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The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class smallmolecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either [PU.1.sup.lo] mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1., Introduction Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by the abnormal clonal proliferation of immature cells following various genetic and epigenetic alterations. Despite efforts to [...]

Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases. BDNF | neuroprotection | small molecule | binding www.pnas.org/cgi/doi/10.1073/pnas.0913572107

A series of 'reversed amidine' substituted heterocycles were prepared to better understand the molecular basis for recognition of the DNA minor groove by heterocyclic cations. The results reveal that amidine derivatives for targeting the minor groove have the amidine carbon linked to a central heterocyclic system whereas in the reverse orientation an amidine nitrogen provides the link.

A general strategy for the rapid structural analysis of DNA binding ligands is described as it is applied to the study of RT29, a benzimidazole-diamidine compound containing a highly twisted diphenyl ether linkage. The high-throughput (HT)/high-resolution (HR) strategy has facilitated the rapid structural analysis of an unstudied DNA-binding compound, RT29.

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity are investigated.

The interaction of G-quadruplex DNA with the macrocyclic compound BOQ1, which possesses two dibenzophenanthroline (quinacridine) subunits, is investigated by a variety of methods. The oligonucleotide 5'A(GGGT2A3)(sub 3)G3, which mimics the human telomeric repeat sequence and forms an intramolecular quadruplex, is used as one model system.

Research has been conducted on the furamidine and related diamidines, drugs active against widespread parasites in the Pneumocystic carinii pathogen. The detailed biochemical and biophysical characterization of the DNA sequence recognition properties of the phenylfuranbenzimidazole diamidine derivative DB293 has been carried out and discussed.

An aromatic dication is described that binds in the minor groove of DNA and recognizes mixed DNA sequences. Successful DNA binding is determined by the structure of the compound.

Specific diphenylfuran derivatives bind very strongly to RNA and this binding correlates with the structure of the compound. The discovery of DB340 dimer and its binding to the minor groove of RNA via RRE sequences serves as a model for RNA-directed drug design.

Research has been conducted on the pepleomycin-mediated DNA cleavage of isolated and cellular DNA. Results indicate that bleomycin amplifiers bind to DNA and enhance drug-mediated DNA degradation that leads to apoptosis.

Researchers demonstrate that fused ring carbazoles can bind to the minor groove of DNA. The 2,7 and 3,6 carbazoles bind in opposite orientations.

Promising clinical trials of antisense therapeutic strategies offer the possibility of highly specific drug targeting. Antisense agents are targeted at a single-stranded section of a cellular RNA, usually a messenger RNA. However, these first generation agents form unstable complexes with target RNA because of their conformations and search for second generation agents is in progress. The structure of the stable N3'->P5' phosphoramidate duplexes is not fully understood. A new study examines the NMR solution structure of an NP duplex with the sequence CGCGAATTCGCG and shows its similarity to classical A-form values.

Investigation was conducted on the binding mode of new 2,7- and 3,6-carbazole dications to AT and GC rich sequences of DNA as part of an attempt to develop new anti-opportunistic infections drugs and examine their interaction with DNA. Based on kinetic and nuclear magnetic resonance spectroscopic data, results incidate that both the fused aromatic cations bind in the minor groove in AT sequences. Molecular models are presented to emonstrate that the carbazoles form new types of minor-groove complexes in AT sequences of DNA.

DNA with modified N3' to P5' phosphoramidate sugar-phosphate backbones act as an analog of structural RNAs, RRE and TAR. The DNA analogs are nuclease-resistant, stable and form A-type duplex helices in solutions. The DNA analogs of HIV-1 RRE IIB and TAR RNA can specifically compete for and bind with HIV-1 Rev and Tat proteins. The binding blocks viral gene regulatory proteins. The relative binding affinities for peptides are determined using gel shift analysis. The analogs can be used for developing new drugs against viral diseases.

The effect of diphenylfuran derivatives on the binding of the regulatory protein Rev to RNA structures such as RRE in the HIV-1 genome was investigated by fluorescence titrations, circular dichroism (CD) and gel mobility shift assays. Synthetic diphenylfuran cations bound to RRE but inhibited Rev-RRE complex formation at concentrations below 1 mumolar. CD spectra indicated that diphenylfuran found RRE at the structured internal loop, resulting in changes in RRE conformation.

Tetracationic azoniacyclophanes (CPnn where nn is the methylene groups from 3 to 6 in linking chains) stabilize DNA irrespective of the size of cyclophane cavity. However, RNA duplex stabilization by CPnn decreases with increasing size of the cyclophane cavity and the solution conditions. The CP66 cyclophane binds the adenine bases of RNA with A-U base pairs. High concentrations of CP66 can also denature the RNA. The cyclophanes are the first known small organic compounds that make RNA duplexes melt by selectively complexing with a base through a cavity complex.

Furamidine is a minor groove binding drug that has been demonstrated to be more potent than pentamidine against the AIDS-associated pathogen Pneumocystis carinii. Its structure is similar to that of the antitrypanosomal drug berenil, the main difference between the two being the substitution of a triazene linker with a furan ring. Crystallographic and spectroscopic analysis of the two drugs are conducted to explain their interaction with the DNA sequence d(CGCGAATTCGCG)2. Results are then used to explain the differences between the two ligands.

Circular dichroism spectroscopy on West Nile virus 3'-terminal RNA sequence revealed the presence of a conserved pseudoknot at the 3' terminus of the flavivirus genomic ribonucleic acid. Ribonuclease probing results also showed the existence of secondary structure consisting of a long stem-loop (SL1) and a shorter stem-loop (SL2). Base pairing by nucleotides was found to take place in the SL2.

Solid-phase methodology may be used to synthesize oligonucleotide N3' to P5' phosphoramidates. These compounds can form stable duplexes with phosphodiester single-stranded DNA and single-stranded RNA. They can form triplexes with RNA-DNA duplexes and double-stranded DNA. Molecular modeling and CD spectra shows that N3' to P5' phosphoramidates are in A form and their sugar puckering and hybridization properties resemble that of RNAs.

A study was conducted on the effect of four triplex-binding ligands on the stability and stringency of triple helix formation using complexes containing predominantly TAT triplets. DNase I footprinting was used to examine the effects. The ligands were found to be capable of stabilizing DNA triplexes, allowing stable complex formation at significantly lower oligonucleotide concentrations.

DNase I footprinting technique at two target sites enables analysis of the influence of a triplex-binding ligand on the formation of DNA triple helices. Complexation with acridine causes unstability of the oligonucleotides. The binding of this compound at T.AT triplets blocks DNA structural change transmission to the nearby duplex. The nature of footprints at one of the target sites is independent of the ligand, while the footprint at the other target site is affected.

NMR, thermal melting and gel electrophoresis studies of two synthetic 23-bp oligonucleotides confirm the formation of B-form hairpins from GT and AC oligonucleotides. Each hairpin has a 3-member loop, a 10-bp stem and two mismatched pairs. One- and two-dimensional nuclear Overhauser effects and comparative study of hairpins having single base substitutions help assign each imino proton. Point substitutions within the loop of the hairpin, outside the binding site, change the conformation of the hairpin.

An automated beta-cyanoethyl phosphoramidite method helps synthesize modified oligothymidylates d(T)12 having 5-(o-carboran-1-yl)-2-deoxyuridine (CDU). The position of the carboranyl nucleotide influences duplex stability duplex and conformation of single- and double-stranded structures. Melting temperature studies and molecular modeling help analyze the influence of CDU on duplex stability. Half-life measurements reveal that some 3'- and 3',5'-CDU-modified oligonucleotides resist toward 5'-exonuclease activity.

The sequence dependence and structure of DNA.RNA duplexes are determined. The determination enables the treatment of several different discuss and antisense therapeutic medicines. The DNA.RNA complexes have all purine or pyrimidine bases, and in some cases, a mixed pyrimidine and purine bases.

A multinuclear NMR analysis and molecular refinement study of a 5-pyrimidine-GA-purine-3' sequence molecular model reveals the presence of a B-type right-handed helix, with the constituent bases possessing routine anti-glycosidic torsional angles. Type I mismatched bases possess edge-to-face hydrogen bonds while type II base pairs have face-to-face hydrogen bonds. The flanking sequences determine the conformation with 5'-pyrimidine-GA-purine-3' sequences which select type I conformation and the 5'-purine-GA-pyrimidine-3' sequences having type II conformation.

The reaction between an oligonucleotide third strand and a duplex nucleic acid series is seen in triple-helical conformations as a therapeutic strategy in the 'antigene' approach. The activity of a selected gene is directly controlled by the triple helix created from the cellular duplex and an incorporated third strand. The nucleic acid triple-helical interactions restrict constancy, limiting the method due to the replacements of methylphosphonate or phosphorothioate in the backbone alterations in the third strand.

The interaction with RNA of a wide variety of compounds that are known to have different interactions with DNA was investigated using thermal melting methods. Groove-binding compounds do not have significant interactions with RNA, while compounds with mixed binding modes can have very strong RNA interactions. A group of classical intercalators and a group of unfused aromatic intercalators contain compounds that intercalate and have strong interactions with RNA. Compounds that bind to DNA by threading intercalation bind to RNA by the same mode, but none have shown selective interactions with RNA.

Molecular modeling studies have revealed that anthracene-9,10-dione derivatives placed at certain substituent positions intercalate with DNA. The studies have also shown the possibility of constructing intercalators with approximating binding constants but with varied binding kinetics. Further observations also verify that the binding modes for the anthracene-9,10-diones involve threading-type and classical DNA intercalation.

The stereoelectronic factors in the interaction of DNA and small aromatic molecules substituted with a short cationic chain was studied. The polarity of the cation-substituted aromatic molecules is important for its binding with DNA. Stronger DNA binding is observed when the positive end of the aromatic dipole favorably interacts with the negative potential of the DNA groove.

A study was conducted on the interaction of 4,6-diamidino-2-phenylindole (DAPI) with RNA(polyApolyU) and DNA(polydApolydT) sequences. Spectral changes of propidium were observed and were found to be identical on binding to the RNA and DNA sequences while different for DAPI binding. Results showed an intercalation binding mode for the DAPI-RNA complex and consequently suggest the formation of a favorable intercalation between RNA and DAPI.

Backbone modifications are incorporated into guanine (G)-rich peptide nucleic acids (PNAs) to improve the selectivity for quadruplex versus duplex formation. The enhanced selectivity is due to decreased affinity for complementary sequences and the improved selectivity of the PNAs is a vital step toward using PNAs for regulating gene expression by G-quadruplex formation.

A host-guest approach was used to determine the crystal structure of CD27, bound to its preferred DNA-binding site 5?-AATT within a self-complementary oligonucleotide, 5?-d(CTTAATTCGAATTAAG) at 1.75 [Angstrom]. The study results provide guidelines to understand the mechanism of the antitrypanosomal activity of studied symmetric diphenylamine compounds.

Six polyamide analogues containing the core triheterocyclic structure IPI were designed and synthesized to understand the role of the N-terminal acylamido which affected sequence specificity and binding affinity. The biophysical results suggested that all six compounds bind within the minor groove at their cognate DNA sequence as stacked, staggered, antiparallel dimers.

The dynamics of the DB921-DNA (where DB291 has a benzimidazole-biphenyl system with terminal amidines) complex and the utility of water in designing compounds to recognize DNA are studied by using a 100 ns molecular dynamics simulation of the complex. The results have helped in understanding how water has coupled the linear DB291 compound to the minor groove for tight binding, without a large unfavorable contribution to the entropy of binding.

The article discusses the synthesis of the synthetic minor groove binding compounds that normally bind to the adjacent A/T base pair sites by jumping over the GC pair. The various benzimidazole derivatives used for the purpose are shown to exhibit the best binding and solution properties.

Arrays of different methods were used to characterize the binding of dicationic diamidines related to DB75 (amidine-phenyl-furan-phenyl-amidine) with alternating and nonalternating AT sequences. The results showed that it is possible to tune the shape of the molecule to match DNA for enhanced affinity and sequence recognition by selecting an appropriate central group.

Two-dimensional (2-D) NMR experiments are performed on both duplex and hairpin to find the effect of DNA structure on adjacent site cooperative binding. The results have confirmed the role of conformational entropy in positive cooperativity in some DNA interactions.

A linear triphenyl diamidine (DB111) and a series of nitrogen tricyclic analogues are prepared. This compound has helped in the development of DNA sequence-specific agents and it is shown to be the first non-polyamide, synthetic compound to recognize a DNA sequence with a majority of GC base pairs.

Molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes and potential therapeutic agents. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed.

A core modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP) is designed and synthesized. Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex.

The synthesis and DNA binding properties of a bis-guanidinium derivative of ethidium designated DB950 are reported using a range of complementary spectroscopic and biochemical methods to determine its affinity for duplex DNA, mode of binding, and sequence selectivity. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties.

A novel DNA-binding template for construction of new therapeutic candidates is proposed. Four bisphenylcarbazoles derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious diatonic carbazoles are designed and their interaction with DNA studied by a combination of biochemical and biophysical methods.

The DNA interactions of a systematic set of diamidine derivatives with a powerful array of methods including DNase I footprinting, biosensor-SPR methods and X-ray crystallography are prepared and explored to develop biologically active minor groove agents. The DNA minor groove is an important target site for enzymes and transcription control proteins.

DNA sequences such as AATT and TTAA have significantly different physical and interaction properties are studied. Footprinting studies with several natural and designed DNA fragments indicate that the synthetic compounds bind at AT sequences in the minor groove and interact more weakly at sites with TpA steps.

Surface plasmon resonance and DNase I footprinting analyses indicate that the consensus sequence 5'-(A/T)-TG-(A/T) participates in the cooperative dimerization of the diamidine DB293. The influence of DNA sequence on drug binding is discussed.

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

Molecular dynamics (MD) simulations were conducted on the CGP 40215A complex with the DNA sequence used in the X-ray studies. It was observed that a particularly favorable position was almost identical to that in the crystal structure with one amidine and the linker- NH groups forming direct H-bonds to DNA.

Very little happens in any biological system unless two or more molecules come together to form a stable complex. When molecules interact through specific molecular contacts, all of the principles [...]