Association of Apolipoprotein E4-related Microvascular Disease in the Alzheimer's Disease Hippocampal CA1 Stratum Radiatum.

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer's disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-ß (Aß) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.

Isolation, Structure Elucidation, and First Total Synthesis of Quinomycins K and L, Two New Octadepsipeptides from the Maowei Sea Mangrove-Derived Streptomyces sp. B475.

Mangrove actinomycetia have been proven to be one of the promising sources for discovering novel bioactive natural products. Quinomycins K (1) and L (2), two rare quinomycin-type octadepsipeptides without intra-peptide disulfide or thioacetal bridges, were investigated from the Maowei Sea mangrove-derived Streptomyces sp. B475. Their chemical structures, including the absolute configurations of their amino acids, were elucidated by a combination of NMR and tandem MS analysis, electronic circular dichroism (ECD) calculation, advanced Marfey's method, and further unequivocally confirmed by the first total synthesis. The two compounds displayed no potent antibacterial activity against 37 bacterial pathogens and had no significant cytotoxic activity against H460 lung cancer cells.

[Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro].

OBJECTIVE: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS: EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, ß-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of ß-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, ß-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/ß-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

The UvrA-like protein Ecm16 requires ATPase activity to render resistance against echinomycin.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.

Unimodular Methylation by Adenylation-Thiolation Domains Containing an Embedded Methyltransferase.

Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.

Quinomycins H1 and H2, new cytotoxic antibiotics from Streptomyces sp. RAL404.

Two new cytotoxic antibiotics designated quinomycins H1 (2) and H2 (3) were isolated from the culture broth of Streptomyces sp. RAL404. The molecular formula of both compounds was established as C52H65N11O13S2 by electrospray ionization mass spectrometry (ESI-MS). Their structures were determined as echinomycin (1) derivatives containing a 3-hydoxyquinaldic acid molecule in place of one of the two quinoxaline-2-carboxylic acid chromophores. Quinomycins H1 (2) and H2 (3) showed selective cytotoxicity against RG-E1-4 cells bearing the adenovirus oncogenes with IC50s of 11 nM and 12 nM, respectively.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Echinomycin, a potential binder of FKBP12, shows minor effect on calcineurin activity.

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor-1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was -10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.

RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening.

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Diversification of echinomycin molecular structure by way of chemoenzymatic synthesis and heterologous expression of the engineered echinomycin biosynthetic pathway.

Echinomycin, a bis-intercalator antitumor cyclic peptide, is biosynthesized by a unique nonribosomal peptide synthetase (NRPS). Successful heterologous expression of the whole gene cluster of echinomycin in Escherichia coli let us to investigate a further application of echinomycin NRPS. To construct a cyclic peptide library, our approach through both chemoenzymatic and rational genetic engineering has been successfully demonstrated. These achievements provided the further support that E. coli-based system can serve as a flexible yet robust platform for producing complex natural products and their analogs.

Enzymatic macrolactonization in the presence of DNA leading to triostin A analogs.

Excised thioesterase domains are versatile catalysts for macrocyclization. However, thioesterase-catalyzed cyclization is often precluded due to the occurrence of hydrolysis and product inhibition. To circumvent these obstacles, we devised an unprecedented strategy: coincubation with DNA to capture the cyclic products possessing DNA-binding properties. In experiments involving echinomycin thioesterase-catalyzed macrolactonization leading to the cyclic triostin A analog TANDEM, we found that the addition of DNA drastically improved the yield of TANDEM (19% --> 67%), with a complete reversal of the cyclization:hydrolysis ratio (1:2 --> 18:1). Furthermore, the applicability of this protocol was demonstrated for a variety of substrates. The results described herein provide insight into the mechanism of echinomycin thioesterase-catalyzed conversions and also pave the way for chemoenzymatic synthesis of the quinoxaline antibiotics and their analogs.

Improved production of triostin A in engineered Escherichia coli with furnished quinoxaline chromophore by design of experiments in small-scale culture.

Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de novo production of triostin A by means of high cell density fed-batch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with small-scale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.

Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations.

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities.

Total syntheses of thiocoraline and BE-22179 and assessment of their DNA binding and biological properties.

Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C(2) symmetric bicyclic octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their relative and absolute stereochemistry were established. Key elements of the approach include the late-stage introduction of the chromophore, symmetrical tetrapeptide coupling, macrocyclization of the 26-membered octadepsipeptide conducted at the single secondary amide site following disulfide formation, and a convergent assemblage of the tetradepsipeptide with introduction of the labile thiol ester linkage in the final coupling reaction under near racemization free conditions. By virtue of the late-stage introduction of the chromophore and despite the challenges this imposes on the synthesis, this approach provides ready access to a range of key chromophore analogues. Thiocoraline and BE-22179 were shown to bind to DNA by high-affinity bisintercalation analogous to echinomycin, but with little or no perceptible sequence selectivity. Both 1 and 2 were found to exhibit exceptional cytotoxic activity (IC(50) = 200 and 400 pM, respectively, L1210 cell line) comparable to echinomycin and one analogue, which bears the luzopeptin chromophore, was also found to be a potent cytotoxic agent.

Recognition elements that determine affinity and sequence-specific binding to DNA of 2QN, a biosynthetic bis-quinoline analogue of echinomycin.

Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments.

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.

Structures of quinoxaline antibiotics.

The crystal structures of three quinoxaline antibiotics-echinomycin 2QN, triostin C and the C222(1) form of triostin A--have been determined, and the structure of the P2(1)2(1)2(1) form of triostin A has been re-refined against our previously reported data. The molecular conformations are compared with those deduced from NMR data and those reported for two complexes of triostin A with oligonucleotides. Although the depsipeptide ring conformations are basically similar, the effective twofold molecular symmetry is violated by the folding of one of the quinoxaline chromophores in echinomycin 2QN and by a rotation of one of the ester planes with the formation of an intramolecular hydrogen bond in triostin C. In the oligonucleotide complexes of triostin A the chirality of the disulfide bridge is inverted. The alanine NH groups are involved in intermolecular hydrogen bonds in all four structures, and (except in echinomycin 2QN) the stacking of the chromophores in the crystal emulates the intercalation involved in DNA complex formation. In echinomycin 2QN, the antibiotic molecules are hydrogen bonded to form a helix along the crystallographic 6(5) screw axes, with a channel of disordered solvent running through the middle of the helix. Crystal data: (1), echinomycin 2QN, C53H66N10O12S2.2.5(C3H6O).2.5(H2O), M(r) = 1289.5, hexagonal, P6(5), a = b = 22.196(15), c = 24.64 (2) A, V = 10,513 (13) A3, Z = 6, Dx = 1.222 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.275 mm-1, T = 193 K, R = 9.0% for 4828 I > 2 sigma (I) and 11.8% for all 7102 unique reflections; (2), triostin C, C54H70N12O12S2.0.67(CHCl3).0.67(H2O), M(r) = 1234.2, orthorhombic, P2(1)2(1)2(1), a = 16.054 (8), b = 17.128 (9), c = 22.706 (12) A, V = 6244 (6) A3, Z = 4, Dx = 1.313 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.239 mm-1, T = 188 K, R = 7.7% for 4678 I > 2 sigma (I) and 14.0% for all 7260 unique reflections; (3), triostin A, C50H62N12O12S2.2(C7H14O2), M(r) = 1347.6, orthorhombic, P2(1)2(1)2(1), a = 20.94 (2), b = 18.53 (2), c = 18.80 (2) A, V = 7292 (13) A3, Z = 4, Dx = 1.228 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.245 mm-1, T = 293 K, R = 6.8% for 2116 I > 2 sigma (I) and 9.3% for all 2928 unique reflections; (4), triostin A, C50H62N12O12S2.HCl.2(C3H7NO), M(r) = 1269.9, monoclinic, C222(1), a = 10.622 (10), b = 17.035 (17), c = 35.21 (3) A, V = 6371 (10) A3, Z = 4, Dx = 1.324 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.199 mm-1, T = 153 K, R = 7.5% for 2164 I > 2 sigma (I) and 13.2% for all 3402 unique reflections. Extensive use was made of restraints on the geometrical and displacement parameters in the successful anisotropic refinement of these structures against weak data.

NMR investigation of Hoogsteen base pairing in quinoxaline antibiotic--DNA complexes: comparison of 2:1 echinomycin, triostin A and [N-MeCys3,N-MeCys7] TANDEM complexes with DNA oligonucleotides.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.

DNA recognition by intercalators and hybrid molecules.

Experiments are described which probe the role of the 2-amino group of guanine as a critical determinant of the recognition of nucleotide sequences in DNA by specific ligands. Homologous samples of tyrT DNA substituted with inosine or 2,6-diaminopurine residues in place of guanosine or adenine respectively yield characteristically modified footprinting patterns when challenged with sequence-selective antibiotics such as echinomycin, actinomycin or netropsin. The capacity of small molecules to recognise particular DNA sequences is exploited in the 'combilexin' strategy to target small molecules to defined sites in DNA. A composite molecule containing a distamycin moiety linked to an intercalating ellipticine derivative has been synthesised and shown to bind tightly to DNA but without much sequence-selectivity. Refinement of this molecule based on predictions from molecular modelling has led to the synthesis of a second generation derivative bearing an additional positive charge: this new hybrid molecule is strongly selective for binding to AT-rich tracts in DNA.