Fabrication of self-healing pectin/chitosan hybrid hydrogel via Diels-Alder reactions for drug delivery with high swelling property, pH-responsiveness, and cytocompatibility.

Self-healing hydrogels with pH-responsiveness could protect loaded drugs from being destroyed till it arrives to the target. The pectin-based hydrogel is a candidate due to the health benefit, anti-inflammation, antineoplastic activity, nontoxicity, and biospecific degradation, et al. However, the abundant existence of water-soluble branched heteropolysaccharide chains influenced its performance resulting in limitation of the potential. In the present study, we prepared a series of self-healing pectin/chitosan hydrogels via the Diels-Alder reaction. Moreover, pectin/chitosan composite hydrogel was prepared as a contrast. By comparison, it can be seen that the Diels-Alder reaction greatly improved the cross-linking density of hydrogels. The self-healing experiments showed excellent self-healing performance. In different swelling mediums, significant transformation in the swelling ratio was shown, indicating well-swelling property, pH- and thermo-responsiveness. The drug loading and release studies presented high loading efficiency and sustained release performance. The cytotoxicity assay that showed a high cell proliferation ratio manifested great cytocompatibility.

Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening pipeline and in-vitro validation assays.

SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2'-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.

Magnetic Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state nuclear magnetic resonance (NMR) spectroscopy and aids in the measurement of residual dipolar couplings via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic alignment properties of styrene maleimide quaternary ammonium (SMA-QA) polymer-based nanodiscs by using 31P and 14N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic alignment of large-sized (≥20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid bilayer normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs is further shown to flip the alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Surface-Functionalized Modified Copper Sulfide Nanoparticles Enhance Checkpoint Blockade Tumor Immunotherapy by Photothermal Therapy and Antigen Capturing.

Nanomaterial-based tumor photothermal therapy (PTT) has attracted increasing attention and been a promising method for cancer treatment because of its low level of adverse effects and noninvasiveness. However, thermotherapy alone still cannot control tumor metastasis and recurrence. Here, we developed surface-functionalized modified copper sulfide nanoparticles (CuS NPs). CuS NPs can not only be used as photothermal mediators for tumor hyperthermia but can adsorb tumor antigens released during hyperthermia as an antigen-capturing agent to induce antitumor immune response. We selected maleimide polyethylene glycol-modified CuS NPs (CuS NPs-PEG-Mal) with stronger antigen adsorption capacity, in combination with an immune checkpoint blocker (anti-PD-L1) to evaluate the effect of hyperthermia, improving immunotherapy in a 4T1 breast cancer tumor model. The results showed that hyperthermia based on CuS NPs-PEG-Mal distinctly increased the levels of inflammatory cytokines in the serum, leading to a tumor immunogenic microenvironment. In cooperation with anti-PD-L1, PTT mediated by CuS NPs-PEG-Mal enhanced the number of tumor-infiltrating CD8+ T cells and inhibited the growth in primary and distant tumor sites of the 4T1 tumor model. The therapeutic strategies provide a simple and effective treatment option for metastatic and recurrent tumors.

Nanoparticle Ligand-Decoration Procedures Affect in Vivo Interactions with Immune Cells.

Ligand-decorated nanoparticles are extensively studied and applied for in vivo drug delivery and molecular imaging. Generally, two different ligand-decoration procedures are utilized; ligands are either conjugated with nanoparticle ingredients and incorporated during nanoparticle preparation, or they are attached to preformed nanoparticles by utilizing functionalized reactive surface groups (e.g., maleimide). Although the two procedures result in nanoparticles with very similar physicochemical properties, formulations obtained through the latter manufacturing process typically contain nonconjugated reactive surface groups. In the current study, we hypothesized that the different ligand-decoration procedures might affect the extent of interaction between nanoparticles and immune cells (especially phagocytes). In order to investigate our hypothesis, we decorated lipidic nanoparticles with a widely used cyclic Arg-Gly-Asp (cRGD) peptide using the two different procedures. As proven from in vivo experiments in mice, the presence of nonconjugated surface moieties results in increased recognition by the immune system. This is important knowledge considering the emerging focus on understanding and optimizing ways to target and track immune cells and the development of nanomedicine-based strategies in the field of immunotherapy.

Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling.

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-Rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag ('dC10*') was then demonstrated in the fluorogenic intracellular labelling of histone H2B in living HeLa cells.

Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells.

SOX9 plays an important role in mesenchymal condensations during the early development of embryonic skeletons. However, its function in the chondrogenic differentiation of adult mesenchymal stem cells (MSCs) has not been fully investigated because SOX9 RNA interference in adult MSCs has seldom been studied. This study used SOX9 gene as the target gene and the quantum dot (QD)-based nanomaterial QD-NH2 (ZnS shell and poly-ethylene glycol (PEG) coating) with a fluorescent tracer function as the gene carrier to transfect siSOX9 into MSCs after sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation in vitro and in vivo. The results showed that QD-SMCC could effectively bind and deliver siRNAs into the MSCs, followed by efficient siRNA escape from the endosomes. The siRNAs released from QD-SMCC retained their structural integrity and could effectively inhibit the targeted gene expression, leading to reduced chondrogenic differentiation of MSCs and delayed cartilage repair. QDs were excreted from living cells instead of dead cells, and the ZnS shell and PEG coating layer greatly reduced the cytotoxicity of the QDs. The transfection efficiency of QD-SMCC was superior to that of polyethylenimine (PEI). In addition, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection. STATEMENT OF SIGNIFICANCE: In this study, we developed functional quantum dot (QD) nanoplexes by sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation of PEG-coated CdSe/ZnS QDs as the gene carrier of siRNA to study the effect of SOX9 RNA interference on the chondrogenic differentiation of MSCs. This study confirmed the importance of SOX9 in chondrogenesis, as evidenced by the findings that SOX9 knockdown significantly inhibited the expression of cartilage-specific markers including acan and col2a1 in MSCs and further delayed cartilage repair. Moreover, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection.

Hybrid palm-oil/styrene-maleimide nanoparticles synthesized in aqueous dispersion under different conditions.

Poly(styrene-co-maleic anhydride) was imidized with ammonium hydroxide and palm oil, resulting in an aqueous dispersion of hybrid nanoparticles with diameters 85-180 nm (dispersed) or 20-50 nm (dried). The reaction conditions were optimized for different precursors by evaluating the relative amount ammonium hydroxide and maximizing the incorporated palm oil up to 70 wt.%. The interactions between palm oil and polymer phase have been studied by TEM, IR, Raman spectroscopy and thermal analysis (TGA, [TM] DSC). From Raman spectra, the amount of imide and reacted oil were quantified. Through concurring effects of imidization and coupling of fatty acids, the imidization needs a slight excess of NH3 relatively to maleic anhydride. The oxidative stability highly depends on oxidative crosslinking of free or non-reacted oil. Comparing the imide content from spectroscopic and thermal analysis suggests that a complex rigid imide phase without strong relaxation behavior has formed in combination with oil.

Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3ß (GSK-3ß) induces the proteasomal degradation of cyclin D1. The addition of GSK-3ß inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3ß.

Synthesis and Evaluation of 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides as Novel GSK-3ß Inhibitors and Anti-Ischemic Agents.

A series of novel 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides were designed, synthesized, and biologically evaluated for their GSK-3ß inhibitory activities. Most compounds showed favorable inhibitory activities against GSK-3ß protein. Among them, compounds 5n, 5o, and 5p significantly reduced GSK-3ß substrate tau phosphorylation at Ser396 in primary neurons, indicating inhibition of cellular GSK-3ß activity. In the in vitro neuronal injury models, compounds 5n, 5o, and 5p prevented neuronal death against glutamate, oxygen-glucose deprivation, and nutrient serum deprivation which are closely associated with cerebral ischemic stroke. In the in vivo cerebral ischemia animal model, compound 5o reduced infarct size by 10% and improved the neurological deficit. The results may provide new insights into the development of novel GSK-3ß inhibitors with potential neuroprotective activity against brain ischemic stroke.

Assessment of different functionalization methods for grafting a protein to an alumina-covered biosensor.

The specificity of biosensors is typically obtained by surface biofunctionalization, which enables the selective binding of biomolecules. This critical step is sensitive to the nature of materials and to the overall experimental conditions. Here, we provide a comprehensive study of several biofunctionalization methods, including the layer-by-layer technique and both the gas-phase and liquid-phase silanizations, and we propose a new maleimide-based protocol for grafting a protein to a sensor covered by alumina. This method was then validated by making a respiratory syncitial virus-specific biosensor.

Targeting single-walled carbon nanotubes for the treatment of breast cancer using photothermal therapy.

This paper focuses on the targeting of single-walled carbon nanotubes (SWNTs) for the treatment of breast cancer with minimal side effects using photothermal therapy. The human protein annexin V (AV) binds specifically to anionic phospholipids expressed externally on the surface of tumour cells and endothelial cells that line the tumour vasculature. A 2 h incubation of the SWNT-AV conjugate with proliferating endothelial cells followed by washing and near-infrared (NIR) irradiation at a wavelength of 980 nm was enough to induce significant cell death; there was no significant cell death with irradiation or the conjugate alone. Administration of the same conjugate i.v. in BALB/c female mice with implanted 4T1 murine mammary at a dose of 0.8 mg SWNT kg(-1) and followed one day later by NIR irradiation of the tumour at a wavelength of 980 nm led to complete disappearance of implanted 4T1 mouse mammary tumours for the majority of the animals by 11 days since the irradiation. The combination of the photothermal therapy with the immunoadjuvant cyclophosphamide resulted in increased survival. The in vivo results suggest the SWNT-AV/NIR treatment is a promising approach to treat breast cancer.

Chemical interrogation of the neuronal kinome using a primary cell-based screening assay.

A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention, small molecule protein kinase inhibitors present a promising therapeutic strategy. Here, we report a robust cell-based phenotypic assay, utilizing primary rat hippocampal neurons, for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening, as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened, revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies.

pH-triggered aggregate shape of different generations lysine-dendronized maleimide copolymers with maltose shell.

Glycopolymers are promising materials in the field of biomedical applications and in the fabrication of supramolecular structures with specific functions. For tunable design of supramolecular structures, glycopolymer architectures with specific properties (e.g., controlled self-assembly) are needed. Using the concept of dendronized polymers, a series of H-bond active giant glycomacromolecules with maleimide backbone and lysine dendrons of different generations were synthesized. They possess different macromolecular size and functionality along the backbone. Their peripheral maltose units lead to solubility under physiological conditions and controlled aggregation behavior. The aggregation behavior was investigated depending on generation number, pH value, and concentration. A portfolio of complementary analytical tools give an insight into the influence of the different parameters in shaping a rod-, coil-, and worm-like molecular structure and their controlled aggregate formation. MD simulation helped us to understand the complex aggregation behavior of the linear polymer chain without dendritic units.

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor.

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.

Methanol extract of Antrodia cinnamomea mycelia induces phenotypic and functional differentiation of HL60 into monocyte-like cells via an ERK/CEBP-ß signaling pathway.

Antrodia cinnamomea (named as Niu-chang-chih), a well-known Taiwanese folk medicinal mushroom, has a spectrum of biological activities, especially with anti-tumor property. This study was carried out for the first time to examine the potential role and the underlying mechanisms of A. cinnamomea in the differentiation of human leukemia HL60 cells. We found that the methanol extract of liquid cultured mycelia of A. cinnamomea (MEMAC) inhibited proliferation and induced G1-phase cell cycle arrest in HL60 cells. MEMAC could induce differentiation of HL60 cells into the monocytic lineage, as evaluated by the morphological change, nitroblue tetrazolium reduction assay, non-specific esterase assay, and expression of CD14 and CD11b surface antigens. In addition, MEMAC activated the extracellular signal-regulated kinase (ERK) pathway and increased CCAAT/enhancer-binding protein ß (C/EBPß) expression. Reverse transcriptase polymerase chain reaction analysis showed that MEMAC upregulated the expression of C/EBPß and CD14 mRNA in HL60 cells. DNA affinity precipitation assay and chromatin immunoprecipitation analyses indicated that MEMAC enhanced the direct binding of C/EBPß to its response element located at upstream of the CD14 promoter. Furthermore, inhibiting ERK pathway activation with PD98059 markedly blocked MEMAC-induced HL60 monocytic differentiation. Consistently, the MEMAC-mediated upregulation of C/EBPß and CD14 was also suppressed by PD98059. These findings demonstrate that MEMAC-induced HL60 cell monocytic differentiation is via the activating ERK signaling pathway, and downstream upregulating the transcription factor C/EBPß and differentiation marker CD14 gene, suggesting that MEMAC might be a potential differentiation-inducing agent for treatment of leukemia.

Development and characterization of immuno-nanocarriers targeting the cancer stem cell marker AC133.

In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG(2000)-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG(2000)-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126nm AC133-LNC with a zeta potential of -22mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133(high)) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133(low) for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines.

Novel cysteine tags for the sequencing of non-tryptic disulfide peptides of anurans: ESI-MS study of fragmentation efficiency.

Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S-S bonds. Long disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described. ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation (HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags [N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation. Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone.

Synthesis, structural, and biological evaluation of bis-heteroarylmaleimides and bis-heterofused imides.

Bis-2,3-heteroarylmaleimides and polyheterocondensed imides joined through nitrogen atoms of the N,N'-bis(ethyl)-1,3-propanediamine linker were prepared from substituted maleic anhydrides and symmetrical diamines in good to satisfactory yields and short reaction times using microwave heating. The novel molecules were shown to inhibit proliferation of human tumor cells (NCI-H460 lung carcinoma) and rat aortic smooth muscle cells (SMCs) with variable potencies. Compound 11a, the most potent one of the series, showed IC(50) values comparable to those observed for the leading molecule elinafide in both cell lines, but with a higher selectivity toward human tumor cells. Compound 11a affected G1/S phase transition of the cell cycle, showed in vitro DNA intercalating activity and in vivo antitumor activity. A thorough structural analysis of the 11a-DNA complex was also made by mean of NMR and computational techniques.