Genetic diversity and historical demography of underutilised goat breeds in North-Western Europe.

In the last decade, several studies aimed at dissecting the genetic architecture of local small ruminant breeds to discover which variations are involved in the process of adaptation to environmental conditions, a topic that has acquired priority due to climate change. Considering that traditional breeds are a reservoir of such important genetic variation, improving the current knowledge about their genetic diversity and origin is the first step forward in designing sound conservation guidelines. The genetic composition of North-Western European archetypical goat breeds is still poorly exploited. In this study we aimed to fill this gap investigating goat breeds across Ireland and Scandinavia, including also some other potential continental sources of introgression. The PCA and Admixture analyses suggest a well-defined cluster that includes Norwegian and Swedish breeds, while the crossbred Danish landrace is far apart, and there appears to be a close relationship between the Irish and Saanen goats. In addition, both graph representation of historical relationships among populations and f4-ratio statistics suggest a certain degree of gene flow between the Norse and Atlantic landraces. Furthermore, we identify signs of ancient admixture events of Scandinavian origin in the Irish and in the Icelandic goats. The time when these migrations, and consequently the introgression, of Scandinavian-like alleles occurred, can be traced back to the Viking colonisation of these two isles during the Viking Age (793-1066 CE). The demographic analysis indicates a complicated history of these traditional breeds with signatures of bottleneck, inbreeding and crossbreeding with the improved breeds. Despite these recent demographic changes and the historical genetic background shaped by centuries of human-mediated gene flow, most of them maintained their genetic identity, becoming an irreplaceable genetic resource as well as a cultural heritage.

Discovering novel clues of natural selection on four worldwide goat breeds.

In goat breeds, the domestication followed by artificial selection for economically important traits have shaped genetic variation within populations, leading to the fixation of specific alleles for specific traits. This led to the formation and evolution of many different breeds specialised and raised for a particular purpose. However, and despite the intensity of artificial selection, natural selection continues acting, possibly leaving a more diluted contribution over time, whose traces may be more difficult to capture. In order to explore selection footprints as response of environmental adaptation, we analysed a total of 993 goats from four transboundary goats breeds (Angora, Boer, Nubian and Saanen) genotyped with the SNP chip 50 K using outlier detection, runs of homozygosity and haplotype-based detection methods. Our results showed that all methods identified footprints on chromosome 6 (from 30 to 49 Mb) for two specific populations of Nubian goats sampled in Egypt. In Angora and Saanen breeds, we detected two selective sweeps using HapFLK, on chromosome 21 (from 52 to 55 Mb) and chromosome 25 (from 1 to 5 Mb) respectively. The analysis of runs of homozygosity showed some hotspots in all breeds. The overall investigation of the selected regions detected combining the different approaches and the gene ontology exploration revealed both novel and well-known loci related to adaptation, especially for heat stress. Our findings can help to better understand the balance between the two selective pressures in commercial goat breeds providing new insights on the molecular mechanisms of adaptation.

Oral ecological environment modifications by hard-cheese: from pH to microbiome: a prospective cohort study based on 16S rRNA metabarcoding approach.

BACKGROUND: The oral ecosystem conditions dental health, and is known to be positively modified by oral hygiene which cannot always be performed between meals, especially outside home. It is therefore important to identify the practices to be adopted to influence the oral environment in an anticariogenic direction. Milk and cheese are considered functional foods and have a role on oral health. There are several mechanisms by which cheese exerts its beneficial effects on teeth. The aim of the present study was to examine whether short term consumption of hard cheese would affect the oral pH and microbial flora of healthy adults modifying ecological oral environment. The Next Generation Sequencing (NGS) approach was applied to study the effect of Italian Grana Padano (GP), as a prototype of typical hard cheese, on the oral microbiota composition. Finally, we explored Streptococcus mutans/sanguinis ratio as a marker of protective biofilm composition. METHODS: Nine oral-healthy adults were instructed to eat 25 gr of GP cheese for 5 consecutive days. Three time points were chosen for supragingival samples collection and pH measurement. 16S rRNA-gene sequences were obtained both from oral samples and GP cheese using the MiSeq platform and analyzed against the expanded Human Oral Microbiome Database (eHOMD). ProgPerm was used to perform statistical analyses to investigate strain differential representation after cheese consumption. RESULTS: Taxonomic analyses of the oral microbiota revealed that Firmicutes was the most abundant phylum, followed by Proteobacteria and Actinobacteria. GP cheese significantly modifies oral pH, causing a shift toward basic conditions which are kept for a few hours. The Streptococcus mutans/Streptococcus sanguinis ratio lowers in the last observed timepoint. CONCLUSION: Our results reveal that a portion of GP cheese eaten after dinner provides important micronutrients (i.e. calcium, vitamins and some aminoacids such as arginine) and changes oral pH toward basic conditions, resulting in a light modification of the oral microbiome towards the reduction of the overall amount of acidophilic bacteria. Furthermore, the S. mutans/S. sanguinis ratio is reduced, contributing to obtain a more protecting environment towards caries establishment and evolution.

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

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.

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

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.

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

A rice GWAS panel of 281 accessions of japonica 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 (OsOFP2, Dlf1, OsMADS56) and seven (SUI1, Sd1, OsCOL4, Nal1, OsphyB, GW5, Ehd1) 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.

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

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.

Segmenting the Human Genome into Isochores.

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.

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

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.

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

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.

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

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.

Image-based surface matching algorithm oriented to structural biology.

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.

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

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.

Virtual screening pipeline and ligand modelling for H5N1 neuraminidase.

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.

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

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.

Cytotoxic alpha-halogenoacrylic derivatives of distamycin A and congeners.

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.

Recent anticancer cytotoxic agents.

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.

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

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.

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

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.

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

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.