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Biomedical subjects

Marco Fragai

Publications and source records attributed to Marco Fragai.

14 recordsLinked to original sources
PubMed2006

Synthesis of bicyclic molecular scaffolds (BTAa): an investigation towards new selective MMP-12 inhibitors.

Starting from 3-aza-6,8-dioxa-bicyclo[3.2.1]octane scaffold (BTAa) a virtual library of molecules was generated and screened in silico against the crystal structure of the Human Macrophage Metalloelastase (MMP-12). The molecules obtaining high score were synthesized and the affinity for the catalytic domain of MMP-12 was experimentally proved by NMR experiments. A BTAa scaffold 20 having a N-hydroxyurea group in position 3 and a p-phenylbenzylcarboxy amide in position 7 showed a fair inhibition potency (IC50 = 149 microM) for MMP-12 and some selectivity towards five different MMPs. These results, taken together with the X-ray structure of the adduct between MMP-12, the inhibitor 20 and the acetohydroxamic acid (AHA), suggest that bicyclic scaffold derivatives may be exploited for the design of new selective matrix metalloproteinase inhibitors (MMPIs).

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2005

Combining in silico tools and NMR data to validate protein-ligand structural models: application to matrix metalloproteinases.

A combination of in silico tools and experimental NMR data is proposed for relatively fast determination of protein-ligand structural models and demonstrated from known inhibitors of matrix metalloproteinases (MMP). The 15N 1H heteronuclear single quantum coherence (HSQC) spectral assignment and the 3D structure, either X-ray or NMR, are needed. In this method, the HSQC spectrum with or without the ligand is used to determine the interaction region of the ligand. Docking calculations are then performed to obtain a set of structural models. From the latter, the nuclear Overhauser effects (NOEs) between the ligand and the protein can be predicted. Guided by these predictions, a number of NOEs can be detected and assigned through a HSQC NOESY experiment. These data are used as structural restraints to reject/refine the initial structural models through further in silico work. For a test protein (MMP-12, human macrophage metalloelastase), a final structure of a protein-ligand adduct was obtained that matches well with the full structural determination. A number of structural predictions were then made for adducts of a similar protein (MMP-1, human fibroblast collagenase) with the same and different ligands. The quality of the final results depended on the type and number of experimental NOEs, but in all cases, a well-defined ligand conformation in the protein binding site was obtained. This protocol is proposed as a viable alternative to the many approaches described in the literature.

Alanine↗
PubMed2005

Conformational variability of matrix metalloproteinases: beyond a single 3D structure.

The structures of the catalytic domain of matrix metalloproteinase 12 in the presence of acetohydroxamic acid and N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid have been solved by x-ray diffraction in the crystalline state at 1.0 and 1.3-A resolution, respectively, and compared with the previously published x-ray structure at 1.2-A resolution of the adduct with batimastat. The structure of the N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid adduct has been solved by NMR in solution. The three x-ray structures and the solution structure are similar but not identical to one another, the differences being sizably higher in the loops. We propose that many of the loops show a dynamical behavior in solution on a variety of time scales. Different conformations of some flexible regions of the protein can be observed as "frozen" in different crystalline environments. The mobility in solution studied by NMR reveals conformational equilibria in accessible time scales, i.e., from 10(-5) s to ms and more. Averaging of some residual dipolar couplings is consistent with further motions down to 10(-9) s. Finally, local thermal motions of each frozen conformation in the crystalline state at 100 K correlate well with local motions on the picosecond time scale. Flexibility/conformational heterogeneity in crucial parts of the catalytic domain is a rule rather than an exception in matrix metalloproteinases, and its extent may be underestimated by inspection of one x-ray structure. Backbone flexibility may play a role in the difficulties encountered in the design of selective inhibitors, whereas it may be a requisite for substrate binding and broad substrate specificity.

Catalytic Domain↗
PubMed2005

Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS.

Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme-inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free.

Catalytic Domain↗
PubMed2004

Persistent contrast enhancement by sterically stabilized paramagnetic liposomes in murine melanoma.

In the present research, we investigated the use of paramagnetic liposomes as contrast agents (CAs) for the detection of solid tumors. The liposomes were sterically stabilized by a polyethylene glycol (PEG) coating, and their size was constrained to approximately 100 nm. Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylene-triaminepentaacetate (DMPE-DTPA) was used as the gadolinium-carrying fatty acid chain. The relaxation properties were characterized through nuclear magnetic relaxation dispersion (NMRD) measurements, and analyzed with the use of theories and computer programs that are adequate for slowly rotating systems. Their relaxivity at 1.5 T was found to be acceptable for in vivo use. We then tested the liposomes against B16-F10 murine melanomas using standard T1-weighted schemes at 1.5 T, and concentrations corresponding to 0.03 mmol/kg of gadolinium (i.e., three to six times lower than the concentration of the small gadolinium complexes in clinical use). The blood half-life was found to be 120 +/- 20 min. The experiments show a good contrast enhancement in the tumor (33% +/- 22%) 2 hr after administration, a further increase (43 +/- 27%) 20 hr after administration, and a decrease (25% +/- 14%) 54 hr after administration. High persistence of the CA was also observed in the liver and intestine, as expected in a hepatobiliar excretion pathway.

Animals↗
PubMed2004

Water accessibility, aggregation, and motional features of polysaccharide-protein conjugate vaccines.

A relaxometric investigation of a nontoxic mutant of diphtheria toxin and of its conjugates with capsular polysaccharides of different groups of Neisseria meningitidis was performed. The insertion of polysaccharides chains alters dramatically the hydrodynamic properties of the protein. The model-free analysis of the (1)H nuclear magnetic relaxation dispersion profiles of their water solutions shows: i), a reduced protein hydration with respect to the carrier protein alone; ii), a much larger flexibility of the conjugates with respect to a compact macromolecule of the same molecular weight; and iii), a strong tendency to aggregate. The above findings are largely independent on the nature of the polysaccharide and thus provide a fairly general picture of the dynamic properties of glycoconjugate proteins.

Bacterial Capsules↗
PubMed2004

NMR-based techniques in the hit identification and optimisation processes.

In this review, the use of general NMR spectroscopy techniques to detect ligand binding and to monitor enzyme kinetics and inhibition, which appear particularly useful in hit identification and validation, is reiterated. Furthermore, the use of NMR-based strategies for lead optimisations that are based on either iterative derivatisations of an initial core structure or on linking fragments that occupy adjacent pockets in the target's binding site will also be described. Several recent examples will be reported and the use of these techniques in cases when the three dimensional structure of the target protein is known will be discussed.

Binding Sites↗
PubMed2002

Mechanistic studies of a calcium-dependent MRI contrast agent.

Intracellular Ca(2+) plays an important role in signal transduction, and we are developing new MRI techniques to study its regulation in living animals. We have reported on an MRI contrast agent (DOPTA-Gd) where the relaxivity of the complex is controlled by the presence or absence of the divalent ion Ca(2+). By structurally modulating inner-sphere access of water to a chelated Gd(3+) ion, we observe a substantial and reversible change in T(1) upon the addition of Ca(2+) and not other divalent ions. Luminescence lifetime and NMRD measurements of the complex have been acquired, and several parameters contribute to the Ca(2+) dependent relaxivity change of DOPTA-Gd. The number of inner-sphere water molecules is more than doubled after the Ca(2+) concentration is increased. This finding strongly supports the proposed conformational change of DOPTA-Gd when Ca(2+) is bound. Relaxometric measurements confirm these results and provide an indication that second-sphere water molecules are probably responsible for paramagnetic relaxation enhancement in the absence of Ca(2+). After Ca(2+) is bound to DOPTA-Gd, the molecule undergoes a substantial conformational change that opens up the hydrophilic face of the tetraazacyclododecane macrocycle. This change dramatically increases the accessibility of chelated Gd(3+) ion to bulk solvent. The design of this class of calcium-activated MR contrast agent was based primarily on the assumption that the number of coordinated inner-sphere water molecules would be the dominating factor in observed relaxivity measurements. This result has been confirmed; however, careful mechanistic studies reveal that additional factors are involved in this process.

Calcium↗
PubMed2002

A paramagnetic probe to localize residues next to carboxylates on protein surfaces.

It is shown that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction. The DO3A ligand is particularly useful in these studies, as it coordinates lanthanides in a heptadentate fashion, leaving two sites available for exogenous donors. A solution of a (15)N-labeled sample protein, calbindin D(9k) (75 residues), was titrated with up to 200% of Gd(III)-DO3A complex, and an inversion recovery (15)N-(1)H HSQC experiment was used to measure the paramagnetic contributions to the longitudinal relaxation rates of the amide protons. Relaxation data were used as distance constraints to estimate the number of interacting complexes and the occupancies of their binding sites. Four preferential interaction sites on the protein surface are found. Inspection of the various carboxylate side chains on the surface of the protein indicates that Gd(III)-DO3A interacts preferentially with carboxylate-rich regions, rather than with isolated carboxylates, suggesting the possibility of chelation of one Gd(III)-DO3A molecule by two carboxylate groups. Gd(III)-DO3A is thus a valuable semi-selective probe for clusters of negative charges on the protein surface.

Algorithms↗