Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogues

Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthesized by insertion of methylene moiety between -S-S- bond. The compound 1a shows remarkable cytotoxicities against human tumor various cell lines, and is active VRE (vancomycin-resistant enterococci) within MIC range 0.5-8 microg/mL. According to the eukaryotic or prokaryotic data, 1a might be a first analogue to replace echinomycin.     

Farnesyltransferase inhibitors: CAAX mimetics based on different biaryl scaffolds

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed as farnesyltransferase (FTase) inhibitors (FTIs) by replacing AA with o-aryl or o-heteroaryl substituted p-hydroxy- or p-aminobenzoic acid, while maintaining the replacement of C with 1,4-benzodioxan-2-ylmethyl or 2-amino-4-thiazolylacetyl residue as in previous CAAX mimetics. Both FTase inhibition and antiproliferative effect were showed by two thiazole derivatives, namely those with 1-naphthyl (10 and 10a) or 3-furanyl (15 and 15a) in the central spacer, and by the benzodioxane derivative with 2-thienyl (6 and 6a) in the same position. Accumulation of unprenylated RAS was demonstrated in cells incubated with 15a. Consistently with FTIs literature, such results delineate the biaryl scaffold not only as a spacer but also as a sensible area of these mimetic molecules, where modifications at the branching aromatic ring are not indifferent and should be matter of further investigation.     

Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope

Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.     

Effect of echinomycin on DNA methylation

Although echinomycin is reported to intercalate and to bind to DNA at CG dinucleotides, the effects of the drug on DNA methylation in vitro and in vivo are much less apparent than are the effects on DNA synthesis and cell growth.     

Surface grafting onto template-assembled synthetic protein scaffolds in molecular recognition

Creating functional biological molecules de novo requires a detailed understanding of the intimate relationship between primary sequence, folding mechanism, and packing topology, and remains up to now a most challenging goal in protein design and mimicry. As a consequence, the use of well-defined robust macromolecules as scaffolds for the introduction of function by grafting surface residues has become a major objective in protein engineering and de novo design. In this article, the concept of scaffolds is demonstrated on some selected examples, illustrating that novel types of functional molecules can be generated. Reengineered proteins and, most notably, de novo designed peptide scaffolds exhibiting molecular function, are ideal tools for structure-function studies and as leads in drug design.     

Quantitative determination of echinomycin by disc agar diffusion assay

Echinomycin is a peptide antibiotic of the quinoxaline group produced by Streptomyces echinatus. In propitious circumstances it can be determined by ultraviolet spectrophotometry, but for work with analogues and congeners a more specific assay is required. A disc agar diffusion technique has been developed and optimised for this purpose. It yields log dose-response curves which are linear over at least a 20-fold range of antibiotic concentration, and the variation of sensitivity with inoculum size, preincubation time, and temperature of incubation has been investigated. Other quinoxaline antibiotics can be assayed by the same technique; they yield quite different log dose-response curves indicative of lower potency associated with triostins as compared to quinomycins. The application of the technique to follow echinomycin production by S. echinatus A8331 in culture in maltose minimal medium is described.     

Rapid synthesis of RGD mimetics with isoxazoline scaffolds on solid phase: identification of alphavbeta3 antagonists lead compounds

Isoxazoline containing RGD mimetics were rapidly synthesized on a solid phase to optimize linkers, regioisomers of isoxazoline scaffolds, and exosite binding groups to yield lead alphavbeta3 antagonists.     

Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments

Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly ¹³C/¹⁵N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.     

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.     

Towards discovery of new leishmanicidal scaffolds able to inhibit Leishmania GSK-3

Abstract

Previous reports have validated the glycogen synthase kinase-3 (GSK-3) as a druggable target against the human protozoan parasite Leishmania. This prompted us to search for new leishmanicidal scaffolds as inhibitors of this enzyme from our in-house library of human GSK-3β inhibitors, as well as from the Leishbox collection of leishmanicidal compounds developed by GlaxoSmithKline. As a result, new leishmanicidal inhibitors acting on Leishmania GSK-3 at micromolar concentrations were found. These inhibitors belong to six different chemical classes (thiadiazolidindione, halomethylketone, maleimide, benzoimidazole, N-phenylpyrimidine-2-amine and oxadiazole). In addition, the binding mode of the most active compounds into Leishmania GSK-3 was approached using computational tools. On the whole, we have uncovered new chemical scaffolds with an appealing prospective in the development and use of Leishmania GSK-3 inhibitors against this infectious protozoan.     

Oxytocin receptor mimetics prepared by molecular imprinting

Summary Oxytocin receptor mimetics were prepared by molecular imprinting using Z-oxytocin as the template. Comparative binding studies with reference polymers showed that the imprinted polymers recognized both Z-oxytocin and unprotected oxytocin selectively. The dissociation constants were 47 μM and 102 μM, respectively, and the density of binding sites was 12 μmol/g. The synthetic oxytocin receptors were easily prepared, possessed high mechanical and chemical stability, and were reused without loss of selectivity and capacity after regeneration by extraction. Abbreviations: B_max, number of binding sites; CLEAR, Cross-Linked Ethoxylate Acrylate Resin; EDMA, ethylene glycol dimethacrylate; FABMS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high-performance liquid chromatography; K_D, dissociation constant; MAA, methacrylic acid; MIP, molecularly imprinted polymer; SPPS, solid-phase peptide synthesis; TRIM, trimethylolpropane trimethacrylate; Z, benzyloxycarbonyl. Abbreviations used for amino acids and the designation of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977–983]. All amino acids were of thel-configuration.     

Oxytocin receptor mimetics prepared by molecular imprinting

Oxytocin receptor mimetics were prepared by molecularimprinting using Z-oxytocin as the template. Comparative binding studies with reference polymersshowed that the imprinted polymers recognized bothZ-oxytocin and unprotected oxytocin selectively. Thedissociation constants were 47 M and 102 M,respectively, and the density of binding sites was12 mol/g. The synthetic oxytocin receptors wereeasily prepared, possessed high mechanical andchemical stability, and were reused without loss ofselectivity and capacity after regeneration byextraction.     

Separation of quinoxaline antibiotics by coil planet centrifugation

Liquid/liquid chromatography has been used for the isolation and purification of quinoxaline-type compounds on a coil planet centrifuge. Three solvent systems have been developed which are capable of high resolution both analytically and preparatively, Separations are described involving naturally occurring antibiotics, biosynthetically produced analogues, and chemically modified derivatives.     

Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised ØX174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.     

Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.     

Complex biomembrane mimetics on the sub-nanometer scale

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain-forming lipid mixtures to asymmetric lipid bilayers. Here, we review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. In particular, we focus on: (1) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function of a size transition from nanoscopic to macroscopic domains; (2) membrane-mediated protein partitioning into lipid domains; (3) the role of the aqueous medium in tuning interactions between membranes and domains; and (4) leaflet-specific structure in asymmetric bilayers and passive lipid flip-flop.     

Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration

Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.     

Hydrogels and electrospun nanofibrous scaffolds of N-methylene phosphonic chitosan as bioinspired osteoconductive materials for bone grafting

In this work, N-methylene phosphonic chitosan (NMPC) based hydrogels and electrospun nanofibrous scaffolds are reported with objective to obtain osteoconductive and osteoinductive matrixes for bone grafting applications. NMPC, a phosphorylated derivative of chitosan, is known to mimic the function of non collagenous phosphoproteins in providing nucleation sites for biomineralization. NMPC hydrogels were prepared by crosslinking between NMPC and genipin. A detailed investigation of physicochemical properties of NMPC solutions is also carried out in order to obtain beads free nanofibers. Both NMPC gels and nanofibers were further evaluated for their biomineralization potential and biocompatibility with human osteoblast like cells. Results indicated that hydrogels and nanofibrous scaffolds NMPC are biocompatible and significantly osteoinductive compared to tissue culture plate controls. However, cells seeded on nanofibrous scaffolds exhibited greater proliferation measured by MTT assay, and higher expression of early markers for osteogenic differentiation proving the superior applicability of nanofibrous scaffolds for bone grafting applications.     

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.