Cyclic pyrrole-imidazole polyamides targeted to the androgen response element

Hairpin pyrrole−imidazole (Py-Im) polyamides are a class of cell-permeable DNA-binding small molecules that can disrupt transcription factor−DNA binding and regulate endogenous gene expression. The covalent linkage of antiparallel Py-Im ring pairs with an γ-amino acid turn unit affords the classical hairpin Py-Im polyamide structure. Closing the hairpin with a second turn unit yields a cyclic polyamide, a lesser-studied architecture mainly attributable to synthetic inaccessibility. We have applied our methodology for solution-phase polyamide synthesis to cyclic polyamides with an improved high-yield cyclization step. Cyclic 8-ring Py-Im polyamides 1−3 target the DNA sequence 5′-WGWWCW-3′, which corresponds to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. We find that cyclic Py-Im polyamides 1−3 bind DNA with exceptionally high affinities and regulate the expression of AR target genes in cell culture studies, from which we infer that the cycle is cell permeable

Next generation hairpin polyamides with (R)-3,4-diaminobutyric acid turn unit

The characterization of a new class of pyrrole−imidazole hairpin polyamides with β-amino-γ-turn units for recognition of the DNA minor groove is reported. A library of eight hairpins containing (R)- and (S)-3,4-diaminobutyric acid (β-amino-γ-turn) has been synthesized, and the impact of the molecules on DNA-duplex stabilization was studied for comparison with the parent γ-aminobutyric acid (γ-turn) and standard (R)-2,4-diaminobutyric acid (α-amino-γ-turn)-linked eight-ring polyamides. For some, but not all, sequence compositions, melting temperature analyses have revealed that both enantiomeric forms of the β-amino-γ-turn increase the DNA-binding affinity of polyamides relative to the (R)-α-amino-γ-turn. The (R)-β-amine residue may be an attractive alternative for constructing hairpin polyamide conjugates. Biological assays have shown that (R)-β-amino-γ-turn hairpins are able to inhibit androgen receptor-mediated gene expression in cell culture similar to hairpins bearing the standard (R)-α-amino-γ-turn, from which we infer they are cell-permeable

Recognition of "mirror-image" DNA by small molecules

May the force be with you: “Mirror-image” hairpin polyamides distinguish the L enantiomer of DNA (L-DNA) in the presence of natural DNA (D-DNA). This specificity is investigated by a molecular force balance at a single-molecule level. The “DNA balance” allows the measurement of rupture forces of match/mismatch diastereomeric complexes in a single experiment

Recognition of "mirror-image" DNA by small molecules

May the force be with you: “Mirror-image” hairpin polyamides distinguish the L enantiomer of DNA (L-DNA) in the presence of natural DNA (D-DNA). This specificity is investigated by a molecular force balance at a single-molecule level. The “DNA balance” allows the measurement of rupture forces of match/mismatch diastereomeric complexes in a single experiment

Insect‐associated bacteria assemble the antifungal butenolide gladiofungin by non‐canonical polyketide chain termination

Genome mining of one of the protective symbionts ( Burkholderia gladioli ) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously‐overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum . By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol‐derived C3 building block. The key role of an A‐factor synthase (AfsA)‐like offloading domain was corroborated by CRISPR‐Cas‐mediated gene editing, which facilitated precise excision within a PKS domain

Next generation hairpin polyamides with (R)-3,4-diaminobutyric acid turn unit

The characterization of a new class of pyrrole−imidazole hairpin polyamides with β-amino-γ-turn units for recognition of the DNA minor groove is reported. A library of eight hairpins containing (R)- and (S)-3,4-diaminobutyric acid (β-amino-γ-turn) has been synthesized, and the impact of the molecules on DNA-duplex stabilization was studied for comparison with the parent γ-aminobutyric acid (γ-turn) and standard (R)-2,4-diaminobutyric acid (α-amino-γ-turn)-linked eight-ring polyamides. For some, but not all, sequence compositions, melting temperature analyses have revealed that both enantiomeric forms of the β-amino-γ-turn increase the DNA-binding affinity of polyamides relative to the (R)-α-amino-γ-turn. The (R)-β-amine residue may be an attractive alternative for constructing hairpin polyamide conjugates. Biological assays have shown that (R)-β-amino-γ-turn hairpins are able to inhibit androgen receptor-mediated gene expression in cell culture similar to hairpins bearing the standard (R)-α-amino-γ-turn, from which we infer they are cell-permeable

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control. © 2021 The Authors. ChemBioChem published by Wiley-VCH Gmb

Quantitative detection of small molecule/DNA complexes employing a force-based and label-free DNA-microarray

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes