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Henrich S.,Molecular and Cellular Modeling Group | Salo-Ahen O.M.H.,Molecular and Cellular Modeling Group | Huang B.,Molecular and Cellular Modeling Group | Rippmann F.,Merck KGaA | And 2 more authors.
Journal of Molecular Recognition | Year: 2010

Given the three-dimensional structure of a protein, how can one find the sites where other molecules might bind to it? Do these sites have the properties necessary for high affinity binding? Is this protein a suitable target for drug design? Here, we discuss recent developments in computational methods to address these and related questions. Geometric methods to identify pockets on protein surfaces have been developed over many years but, with new algorithms, their performance is still improving. Simulation methods show promise in accounting for protein conformational variability to identify transient pockets but lack the ease of use of many of the (rigid) shape-based tools. Sequence and structure comparison approaches are benefiting from the constantly increasing size of sequence and structure databases. Energetic methods can aid identification and characterization of binding pockets, and have undergone recent improvements in the treatment of solvation and hydrophobicity. The "druggability" of a binding site is still difficult to predict with an automated procedure. The methodologies available for this purpose range from simple shape and hydrophobicity scores to computationally demanding free energy simulations. © 2009 John Wiley & Sons, Ltd.

Salo-Ahen O.M.H.,Molecular and Cellular Modeling Group | Battini R.,University of Modena and Reggio Emilia | Ponterini G.,University of Modena and Reggio Emilia | Wade R.C.,Molecular and Cellular Modeling Group
Protein Engineering, Design and Selection | Year: 2010

Owing to its central role in DNA synthesis, human thymidylate synthase (hTS) is a well-established target for chemotherapeutic agents, such as fluoropyrimidines. The use of hTS inhibitors in cancer therapy is limited by their toxicity and the development of cellular drug resistance. Here, with the aim of shedding light on the structural role of the A-helix in fluoropyrimidine resistance, we have created a fluoropyrimidine-resistant mutant by making a single point mutation, Glu30Trp. We postulated that residue 30, which is located in the A-helix, close to but outside the enzyme active site, could have a long-range effect on inhibitor binding. The mutant shows 100 times lower specific activity with respect to the wild-type hTS and is resistant to the classical inhibitor, FdUMP, as shown by a 6-fold higher inhibition constant. Circular dichroism experiments show that the mutant is folded. The results of molecular modeling and simulation suggest that the Glu30Trp mutation gives rise to resistance by altering the hydrogen-bond network between residue 30 and the active site.

Henrich S.,Molecular and Cellular Modeling Group | Feierberg I.,Astrazeneca | Wang T.,Molecular and Cellular Modeling Group | Wang T.,University of California at Davis | And 3 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2010

A major challenge in drug design is to obtain compounds that bind selectively to their target receptors and do not cause side-effects by binding to other similar receptors. Here, we investigate strategies for applying COMBINE (COMparative BINding Energy) analysis, in conjunction with PIPSA (Protein Interaction Property Similarity Analysis) and Iigand docking methods, to address this problem. We evaluate these approaches by application to diverse sets of inhibitors of three structurally related serine proteases of medical relevance: thrombin, trypsin, and urokinase-type plasminogen activator (uPA). We generated target-specific scoring functions (COMBINE models) for the three targets using training sets of ligands with known inhibition constants and structures of their receptor-ligand complexes. These COMBINE models were compared with the PIPSA results and experimental data on receptor selectivity. These scoring functions highlight the ligand-receptor interactions that are particularly important for binding specificity for the different targets. To predict target selectivity in virtual screening, compounds were docked into the three protein binding sites using the program GOLD and the docking solutions were re-ranked with the target-specific scoring functions and computed electrostatic binding free energies. Limits in the accuracy of some of the docking solutions and difficulties in scoring them adversely affected the predictive ability of the target specific scoring functions. Nevertheless, the target-specific scoring functions enabled the selectivity of ligands to thrombin versus trypsin and uPA to be predicted. © 2009 Wiley-Liss Inc.

Cardinale D.,University of Modena and Reggio Emilia | Salo-Ahen O.M.H.,Molecular and Cellular Modeling Group | Ferrari S.,University of Modena and Reggio Emilia | Ponterini G.,University of Modena and Reggio Emilia | And 6 more authors.
Current Medicinal Chemistry | Year: 2010

Many enzymes and proteins are regulated by their quaternary structure and/or by their association in homoand/ or hetero-oligomer complexes. Thus, these protein-protein interactions can be good targets for blocking or modulating protein function therapeutically. The large number of oligomeric structures in the Protein Data Bank (http://www.rcsb.org/) reflects growing interest in proteins that function as multimeric complexes. In this review, we consider the particular case of homodimeric enzymes as drug targets. There is intense interest in drugs that inhibit dimerization of a functionally obligate homodimeric enzyme. Because amino acid conservation within enzyme interfaces is often low compared to conservation in active sites, it may be easier to achieve drugs that target protein interfaces selectively and specifically. Two main types of dimerization inhibitors have been developed: peptides or peptidomimetics based on sequences involved in protein-protein interactions, and small molecules that act at hot spots in protein-protein interfaces. Examples include inhibitors of HIV protease and HIV integrase. Studying the mechanisms of action and locating the binding sites of such inhibitors requires different techniques for different proteins. For some enzymes, ligand binding is only detectable in vivo or after unfolding of the complexes. Here, we review the structural features of dimeric enzymes and give examples of inhibition through interference in dimer stability. Several techniques for studying these complex phenomena will be presented. © 2010 Bentham Science Publishers Ltd.

Ami D.,Fondazione IRCCS Policlinico San Matteo | Mereghetti P.,University of Heidelberg | Mereghetti P.,Molecular and Cellular Modeling Group | Natalello A.,University of Milan Bicocca | And 4 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2011

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050-2800cm-1) and protein (1700-1600cm-1) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm-1. The use of the multivariate principal component-linear discriminant analysis (PCA-LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data. © 2011 Elsevier B.V.

Lamperti C.,Fondazione Instituto Neurologico Carlo Besta IRCCS | Diodato D.,Fondazione Instituto Neurologico Carlo Besta IRCCS | Lamantea E.,Fondazione Instituto Neurologico Carlo Besta IRCCS | Carrara F.,Fondazione Instituto Neurologico Carlo Besta IRCCS | And 4 more authors.
Neuromuscular Disorders | Year: 2012

We report a 35-year-old woman presenting a stroke-like episode with transitory aphasia followed by generalized tonic-clonic seizures. She had severe hearing loss and suffered from frequent episodes of migraine. Although a brain MRI disclosed a T2-hyperintense lesion in the left parietal lobe, she had hardly any long-term sequela. Exercise intolerance, myalgias and limb-girdle muscle weakness indicated a slowly progressive myopathy. Extra-neurological features included short stature, and secondary amenorrhea with low gonadotropin levels, indicating secondary hypogonadism. However, she had three mutation-free, healthy children by ovarian stimulation. A muscle biopsy showed ragged-red, cytochrome c oxidase-negative fibers, and an isolated defect of cytochrome c oxidase activity in muscle mitochondria. Sequence analysis of muscle mtDNA revealed a previously unreported heteroplasmic m.6597C>A transversion in the MTCOI gene, encoding subunit I of cytochrome c oxidase, corresponding to p.Q232K aminoacid change. Analysis on transmitochondrial cybrids demonstrated that the mutation is indeed associated with COX deficiency, i.e. pathogenic. © 2012 Elsevier B.V.

PubMed | Molecular and Cellular Modeling Group
Type: Journal Article | Journal: Journal of chemical theory and computation | Year: 2015

The secondary structure propensities observed in protein simulations depend heavily on the force field parameters used. The existing empirical force fields often have difficulty in balancing the relative stabilities of helical and extended conformations. The resultant secondary structure bias may not be apparent in short simulations at room temperature starting from the native folded states. However, it can manifest itself dramatically at high temperatures and lead to large deviations from experimentally observed secondary structure propensities. Motivated by thermal unfolding simulations of several WW domains, which have a three-stranded -sheet structure, we chose the FBP28 WW domain as a well-characterized system to investigate several AMBER force fields as well as parametrization of the NPSA (Neutralized, Polarized ionizable side chains with a solvent-accessible Surface Area-dependent term) implicit solvent model. The ff94 force field and two variants with altered parameters for the backbone torsion term were found to convert the native -sheet structure directly to a single helix at high temperatures, whereas the ff96 force field produced significant non-native -sheet content at high temperatures. The ff03 force field was able to reproduce the -sheet-coil transition and experimentally observed unfolding pathways with both an explicit water solvent and the NPSA implicit solvent model at relatively low temperatures. However, the protein domain became predominantly helical after unfolding. Modification of the solvation parameter in the NPSA implicit solvent model was not sufficient to remedy this problem. The results imply that the intrinsic secondary structure bias in a force field cannot easily be solved by modifying a single parameter such as backbone torsion potential or a solvation parameter of a solvent model. Nevertheless, the results show that the AMBER ff03 force field together with an explicit solvent model or the NPSA implicit solvent model is a useful tool for studying the unfolding of both - and -sheet structure protein domains, and an integrative consideration of all force field parameters is likely to be necessary for a complete solution.

PubMed | Molecular and Cellular Modeling Group
Type: Journal Article | Journal: Journal of chemical theory and computation | Year: 2015

In protein unfolding simulations, elevated temperature, significantly exceeding the melting temperature Tm, provides an important means to accelerate unfolding to a computationally accessible time range. This procedure is based on the assumption that protein thermal unfolding has Arrhenius behavior and therefore that increasing temperature does not alter the protein unfolding pathways. However, in nature, proteins can show non-Arrhenius behavior and, in practice, overly fast unfolding in high-temperature simulations can result in difficulties in identifying unfolding intermediates and distinguishing their relative stabilities. In this paper, we describe simulations of two WW domains, small protein domains that have a three-stranded -sheet structure. Simulations were carried out at several temperatures ranging from 300 K to 500 K, starting from folded structures. The results demonstrate the temperature dependence of the unfolding pathways, showing that to obtain unfolding pathways corresponding to those observed in experiments, the elevation of the simulation temperature has to be controlled. Based on trajectory analysis, we proposed a qualitative criterion for judging when an elevated temperature is acceptable or not, namely, that the temperature must be such that the native folded state is sampled substantially before protein unfolding begins. While depending on force field parameters and protein fold complexity, this criterion can be quantified to obtain the upper bound of an acceptable elevated temperature, which was observed to be dependent on the thermostabilities of the two WW domain proteins.

PubMed | Molecular and Cellular Modeling Group
Type: Journal Article | Journal: Drug discovery today. Technologies | Year: 2014

The three-dimensional structures of proteins are being solved apace, yet this information is often underused in quantitative structure-activity relationship (QSAR) studies. Here, we describe and compare methods for exploiting protein structures to derive 3D-QSARs. These methods can facilitate molecular design and lead optimization and should increasingly become a standard component of the drug designers repertoire.:

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