Publications by authors named "Paolo Cozzi"

19 Publications

  • Page 1 of 1

Water management and phenology influence the root-associated rice field microbiota.

FEMS Microbiol Ecol 2020 09;96(9)

Institute for Sustainable Plant Protection (IPSP), National Research Council (CNR), Viale P.A. Mattioli 25, I-10125 Torino, Italy.

Microbial communities associated with plants are greatly influenced by water availability in soil. In flooded crops, such as rice, the impact of water management on microbial dynamics is not fully understood. Here, we present a comprehensive study of the rice microbiota investigated in an experimental field located in one of the most productive areas of northern Italy. The microbiota associated with paddy soil and root was investigated using 454 pyrosequencing of 16S, ITS and 18S rRNA gene amplicons under two different water managements, upland (non-flooded, aerobic) and lowland (traditional flooding, anaerobic), at three plant development stages. Results highlighted a major role of the soil water status in shaping microbial communities, while phenological stage had low impacts. Compositional shifts in prokaryotic and fungal communities upon water management consisted in significant abundance changes of Firmicutes, Methanobacteria, Chloroflexi, Sordariomycetes, Dothideomycetes and Glomeromycotina. A vicariance in plant beneficial microbes and between saprotrophs and pathotrophs was observed between lowland and upland. Moreover, through network analysis, we demonstrated different co-abundance dynamics between lowland and upland conditions with a major impact on microbial hubs (strongly interconnected microbes) that fully shifted to aerobic microbes in the absence of flooding.
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http://dx.doi.org/10.1093/femsec/fiaa146DOI Listing
September 2020

Durum wheat genome highlights past domestication signatures and future improvement targets.

Nat Genet 2019 05 8;51(5):885-895. Epub 2019 Apr 8.

CREA-Research Centre for Genomics and Bioinformatics, Fiorenzuola d'Arda, Italy.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.
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http://dx.doi.org/10.1038/s41588-019-0381-3DOI Listing
May 2019

Genome-Wide Analysis of Rice Performance under Limited Water and Permanent Flooding Conditions.

Front Plant Sci 2017 30;8:1862. Epub 2017 Oct 30.

Research Centre for Cereal and Industrial Crops, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Vercelli, Italy.

A rice GWAS panel of 281 accessions of rice was phenotypically characterized for 26 traits related to phenology, plant and seed morphology, physiology and yield for 2 years in field conditions under permanent flooding (PF) and limited water (LW). A genome-wide analysis uncovered a total of 160 significant marker-trait associations (MTAs), of which 32 were LW-specific, 59 were PF-specific, and 69 were in common between the two water management systems. LW-specific associations were identified for several agronomic traits including days to maturation, days from flowering to maturation, leaf traits, plant height, panicle and seed traits, hundred grain weight, yield and tillering. Significant MTAs were detected across all the 12 rice chromosomes, while clusters of effects influencing different traits under LW or in both watering conditions were, respectively, observed on chromosomes 4, 8, and 12 and on chromosomes 1, 3, 4, 5, and 8. The analysis of genes annotated in the Nipponbare reference sequence and included in the regions associated to traits related to plant morphology, grain yield, and physiological parameters allowed the identification of genes that were demonstrated to affect the respective traits. Among these, three () and seven () candidate genes were, respectively, identified to co-localize with LW-specific associations and associations in common between the two water treatments. For several LW-specific MTAs, or in common among the two treatments, positional co-localizations with previously identified QTLs for rice adaptation to water shortages were observed, a result that further supports the role of the loci identified in this work in conferring adaptation to LW. The most robust associations identified here could represent suitable targets for genomic selection approaches to improve yield-related traits under LW.
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http://dx.doi.org/10.3389/fpls.2017.01862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670151PMC
October 2017

Genome-Wide Association Study for Traits Related to Plant and Grain Morphology, and Root Architecture in Temperate Rice Accessions.

PLoS One 2016 26;11(5):e0155425. Epub 2016 May 26.

PTP Science Park, 26900 Lodi, Italy.

Background: In this study we carried out a genome-wide association analysis for plant and grain morphology and root architecture in a unique panel of temperate rice accessions adapted to European pedo-climatic conditions. This is the first study to assess the association of selected phenotypic traits to specific genomic regions in the narrow genetic pool of temperate japonica. A set of 391 rice accessions were GBS-genotyped yielding-after data editing-57000 polymorphic and informative SNPS, among which 54% were in genic regions.

Results: In total, 42 significant genotype-phenotype associations were detected: 21 for plant morphology traits, 11 for grain quality traits, 10 for root architecture traits. The FDR of detected associations ranged from 3 · 10-7 to 0.92 (median: 0.25). In most cases, the significant detected associations co-localised with QTLs and candidate genes controlling the phenotypic variation of single or multiple traits. The most significant associations were those for flag leaf width on chromosome 4 (FDR = 3 · 10-7) and for plant height on chromosome 6 (FDR = 0.011).

Conclusions: We demonstrate the effectiveness and resolution of the developed platform for high-throughput phenotyping, genotyping and GWAS in detecting major QTLs for relevant traits in rice. We identified strong associations that may be used for selection in temperate irrigated rice breeding: e.g. associations for flag leaf width, plant height, root volume and length, grain length, grain width and their ratio. Our findings pave the way to successfully exploit the narrow genetic pool of European temperate rice and to pinpoint the most relevant genetic components contributing to the adaptability and high yield of this germplasm. The generated data could be of direct use in genomic-assisted breeding strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881974PMC
July 2017

Segmenting the Human Genome into Isochores.

Evol Bioinform Online 2015 25;11:253-61. Epub 2015 Nov 25.

National Research Council, Institute for Biomedical Technologies, Segrate, Milan, Italy. ; Science Department, Rome 3 University, Rome, Italy.

The human genome is a mosaic of isochores, which are long (>200 kb) DNA sequences that are fairly homogeneous in base composition and can be assigned to five families comprising 33%-59% of GC composition. Although the compartmentalized organization of the mammalian genome has been investigated for more than 40 years, no satisfactory automatic procedure for segmenting the genome into isochores is available so far. We present a critical discussion of the currently available methods and a new approach called isoSegmenter which allows segmenting the genome into isochores in a fast and completely automatic manner. This approach relies on two types of experimentally defined parameters, the compositional boundaries of isochore families and an optimal window size of 100 kb. The approach represents an improvement over the existing methods, is ideally suited for investigating long-range features of sequenced and assembled genomes, and is publicly available at https://github.com/bunop/isoSegmenter.
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http://dx.doi.org/10.4137/EBO.S27693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662427PMC
December 2015

Characterization of HIV-1 entry inhibitors with broad activity against R5 and X4 viral strains.

J Transl Med 2015 Apr 2;13:107. Epub 2015 Apr 2.

Unit of Molecular Immunology, Division of Genetics and Cell Biology, San Raffaele Hospital, Via Olgettina 60, 20132, Milan, Italy.

Background: Combined antiretroviral therapy has drastically reduced mortality and morbidity of HIV-infected individuals. Nevertheless long-term toxicity and appearance of viral resistance hampers the prolonged effectiveness of combination therapy, requiring a continuous input of drugs to replace those utilized in combination regimens. We here investigated the anti-HIV activity of novel derivatives of the suradista chemical class.

Methods: Compounds were tested on acute HIV-1 infection of activated peripheral blood mononuclear cells. HIV production was monitored by enzyme-linked immunosorbent assay measuring the protein p24 released in culture supernatants. Fusion assays were carried out to study the mechanism of action of these compounds. A modified version of a previously established recombinant vaccinia virus-based assay was used measuring activation of a reporter gene upon fusion of two distinct cell populations. Flow cytometry was performed in competition assays for the binding of several antibodies targeting different sites of the viral envelope glycoprotein gp120, or the receptor CD4, or the coreceptors CXCR4 and CCR5.

Results: Four compounds inhibited replication of a prototypic R5 (BaL) and X4 (IIIB) laboratory-adapted HIV-1 strain at low micromolar concentrations, in the absence of cytotoxicity. Approximately a ten fold greater activity was achieved against the X4 as compared to the R5 strain. The compounds blocked X4 and R5 HIV-1 fusion, a step of viral entry. This activity appeared specific for HIV-1, as entry of human herpesvirus 6 (HHV-6) and influenza virus was not substantially affected. Further investigation of the inhibitory mechanism revealed that these new molecules target the viral envelope, rather than the coreceptors, as previously shown for a congener of the same class characterized by a long plasmatic half-life. Indeed ND-4043, the most active compound, specifically competed with binding of monoclonal antibodies against the CD4-binding site (CD4-BS) and coreceptor-binding site (CoR-BS) of gp120. These compounds displayed broad anti-HIV activity, as they inhibited various primary R5, X4 and, importantly, dualtropic R5X4 HIV-1 isolates. Of the four derivatives tested, the dimeric compounds were consistently more potent than the monomeric ones.

Conclusions: Given their unique features, these molecules represent promising candidates for further development and exploitation as anti-HIV therapeutics.
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http://dx.doi.org/10.1186/s12967-015-0461-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399250PMC
April 2015

SNPchiMp v.3: integrating and standardizing single nucleotide polymorphism data for livestock species.

BMC Genomics 2015 Apr 10;16:283. Epub 2015 Apr 10.

Bioinformatics and Biostatistical Genomics group, Fondazione Parco Tecnologico Padano, Via Einstein, Loc. Cascina Codazza, 26900, Lodi, Italy.

Background: In recent years, the use of genomic information in livestock species for genetic improvement, association studies and many other fields has become routine. In order to accommodate different market requirements in terms of genotyping cost, manufacturers of single nucleotide polymorphism (SNP) arrays, private companies and international consortia have developed a large number of arrays with different content and different SNP density. The number of currently available SNP arrays differs among species: ranging from one for goats to more than ten for cattle, and the number of arrays available is increasing rapidly. However, there is limited or no effort to standardize and integrate array- specific (e.g. SNP IDs, allele coding) and species-specific (i.e. past and current assemblies) SNP information.

Results: Here we present SNPchiMp v.3, a solution to these issues for the six major livestock species (cow, pig, horse, sheep, goat and chicken). Original data was collected directly from SNP array producers and specific international genome consortia, and stored in a MySQL database. The database was then linked to an open-access web tool and to public databases. SNPchiMp v.3 ensures fast access to the database (retrieving within/across SNP array data) and the possibility of annotating SNP array data in a user-friendly fashion.

Conclusions: This platform allows easy integration and standardization, and it is aimed at both industry and research. It also enables users to easily link the information available from the array producer with data in public databases, without the need of additional bioinformatics tools or pipelines. In recognition of the open-access use of Ensembl resources, SNPchiMp v.3 was officially credited as an Ensembl E!mpowered tool. Availability at http://bioinformatics.tecnoparco.org/SNPchimp.
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http://dx.doi.org/10.1186/s12864-015-1497-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399246PMC
April 2015

SNPranker 2.0: a gene-centric data mining tool for diseases associated SNP prioritization in GWAS.

BMC Bioinformatics 2013 14;14 Suppl 1:S9. Epub 2013 Jan 14.

Consiglio Nazionale delle Ricerche - Istituto di Tecnologie Biomediche (CNR-ITB), Via F,lli Cervi 93, 20090 Segrate (MI), Italy.

Background: The capability of correlating specific genotypes with human diseases is a complex issue in spite of all advantages arisen from high-throughput technologies, such as Genome Wide Association Studies (GWAS). New tools for genetic variants interpretation and for Single Nucleotide Polymorphisms (SNPs) prioritization are actually needed. Given a list of the most relevant SNPs statistically associated to a specific pathology as result of a genotype study, a critical issue is the identification of genes that are effectively related to the disease by re-scoring the importance of the identified genetic variations. Vice versa, given a list of genes, it can be of great importance to predict which SNPs can be involved in the onset of a particular disease, in order to focus the research on their effects.

Results: We propose a new bioinformatics approach to support biological data mining in the analysis and interpretation of SNPs associated to pathologies. This system can be employed to design custom genotyping chips for disease-oriented studies and to re-score GWAS results. The proposed method relies (1) on the data integration of public resources using a gene-centric database design, (2) on the evaluation of a set of static biomolecular annotations, defined as features, and (3) on the SNP scoring function, which computes SNP scores using parameters and weights set by users. We employed a machine learning classifier to set default feature weights and an ontological annotation layer to enable the enrichment of the input gene set. We implemented our method as a web tool called SNPranker 2.0 (http://www.itb.cnr.it/snpranker), improving our first published release of this system. A user-friendly interface allows the input of a list of genes, SNPs or a biological process, and to customize the features set with relative weights. As result, SNPranker 2.0 returns a list of SNPs, localized within input and ontologically enriched genes, combined with their prioritization scores.

Conclusions: Different databases and resources are already available for SNPs annotation, but they do not prioritize or re-score SNPs relying on a-priori biomolecular knowledge. SNPranker 2.0 attempts to fill this gap through a user-friendly integrated web resource. End users, such as researchers in medical genetics and epidemiology, may find in SNPranker 2.0 a new tool for data mining and interpretation able to support SNPs analysis. Possible scenarios are GWAS data re-scoring, SNPs selection for custom genotyping arrays and SNPs/diseases association studies.
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http://dx.doi.org/10.1186/1471-2105-14-S1-S9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548692PMC
August 2013

Image-based surface matching algorithm oriented to structural biology.

IEEE/ACM Trans Comput Biol Bioinform 2011 Jul-Aug;8(4):1004-16

Institute for Biomedical Technologies, Italian National Research Council, Segrate (Milan), Italy.

Emerging technologies for structure matching based on surface descriptions have demonstrated their effectiveness in many research fields. In particular, they can be successfully applied to in silico studies of structural biology. Protein activities, in fact, are related to the external characteristics of these macromolecules and the ability to match surfaces can be important to infer information about their possible functions and interactions. In this work, we present a surface-matching algorithm, based on encoding the outer morphology of proteins in images of local description, which allows us to establish point-to-point correlations among macromolecular surfaces using image-processing functions. Discarding methods relying on biological analysis of atomic structures and expensive computational approaches based on energetic studies, this algorithm can successfully be used for macromolecular recognition by employing local surface features. Results demonstrate that the proposed algorithm can be employed both to identify surface similarities in context of macromolecular functional analysis and to screen possible protein interactions to predict pairing capability.
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http://dx.doi.org/10.1109/TCBB.2010.21DOI Listing
September 2011

Fine mapping of AHI1 as a schizophrenia susceptibility gene: from association to evolutionary evidence.

FASEB J 2010 Aug 6;24(8):3066-82. Epub 2010 Apr 6.

Genomics and Bioinformatics Unit, University of Milan-Fondazione Filarete, University of Milan, Milan, Italy.

In previous studies, we identified a locus for schizophrenia on 6q23.3 and proposed the Abelson helper integration site 1 (AHI1) as the candidate gene. AHI1 is expressed in the brain and plays a key role in neurodevelopment, is involved in Joubert syndrome, and has been recently associated with autism. The neurodevelopmental role of AHI1 fits with etiological hypotheses of schizophrenia. To definitively confirm our hypothesis, we searched for associations using a dense map of the region. Our strongest findings lay within the AHI1 gene: single-nucleotide polymorphisms rs11154801 and rs7759971 showed significant associations (P=6.23E-06; P=0.84E-06) and haplotypes gave P values in the 10E-8 to 10E-10 range. The second highest significant region maps close to AHI1 and includes the intergenic region between BC040979 and PDE7B (rs2038549 at P=9.70E-06 and rs1475069 at P=6.97E-06), and PDE7B and MAP7. Using a sample of Palestinian Arab families to confirm these findings, we found isolated signals. While these results did not retain their significance after correction for multiple testing, the joint analysis across the 2 samples supports the role of AHI1, despite the presence of heterogeneity. Given the hypothesis of positive selection of schizophrenia genes, we resequenced a 11 kb region within AHI1 in ethnically defined populations and found evidence for a selective sweep. Network analysis indicates 2 haplotype clades, with schizophrenia-susceptibility haplotypes clustering within the major clade. In conclusion, our data support the role of AHI1 as a susceptibility gene for schizophrenia and confirm it has been subjected to positive selection, also shedding light on new possible candidate genes, MAP7 and PDE7B.
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http://dx.doi.org/10.1096/fj.09-152611DOI Listing
August 2010

Virtual screening pipeline and ligand modelling for H5N1 neuraminidase.

Biochem Biophys Res Commun 2009 Jun 14;383(4):445-9. Epub 2009 Apr 14.

CNR-Institute for Biomedical Technologies, Via Fratelli Cervi 93, 20090 Segrate (MI), Italy.

The H5N1 virus neuraminidase structure was solved in two different conformations depending on the inhibitor concentration. In the absence of oseltamivir or at a low concentration, the neuraminidase structure assumes an open form that closes at a high oseltamivir concentration due to the shift of the so-called 150-loop near the active site. Although the close conformation is similar to all the other structurally known neuraminidase types, it doesn't appear to be the most likely physiological condition for N1. To investigate the specific ligand binding properties of the open form, we screened by docking simulation, a large dataset of ligands and compared the results with closed form. The virtual screening procedure was implemented in a docking pipeline that also performs a step-by-step, target specific, filtering approach for data reduction. The selected ligands display binding ability involving multiple sites of interaction including the active site and an adjacent cavity made available by the 150-loop shift. Two ligands are especially interesting and are proposed as substituents to design oseltamivir derivatives specifically suited for the open conformation.
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http://dx.doi.org/10.1016/j.bbrc.2009.04.030DOI Listing
June 2009

Cinnamoyl nitrogen mustard derivatives of pyrazole analogues of tallimustine modified at the amidino moiety: design, synthesis, molecular modeling and antitumor activity studies.

Bioorg Med Chem 2004 Jul;12(14):3911-21

Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy.

The design, synthesis and in vitro activities of a series of cinnamoyl nitrogen mustard pyrazole analogues of tallimustine 8-13, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the amidino moiety, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. A selected series of compounds have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukemia cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity. Our preliminary results indicated that the compounds of this series have a pattern of alkylation similar to that of tallimustine, but they seem to be less reactive overall in alkylating naked DNA.
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http://dx.doi.org/10.1016/j.bmc.2004.04.045DOI Listing
July 2004

Cytotoxic alpha-halogenoacrylic derivatives of distamycin A and congeners.

J Med Chem 2004 May;47(10):2611-23

Pharmacia Italia S.p.A., Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

The mechanism of action of many antitumor agents involves DNA damage, either by direct binding of the drug to DNA or to DNA-binding proteins. However, most of the DNA-interacting agents have only a limited degree of sequence specificity, which implies that they may hit all the cellular genes. DNA minor groove binders, among which the derivatives of distamycin A play an important role, could provide significant improvement in cancer management, increasing gene specificity, due to high selectivity of interaction with thymine-adenine (TA) rich sequences. We now report and discuss the synthesis, the in vitro and in vivo activities, and some mechanistic features of alpha-halogenoacrylamido derivatives of distamycin A. The final result of this work was the selection of brostallicin 17 (PNU-166196). Brostallicin, presently in phase II clinical trials, shows a broad spectrum of antitumor activity and an apoptotic effect higher than distamycin derivative tallimustine. An important in vitro toxicological feature of brostallicin is the very good ratio between myelotoxicity on human haematopoietic progenitor cells and cytotoxicity on tumor cells, in comparison with clinically tested DNA minor groove binders. A peculiarity of brostallicin is its in vitro reactivity in the DNA alkylation assays only in the presence of glutathione. Moreover brostallicin's antitumor activity, both in in vitro and in vivo tumor models, is higher in the presence of increased levels of glutathione/glutathione-S-tranferases. These findings contribute to the definition of brostallicin as a novel anticancer agent that differs from other minor groove binders and alkylating agents for both the profile of activity and the mechanism of action and to classify the alpha-bromoacrylamido derivatives of distamycin as a new class of cytotoxics. Moreover, due to its interaction with glutathione, brostallicin may have a role for the tailored treatment of tumors characterized by constitutive or therapy-induced overexpression of glutathione/glutathione-S-tranferase levels.
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http://dx.doi.org/10.1021/jm031051kDOI Listing
May 2004

Recent anticancer cytotoxic agents.

Curr Med Chem Anticancer Agents 2004 Mar;4(2):93-121

Pharmacia Italia, Gruppo Pfizer Inc, Discovery Research Oncology, Department of Chemistry, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

In spite of the impressive progress in diagnosis, surgery and therapy that occurred since the Sixties, the overall cancer mortality is still high and the medical need is largely unmet. A number of innovative strategies, aimed to target malignant abnormalities of tumor cells are in development and begin to give important results. In alternative, angiogenesis inhibition has been addressed with the aim to limit the tumor ability to grow and metastasize. However, it will likely take some years to fully define the therapeutic role of different innovative drugs. Therefore, cytotoxic drugs will continue to represent a chief part of the therapy in the forthcoming years, possibly in combination with innovative agents addressing molecular targets. Most important traditional chemotherapeutic drugs or investigational anticancer agents were derived from natural sources also through synthetic structural modifications. In the Nineties, taxanes and camptothecins represented important success stories of this approach, while among DNA interacting agents anthracyclines continued to represent a structural platform for discovering new drugs and DNA minor groove binders represented a new field of investigation. Combinatorial chemistry combined with high-throughput screening programs are an important source of totally synthetic new agents, however, it should not be disregarded the fact that nature already performed combinatorial chemistry and leads selection through the ages. New natural or semisynthetic agents acting as tubulin stabilizers or DNA interactive agents of various mechanisms of action are presently investigated and will probably continue to give important contribution to cancer therapy in the near future. In this review, the medicinal chemistry and the development status of these anticancer cytotoxic agents are focused and discussed.
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http://dx.doi.org/10.2174/1568011043482061DOI Listing
March 2004

Solid-phase synthesis of a small library of 3-phenylthio-3-nicotinyl propionic acid derivatives acting as antagonists of the integrin alphaVbeta3.

Bioorg Med Chem Lett 2004 Feb;14(3):657-61

Department of Chemistry, Pharmacia Italia SpA-Gruppo Pfizer, Discovery Research Oncology, Viale Pasteur 10, 20014 (MI), Nerviano, Italy.

We describe the synthesis of a series of low molecular weight inhibitors of the alphavbeta3 integrin obtained by modifying a high-throughput screening hit with micromolar activity. A solid phase synthesis to prepare 3-phenylthio-3-nicotinyl propionic acid derivatives, exemplified by 13c, was set up. Compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit cell adhesion mediated by vitronectin have been obtained.
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http://dx.doi.org/10.1016/j.bmcl.2003.11.045DOI Listing
February 2004

The discovery of a new potential anticancer drug: a case history.

Authors:
Paolo Cozzi

Farmaco 2003 Mar;58(3):213-20

Department of Chemistry, Pharmacia, Global Chemistry, Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

DNA minor groove binders (MGB) represent a class of anticancer agents whose DNA sequence specificity was hypothesized to lead to high selectivity of action. Tallimustine (TAM), a benzoyl nitrogen mustard derivative of distamycin A (DST), showed excellent antitumor activity in preclinical tests, but also a severe myelotoxicity. Novel nitrogen mustard, nitrogen half-mustard and sulfur mustard derivatives of DST showing excellent activity were recently identified and SAR reported. In particular nitrogen half-mustard and sulfur mustard derivatives, as one-arm alkylating agents, represent interesting structural novelties. A further new class of cytotoxic anticancer agents is that of alpha-halogenoacrylamido derivatives of DST-like oligopeptides, which show an activity profile substantially improved in comparison to TAM. In particular brostallicin (PNU-166196), alpha-bromo-acrylamido tetra-pyrrole derivative ending with a guanidino moiety, showed high cytotoxic potency and myelotoxicity dramatically reduced in comparison to TAM and other MGB. Brostallicin binds to the minor groove but appears unreactive in classical in vitro DNA alkylation assays. About the apparent lack of DNA alkylation we speculated that an intracellular nucleophile, e.g. glutathione (GSH), could activate the reactivity of the compound leading to alkylation of DNA in vivo. Evidence of both covalent interaction of brostallicin with plasmidic DNA in the presence of GSH and of enhanced cytotoxicity in cancer cells characterized by high levels of GSH were obtained. Brostallicin was selected for clinical development and is now undergoing Phase II studies.
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http://dx.doi.org/10.1016/S0014-827X(03)00014-4DOI Listing
March 2003

Synthesis and growth inhibition activity of alpha-bromoacrylic heterocyclic and benzoheterocyclic derivatives of distamycin A modified on the amidino moiety.

Bioorg Med Chem 2003 Mar;11(6):965-75

Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy.

The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of alpha-bromoacrylic derivatives of distamycin.
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http://dx.doi.org/10.1016/s0968-0896(02)00533-3DOI Listing
March 2003

Cytotoxic alpha-bromoacrylic derivatives of low molecular weight.

Bioorg Med Chem Lett 2002 Jun;12(11):1467-71

Chemistry Department, Pharmacia Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

In vitro and in vivo activities of a small series of alpha-bromoacrylic derivatives of low molecular weight (MW) are described and compared with those of alpha-bromoacrylic derivatives of distamycin-like frames. Low MW compounds, when lacking of a strong basic moiety, are potent cytotoxics, while analogues bearing a strong basic moiety are not. This suggests the existence of an active transport mechanism for distamycin-derived cytotoxics characterized by strong basic amidino or guanidino moieties. Low MW compounds are inactive in vivo, possibly because of the metabolic lability of alpha-bromoacrylic moiety. The same moiety is however present in a series of potent anticancer distamycin-like minor groove binders, for example, PNU-166196 (brostallicin), a fact that underlines the features of the latter.
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http://dx.doi.org/10.1016/s0960-894x(02)00177-4DOI Listing
June 2002

Brostallicin, a novel anticancer agent whose activity is enhanced upon binding to glutathione.

Cancer Res 2002 Apr;62(8):2332-6

Laboratory of Molecular Pharmacology, Department of Oncology, Mario Negri Institute of Pharmacological Research, via Eritrea 62, 20157 Milan, Italy.

Brostallicin (PNU-166196) is a synthetic alpha-bromoacrylic, second-generation DNA minor groove binder structurally related to distamycin A, presently in Phase II trials in Europe and the United States. The compound shows broad antitumor activity in preclinical models and dramatically reduced in vitro myelotoxicity in human hematopoietic progenitor cells compared with that of other minor groove binders. Brostallicin showed a 3-fold higher activity in melphalan-resistant L1210 murine leukemia cells than in the parental line (IC(50) = 0.46 and 1.45 ng/ml, respectively) under conditions in which the cytotoxicity of conventional antitumor agents was either unaffected or reduced. This melphalan-resistant cell line has increased levels of glutathione (GSH) in comparison with the parental cells. Conversely, GSH depletion by buthionine sulfoximine in a human ovarian carcinoma cell line (A2780) significantly decreased both the cytotoxic and the proapoptotic effects of brostallicin. In one experiment, human glutathione S-transferase pi (GST-pi) cDNA was transfected into A2780 cells, and four clones of A2780 with different expression levels of GST-pi were generated (i.e., two clones with high and two clones with low GST-pi expression). A 2-3-fold increase in GST-pi levels resulted in a 2-3-fold increase in cytotoxic activity of brostallicin. Similar results were obtained for GST-pi-transfected human breast carcinoma cells (MCF-7). Brostallicin showed 5.8-fold increased cytotoxicity in GST-pi-transfected versus empty vector-transfected cells with low GST-pi expression. In an in vivo experiment, A2780 clones were implanted into nude mice. The antitumor activity of brostallicin was higher in the GST-pi-overexpressing tumors without increased toxicity. Regarding the mechanism of action, brostallicin interacts reversibly with the DNA minor groove TA-rich sequences but appears unreactive in classical in vitro DNA alkylation assays. We speculated that an intracellular reactive nucleophilic species, e.g., GSH, could react with the alpha-bromoacrylamide moiety functions. Experiments on the interaction with plasmid DNA showed a change of the DNA topology from supercoiled to circular form (nicking) in the presence of GSH, whereas no change was found in its absence. In vitro incubations of brostallicin were performed with the human recombinant GST isoenzymes A1-1, M1-1, and P1-1 (alpha, mu and pi isoenzymes, respectively) in the presence of GSH. The decrease in brostallicin levels was monitored in these incubations; the rate of loss (and therefore brostallicin metabolism) was significantly higher for the M1-1 and P1-1 isoenzymes than for the A1-1 isoenzyme.
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April 2002