable monomer system and an initiator of radical polymerization) with the use of a coupling agent. The performances and the biocompatibility of these materials depend largely on the extent of the polymerization: as a matter of fact the residual monomeric material – useless for the properties of the polymer – is also the main responsible of the toxicity. Some studies are carried out regarding the relationship between the degree of conversion and the different intensity of light sources. In particular the concentration of the released monomer is measured by HPLC and their conversion degree is evaluated using the FTIR spectroscopy; the thermodynamic behavior is analyzed through the differential scanning calorimetry. Cellular toxicity tests are used to evaluate the possible damages caused by the interaction of the monomers with the tissues: alterations of the plasmatic membrane and mitochondrial damages are detected respectively by NRU and MTT test. The evaluation of the macrophagic functionality, after the exposition of the cellular line to the monomers, is performed by chemiluminescence technique. The Michael addition between glutathione and methacrylic monomers (a step of their metabolism) is also studied to clarify one of the cytotoxicity mechanisms. New researches about the interactions among mesenchymal stem cells (MSCs) and several scaffold types are now in progress; in particular the MSCs differentiation toward osteoblasts will be evaluated using biochemical assays and RT-PCR.
2) Chemical biotechnologies (bioconversions, enzyme technology and analytical methodologies)
New Applications of Biocatalysis in Chemical Processes
Giuseppe Pedrocchi-Fantoni, Mattia Valentino, Fiorenza Viani, Paola D’Arrigo and Stefano Servi
The main research activities of the group are focused on the use of biocatalysis in different areas including:
The main objective of this work is the development of a novel chemo-enzymatic process for the depolymerisation and transformation of lignin to value macromolecular and monomer product streams. This activity includes i) identification, screening and engineering of lignin active enzymes to create a tool box for selective lignin (or lignin analogues) transformations; ii) development of an integrated process combining chemical depolymerisation with targeted enzymatic transformation; iii) isolation and characterization of lignin degradation products improving their functionalization for the obtainment of macromolecular products.
The aim of this work is to investigate innovative reaction media such as ionic liquids for enzymatic biotransformations especially in phospholipids transformations catalyzed by phospholipase D (PLD) from Streptomyces PMF and in the cellulose depolimerization field in order to set-up a one-pot batch based on homogeneous phase for the dissolution and depolymerisation processes.
The work focuses on the screening, for biotechnological applications, of potential solid supports for the immobilization of enzymes belonging to the family of the L-amino acid oxidases (LAAOs, EC 126.96.36.199). Studies of the enzyme precipitation and cross linked aggregates formation are currently studied.
Transaminases from different sources and possessing different selectivities have been expressed, isolated and characterized in their selectivity towards target substrates. Moreover a Dynamic Kinetic Resolution platform for the enantioselective hydrolysis or aminolysis of thioesters in organic solvents is under investigation.
Activity and conformational studies of enzymes used for biotechnological applications
The use of enzymes for applications such as organic synthesis, biosensors, detergents, components of advanced materials is a topic of major interest in the pharmaceutical, biomedical, agrochemical and food industry. In fact, enzymatic properties such as chemo-, regio- and enatioselectivity can be profitably exploited to obtain chemical compounds by means of biocatalytic processes, to monitor the presence of an analyte in samples of different origin (e.g., agri-food industry, clinical samples), to prepare materials (plastic, textiles) with added value (e.g., endowed of antimicrobial properties), or to be formulated for applications as special detergents. Nevertheless, to render enzymes exploitable in any of the above applications, a critical factor that has to be considered is their catalytic efficiency in the formulation or in the device in which the enzyme is included. To this end, our research aims to optimize the catalytic activity of enzymes immobilized on different materials or used in non natural media (e.g., organic solvents). This task is pursued also through extensive conformational studies that are carried out by means of spectroscopic techniques such as FT/IR, circular dichroism and fluorescence.
Isolation, characterization and use of enzymes for the selective modification of functional groups, for the biocatalyzed formation of carbon-carbon bonds and for the synthesis and modification of polymers
Daniela Monti, Elisabetta Brenna, Gianluca Ottolina, Yves Galante, and Sergio Riva
The current research activities, that relates to biocatalysis (that is, the exploitation of enzymes in organic synthesis), refer to:
We have been studying the performances of lipases/proteases, glycosyltransferases and glycosidases for the modification of natural bioactive glycosides and of glutarylacylases for the modification of b-lactam antibiotics. These groups of enzymes are exploited in different synthetic applications, following the optimization of reaction conditions.
Cloning and recombinant expression of novel enzymes (hydroxysteroid dehydrogenases, alcohol dehydrogenases, ene-reductases, epoxide hydrolases, etc.) is carried out starting either from selected microbial strains available from public collections or from metagenomic libraries. Tailor-made variants are rationally designed and produced by site-directed mutagenesis.
Hydroxysteroid dehydrogenases suitable for the regio- and stereoselective modification of bile acids are applied in different synthetic processes together with innovative NAD(P)(H) cofactor regeneration systems. Recombinant ene-reductases are used for the regio- and stereoselective reduction of alpha-beta unsaturated alkenes aimed to the synthesis of chiral biologically active molecules, such as active pharmaceutical ingredients, flavours and fragrances. Monooxygenases, such as cyclohexanonemonooxygenase from Acinetobacter calcoaceticus (CHMO) are exploited for the preparation of chiral synthons such as enantiomerically pure or enriched organic sulfoxides, and lactones.
We have been exploiting laccases for the radical coupling of phenolic derivatives and
hydroxynitrile lyases for the stereoselective formation of cyanohydrins from aldehydes.
Chemical processes towards a second generation biorefinery: biocatalyzed transformation of waste biomasses
Gianluca Ottolina, Giuseppe Pedrocchi Fantoni and Sergio Riva
The activity of the “biorefinery group” is focused on the transformation of biomass feedstocks to higher value eco-friendly industrial products with a large market potential. Biomass is mainly considered a renewable source of lignocellulosic raw material. Biorefineries combine and integrate various processes, such as biomass pretreatment, fractionation, conversion and purification, also exploiting biocatalysis and fermentation processes.
In particular, the research activities are organized along two areas: biomass destructuration and lignin valorization by chemo-enzymatic upgrading. Biomass destructuration, obtained by organosolv processes, has been recognized as an important tool for the pretreatment of lignocellulosic materials and for the separation of their main components cellulose, hemicelluloses and lignin. Enzymatic hydrolysis of the polysaccharidic components furnishes sugar syrups suitable for targeted fermantations.
Microscale separations and analyses
Marcella Chiari, Marina Creich and Francesco Damin
The aim of the research group is to develop small-size and high-versatility analytical systems for for genomics and proteomics and to validate them in actual medical applications, thus setting the basis for the evolution towards low-cost industrial products. The group has an internationally recognized know-how in the production of polymeric coatings for analytical devices, microchip electrophoresis and microarrays on different materials.
The research includes: 1) synthesis of copolymers for coating microfluidics devices and microarrays; 2) development of polymers to be used as sieving matrix in electrophoresis; 3) development of new methods for the attachment of biological molecules (nucleic acids, peptides or proteins) on the surface of solid support for microarrays and biosensors (glass, silicon, gold or plastic materials); 4) development of materials and protocols for biosensing and point of care devices
Know-how of the group: 1) synthesis and characterization of polymers for tailoring of surface properties in analytical microdevices; 2) synthesis and characterization of polymers to be used in electrophoresis as dynamic coatings and as sieving matrix for DNA and proteins; 3) DNA and protein microarrays; 4) electrophoretic techniques for protein and DNA separations in slab gel and in capillary electrophoresis; 5) microchip electrophoresis.
3) Mechanisms of bioregulation (molecular basis of biological regulation and experimental and theoretical studies of molecular recognition)
Correlation of structure and function in hemoproteins
Bruno Giardina, Andrea Brancaccio, Maria Cristina De Rosa, Maria Elisabetta Clementi
Neuroglobin (Ngb) is one of the most recent partner to having joined the ever expanding family of cellular globins. It has been proposed that Ngb may play a role as an endogenous neuroprotectant since it was found that its up-regulation is induced by hypoxic-ischemic injury. However, recently it was observed that the absence of Ngb does not particularly affect neuronal survival during severe hypoxia although it may affect the transcriptional regulation of the glycolytic pathway. Considering the described intriguing biochemical properties of such a hexacoordinate globin and above all its preferential high expression in fundamental regions of brain (frontal cortex and talamo) in which oxygen supply must be carefully preserved and regulated, our hypothesis considers neuroglobin as a multifunctional sensor (for oxygen, nitric oxide, ROS) that acts at cellular level to support an adequate neuronal oxidative metabolism in response to various insults. In order to verify the role of Ngb we will investigate the effects induced by some molecules (17beta-estradiol , beta amyloide peptide, etc) on the expression levels of neuroglobin in human neuroblastoma cell lines, and the effects of this induction/repression on some fundamental indexes of mithocondrial respiration. Normal and dystrophic mice will be also analyzed in order to determine whether there is a correlation between inflammation processes and the expression levels of globins. Moreover, immunoprecipitation, mass spectrometry and computational biochemistry techniques will be employed for identifying specific globin ligands.
Molecular and biochemical mechanisms in neurodegenerative processes: the role of amyloid beta peptide in Alzheimer disease
Maria Elisabetta Clementi, Beatrice Sampaolese
Alzheimer disease (AD) is the most common form of late-life dementia. At molecular level, impairment of mitochondrial metabolism in AD patients has been well documented; in addition, several in vitro studies have reported that Amyloid peptide (Aß) affect mitochondrial DNA and proteins, leading to an alterated electronic transport chain and ultimately mitochondrial dysfunction. Although the classical view is that Aß; is deposited extracellularly, emerging evidence from transgenic mice and human patients indicates that this peptide can also accumulate intraneuronally. The intracellular localization of Aß in the AD brains determines extensive oxidative stress. In this scenario we investigate the toxic effects of amyloid peptide on neuronal cell culture and in particular on the mitochondrial functions. Moreover, the role of redox state of Ab in the early events of neurodegeneration (i. e. membrane colonization, ionic transport, mitochondrial activity, transcriptional regulation) will be deepened.
Computational simulation and drug design
Maria Cristina De Rosa
Advances in computer technology offer great opportunities for new explorations of protein structure and dynamics. Modeling tools can often predict the structure and shed some light on the function and its underlying mechanism. They can also provide insight to design experiments and suggest possible leads for drug design.
Specific aims of this research are:
Expertise: homology modelling, fold recognition and threading, molecular dynamics simulations, molecular docking, virtual screening.
Biochemistry of extracellular matrix
Andrea Bancaccio, Francesca Sciandra
Dystroglycan (DG) is an adhesion molecule that connects the extracellular matrix to the cytoskeletal elements of the cells. DG is composed of two subunits, α and β. A large number of ligands for both α- and β-DG have been identified indicating its functional versatility. It is crucial for skeletal muscle stability, it is involved in the maintenance of post-synaptic elements at the neuromuscular junction and in the central nervous system, in early embryogenesis and in infections caused by some pathogens. Our main research aim is to collect detailed information on the structural and functional properties of DG.
Specific subdomains of DG are expressed as recombinant proteins and used to carry out solid-phase binding assays, multidimensional NMR in solution and crystallization experiments. Recombinant and/or synthetic peptides are also used for the preparation of polyclonal and monoclonal antibodies, directed versus the dystroglycan core protein, to develop new tools both for the immunodetection of DG in tissues affected by different pathologies.
Recently, we have also produced a knock-in mouse expressing a DG mutated in the amino acids crucial for the interaction between α and β-DG. The novel knock-in mouse will help to elucidate the consequences of inhibiting the cross-talk between the two DG subunits for the extracellular matrix organization and tissues stability. Moreover, our knock-in mouse would become a novel animal model for the study of the molecular mechanisms of muscular dystrophy.
Proteomics and peptidomics of biological fluids
Alberto Vitali, Claudia Desiderio, Maria Patamia,. Massimo Castagnola
Biological fluids are a goldmine of molecules possessing a wide range of biological activities, the majority of which are to be explored yet. The proteomic analysis of biological fluids aided by high-resolution mass-spectrometry instruments is hence mandatory in order to define the structural characteristics and to qualitatively and quantitatively evaluate the content of target proteins and peptides. In this view the main areas of interests of this research theme are:
Proteomics and peptidomics of biological fluids (saliva, cerebrospinal fluid, tumoral intracystic fluids) and tissues (salivary glands, brain tissues, bioptic samples) from humans suffering different pathological conditions (metabolic diseases, premature born pediatric brain tumors and neurodegenerative diseases) for the characterization and identification of candidate biomarkers for prognostic and diagnostic purposes.
Investigation of the biological role of salivary peptides. The peptidomic analysis of saliva led us toidentify a wide gamma of small proline-rich peptides (PRPs) generated by post-tranlsational events at the expenses of larger proteins. The attribution of specific function to single or more components is demanding, as these peptides probably exert multiple and integrated functions. Structural and functional studies on specific cell lines are carried out.
Animal fluids such as snake venoms are a rich source of molecules such as neurotoxin, phospholipase A2, nerve growth factor and various enzymes acting on blood coagulation factors. The biodiversity of venoms and toxins make them a unique source from which novel therapeutics may be developed. Proteomic analysis should prompt a useful understanding for the mode and mechanism of toxin action and develop prototypes of pharmaceutical agents based on the structure of these compounds.
As complement and integration to the above studies: development and validation of analytical methods by Capillary Electrophoresis in coupling with mass spectrometry detection (CE-MS) for specific applications of biomedical-clinical interest: (analysis of amino acids and derivatives, methylarginines, pharmaceutical compounds, chiral analysis, peptides biomarkers in biological fluids and complex matrices).
Structural studies of organic substances and non covalent complexes by NMR spectrometry
Giovanni Fronza, Walter Panzeri
The research is addressed to the structural and conformational chacterization of biologically active organic molecules and of supramolecular associations, such as the non-covalent inclusion (or host-guest) complexes with cyclodextrins. Such complexes are of current interest to pharmaceutical, cosmetic and food industries whereas they can improve hydrosolubilty, stability, volatility, release control, bioavailability and palatability of guest molecules, enhancing the opportunity of application. Fundamental studies on structure and physico-chemical properties of such associations are achieved in solution by NMR and in the gas phase by mass spectrometry. Theoretical investigation aiming to provide support to the experimental mass spectral results is performed by molecular modelling methods.
Computational studies of folding and dynamics of peptides and proteins
Giorgio Colombo, Giulia Morra and Massimiliano Meli
Our research is focused on understanding the relationships between protein structure, dynamics and function at the atomic level.
Complex biophysical processes such as protein folding and molecular recognition underlie the behavior of all biological systems. Although functional mechanisms can be generally understood at a macroscopic level, insights into the microscopic origins of a given phenotype are often lacking.
The overarching goal of our lab is to understand the macroscopic behavior of complex systems from the analysis of atomic and molecular interactions and, on the basis of this knowledge, to design new chemical and biochemical modulators of biological functions.
Studies in our group explore the links between protein sequence, structure, conformational dynamics and molecular recognition to identify the molecular determinants of allosteric phenomena and protein interactions responsible for defined biological (mis)functions. These objectives are pursued through a combination of existing and newly developed computational techniques, mainly based on Molecular Dynamics simulations.
The strength of our research relies on the integration of computational methodologies with collaborative experimental approaches to provide valuable mechanistic interpretations and validations of theoretical predictions at the molecular level. Insights gained from these studies are used to guide the development of new methods for the design of peptide-based molecules for immunological applications, for the selection of small molecule modulators of protein function and dynamics, for drug-screening and protein design.