Synthesis and biological evaluation of biaryl alkyl ethers as inhibitors of IDO1.

Starting from the dialkylaniline indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor lead 3 (IDO1 HeLa IC50 = 7.0 nM), an iterative process of synthesis and screening led to cyclized analog 21 (IDO1 HeLa IC50 = 3.6 nM) which maintained the high potency of 3 while addressing issues of lipophilicity, cytochrome P450 (CYP) inhibition, hERG (human potassium ion channel Kv11.1) inhibition, Pregnane X Receptor (PXR) transactivation, and oxidative metabolic stability. An x-ray crystal structure of a biaryl alkyl ether 11 bound to IDO1 was obtained. Consistent with our earlier results, compound 11 was shown to bind to the apo form of the enzyme.

In the sense of transcription regulation by G-quadruplexes: asymmetric effects in sense and antisense strands.

G-Quadruplexes occupy important regulatory regions in the genome. DNA G-quadruplexes in the promoter regions and RNA quadruplexes in the UTRs (untranslated regions) have been individually studied and variously implicated at different regulatory levels of gene expression. However, the formation of G-quadruplexes in the sense and antisense strands and their corresponding roles in gene regulation have not been studied in much detail. In the present study, we have elucidated the effect of strand asymmetry in this context. Using biophysical methods, we have demonstrated the formation of stable G-quadruplex structure in vitro using CD and UV melting. Additionally, ITC was employed to demonstrate that a previously reported selective G-quadruplex ligand was able to bind and stabilize the G-quadruplex in the present sequence. Further, we have shown using reporter constructs that although the DNA G-quadruplex in either strand can reduce translation efficiency, transcriptional regulation differs when G-quadruplex is present in the sense or antisense strand. We demonstrate that the G-quadruplex motif in the antisense strand substantially inhibits transcription, while when in the sense strand, it does not affect transcription, although it does ultimately reduce translation. Further, it is also shown that the G-quadruplex stabilizing ligand can enhance this asymmetric transcription regulation as a result of the increased stabilization of the G-quadruplex.

Morphological effects of G-quadruplex stabilization using a small molecule in zebrafish.

Zebrafish (Danio rerio) embryos are transparent and advantageous for studying early developmental changes due to ex utero development, making them an appropriate model for studying gene expression changes as a result of molecular targeting. Zebrafish embryos were injected with a previously reported G-quadruplex selective ligand, and the phenotypic changes were recorded. We report marked discrepancies in the development of intersegmental vessels. In silico analysis determined that the putative G-quadruplex motif occur in the upstream promoter region of the Cdh5 (N-cadherin) gene. A real-time polymerase chain reaction-based investigation indicated that in zebrafish, CDH-2 (ZN-cad) was significantly downregulated in the ligand-treated embryos. Biophysical characterization of the interaction of the ligand with the G-quadruplex motif found in this promoter yielded strong binding and stabilization of the G-quadruplex with this ligand. Hence, we report for the first time the phenotypic impact of G-quadruplex targeting with a ligand in a vertebrate organism. This study has unveiled not only G-quadruplex targeting in non-human animal species but also the potential that G-quadruplexes can provide a ready tool for understanding the phenotypic effects of targeting certain important genes involved in differentiation and developmental processes in a living eukaryotic organism.

Formal total synthesis of (+)-neopeltolide.

This paper describes the formal total synthesis of (+)-neopeltolide, a cytotoxic macrolide isolated from the marine sponge Neopeltidae. The key features of the synthesis include an asymmetric Evans alkylation to fix the C9-methyl center, Jacobsen hydrolytic kinetic resolution of terminal epoxides followed by their regioselective opening to fix the stereocenters at the C11 and C13 positions, respectively, a Pd-catalyzed oxa-Michael reaction to construct the tetrahydropyran ring, and Yamaguchi macrolactonization to form the macrocyclic core of the molecule.

Hybrids of amino acids and acetylenic DNA-photocleavers: optimising efficiency and selectivity for cancer phototherapy.

Hybrid agents which combine potent DNA-photocleavers with tunable amino acids or small peptides were designed to improve selectivity of Nature's most potent class of antibiotics towards cancer cells. The ability of these compounds to photocleave DNA is controlled by their incorporation into hybrid architectures with functional elements derived from natural amino acids. These conjugates are highly effective at inducing double-strand DNA cleavage and, in some cases, rival or even surpass both naturally occurring DNA cleavers and anticancer agents that are currently in clinical use. The possibility of triggering their activity in a photochemical and pH-sensitive fashion allows for a high degree of selectivity over activation. The conjugates were shown to penetrate cell membranes and induce efficient intracellular DNA cleavage. Initial in vitro tests against a variety of cancer cell lines confirm the potential of these compounds as anticancer agents at low nanomolar concentrations.

Dissecting alkynes: full cleavage of polarized C≡C moiety via sequential bis-Michael addition/retro-Mannich cascade.

The reaction of diaryl ketoalkynes with 1,2-diamino ethane leads to the full scission of the triple bond with the formation of acetophenone and imidazoline fragments. In this transformation, one of the alkyne carbons undergoes formal reduction with the formation of three C-H bonds, whereas the other carbon undergoes formal oxidation via the formation of three C-N bonds (one π and two σ). Computational analysis confirmed that the key fragmentation step proceeds via a six-membered TS in a concerted manner. Both amines are involved in the fragmentation: the N-H moiety of one amine transfers a proton to the developing negative charge at the enolate oxygen, while the other amine provides direct stereoelectronic assistance to the C-C bond cleavage via a hyperconjugative n(N) → σ*(C-C) interaction.

Selective targeting of G-quadruplex using furan-based cyclic homooligopeptides: effect on c-MYC expression.

Quadruplex-specific molecules can serve as suitable drugs in cancer therapy. We have synthesized a pair of furan-based cyclic homooligopeptides, ligand 1 and ligand 2, to specifically target G-quadruplexes. We have shown by CD spectroscopy and UV melting that these ligands can effectively induce G-quadruplex structures in the G-rich 22-mer c-MYC DNA sequence and further stabilize the structure. Equilibrium binding constants measured by isothermal titration calorimeter methods indicate a high affinity of the ligands for the quadruplex structures (K ∼ 10(7) M(-1)) and no affinity for the duplex DNA, demonstrating that these ligands are selective for G-quadruplex structures. Surface plasmon resonance was also used to compute the binding while fluorescence resonance energy transfer-based assay was additionally used to confirm the selectivity. Moreover, using real time PCR we observed up to 90% downregulation of c-MYC transcripts after 24 h of ligand treatment in HeLa cells. Using a luciferase assay we show the downregulation of the protein levels. Fluorescent-assisted cell sorter-based cell cycle analysis showed a prominent arrest of cells in the sub-G1 stage upon treatment of ligands that leads toward apoptosis. Altogether, these experiments support the hypothesis that the present molecules are effective in specifically binding and stabilizing quadruplexes and provide a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.

Furan based cyclic homo-oligopeptides bind G-quadruplex selectively and repress c-MYC transcription.

We demonstrate the synthesis and selective binding of two novel furan based tricyclic homo-oligopeptides to G-quadruplex and using Real Time PCR show its repressive effect on c-MYC transcription. CD spectroscopy and FRET melting studies show that these ligands can induce G-quadruplex structures in the G rich 22 mer c-MYC DNA sequence and further stabilize the structure. Using real time polymerase chain reaction we observed that up to 70% downregulation of c-MYC transcripts upon ligand treatment in HeLa cells.

Furan based cyclic oligopeptides selectively target G-quadruplex.

We report the binding properties of 18- and 24-membered cyclic oligopeptides developed from a novel furan amino acid, 5-(aminomethyl)-2-furancarboxylic acid, to G-quadruplex. Comparative analysis of the binding data of these ligands with G-quadruplex and double-strand DNA shows that 24-membered cyclic peptides are highly selective for telomeric G-quadruplex structures and thus can be used as a scaffold to target quadruplex structures at the genomic level.

SMI-Ribosome inactivating protein conjugates selectively inhibit tumor cell growth.

Cell-targeting conjugates of Saporin 6, a ribosome inactivating protein (RIP), were prepared using the Saporin Ala 157 Cys mutant, a small molecule inhibitor (SMI) of integrins αvß3/αvß5, and a potent cytotoxin, auristatin F (AF). The conjugates selectively and potently inhibited proliferation of tumor cells expressing the target integrins. We anticipate that the small molecule-RIP bioconjugate approach can be broadly applied using other small molecule drugs.

Optimizing Protonation States for Selective Double-Strand DNA Photocleavage in Hypoxic Tumors: pH-Gated Transitions of Lysine Dipeptides.

We report pH-switching properties of the new family of dipeptide-acetylene conjugates where pH-gated light-activated double-strand (ds) DNA cleavage is controlled by variations in electronic and geometric parameters. The conjugates have higher activities at the slightly acidic pH values that separate normal and cancerous tissue (pH < 7). This favorable pH dependence originates from several elements of structural design. Basicities of the two amines determine the threshold pH range where the changes in binding and reactivity are observed, whereas the distance between the two amino groups and the hydrophobic aryl alkyne moiety can further modulate DNA binding. The changes of the protonation state from a neutral molecule to a dication results in dramatically increased efficiency of ds DNA photocleavage, the most therapeutically valuable type of DNA cleavage.

An ex vivo comparative analysis on shaping ability of four NiTi rotary endodontic instruments using spiral computed tomography.

OBJECTIVE: Analysis of shaping ability of four different rotary endodontic instruments using spiral computed tomography (CT). MATERIALS AND METHODS: Eighty freshly extracted human mandibular first molars were used in the present study. Samples were randomly divided into four experimental groups with twenty samples in each group. Images of mesiobuccal canal of each sample were obtained pre- and post-operatively using spiral CT. All samples were prepared using their respective endodontic file systems (group I - ProTaper, group II - K3, group III - RaCe, and group IV - Mtwo). Image analyses were done using image analysis software for evaluation of canal transportation and centering ability. Data was then statistically analyzed using analysis of variance. RESULTS: There was no statistically significance in transportation in their intergroup difference at any of the three locations (coronal, middle, and apical third). In centering ability there was no statistically significance in the coronal and middle third of the intergroup. However, there was a statistically significance of (P = 0.044) at the apical third between all the groups. CONCLUSION: Canals prepared with ProTaper had more canal transportation at all the three levels of root canal (coronal, middle, and apical third). Canals prepared with Mtwo were well centered at coronal and middle third whereas with RaCe canals were centered only at the apical third. All instruments showed some degree of canal aberrations in terms of shaping ability.

Inter- versus intra-molecular cyclization of tripeptides containing tetrahydrofuran amino acids: a density functional theory study on kinetic control.

Density functional B3LYP method was used to investigate the preference of intra- and inter-molecular cyclizations of linear tripeptides containing tetrahydrofuran amino acids. Two distinct model pathways were conceived for the cyclization reaction, and all possible transition states and intermediates were located. Analysis of the energetics indicate intermolecular cyclization being favored by both thermodynamic and kinetic control. Geometric and NBO analyses were performed to explain the trends obtained along both the reaction pathways. Conceptual density functional theory-based reactive indices also show that reaction pathways leading to intermolecular cyclization of the tripeptides are relatively more facile compared to intramolecular cyclization.

Preparation of a responsive carbohydrate-coated biointerface based on graphene/azido-terminated tetrathiafulvalene nanohybrid material.

A one-step method for the reduction of graphene oxide (GO) to reduced graphene oxide (rGO) is reported taking advantage of the electron-donor properties of an azido-terminated tetrathiafulvalene (TTF-N(3)). The resulting graphene/TTF-N(3) nanohybrid material is characterized by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) spectroscopy, and by electrical and electrochemical means. The accessibility of the azide function to chemoselective modification by any alkyne-terminated partner molecule via Cu(I)-catalyzed "click" chemistry is demonstrated. In a proof of principle and motivated by the importance of glycan-modified materials, many alkynyl-terminated mannose units were grated onto graphene/TTF-N(3). The TTF-mannose units could be released efficiently from the graphene matrix by chemical oxidation of TTF-mannose surface units to TTF(2+)-mannose, using Fe(ClO(4))(3) or the electron-deficient tetracationic cyclophane cyclobis(paraquat-p-phenylene) (CBPQT(4+)).

Engineering pH-gated transitions for selective and efficient double-strand DNA photocleavage in hypoxic tumors.

We describe a family of hybrid compounds for the most efficient light-activated double-strand (ds) DNA cleavage known to date. This family represents the second generation of "switchable" molecular systems for pH-gated ds DNA-cleavage which combine a potent DNA-photocleaver and a pH-regulated part derived from a dipeptide. Design of the pH-switchable part utilizes amino groups of different basicity. Whereas the basic amino groups are protonated throughout the biologically relevant pH range, the pH-gating amines undergo protonation at the pH threshold which separates cancer and normal cells. Control over the reactivity and selectivity is achieved via transformation of the initial protonation state (a monocation or a dication) into a trication at the acidic pH. This change leads to an extraordinary increase in the efficiency of ds DNA cleavage leading to the ds:ss ratios comparable with the most efficient nonenzymatic ds DNA cleavers. Statistical analysis reveals that these high ds:ss ratios result from the combination of several factors: (a) true double-stranded cleavage, and (b) conversion of single-stranded (ss)-scission into ds cleavage. Considerable part of ds cleavage is also produced via the combination of ss cleavage events.

Conformational analysis of some C2-symmetric cyclic peptides containing tetrahydrofuran amino acids.

Cyclic oligomers of tetrahydrofuran amino acids, cyclo-(Taa1-Leu-Val)2 (left), cyclo-(Taa2-Leu-Val)2 (middle), and cyclo-(Taa2-Phe-Leu)2 (right), displayed well-defined intramolecularly hydrogen-bonded structures with distorted "beta-beta corner" motifs similar to the tennis ball seam.