Entropy-driven binding of gut bacterial ß-glucuronidase inhibitors ameliorates irinotecan-induced toxicity.

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of ß-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial ß-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 µM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers.

We report the syntheses of chemical building blocks of a particular class of chiral PNAs, called miniPEG-containing (R)-gamma PNAs (or (R)-MPγPNAs). The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of racemization in the alkylation and reductive amination steps. The methodology is general and robust, ideally suited for large-scale monomer productions with most synthetic steps providing excellent chemical yields without the need for purification other than a simple workup and precipitation.

Substituent Position of Iminocyclitols Determines the Potency and Selectivity for Gut Microbial Xenobiotic-Reactivating Enzymes.

Selective inhibitors of gut bacterial ß-glucuronidases (GUSs) are of particular interest in the prevention of xenobiotic-induced toxicities. This study reports the first structure-activity relationships on potency and selectivity of several iminocyclitols (2-7) for the GUSs. Complex structures of Ruminococcus gnavus GUS with 2-7 explained how charge, conformation, and substituent of iminocyclitols affect their potency and selectivity. N1 of uronic isofagomine (2) made strong electrostatic interactions with two catalytic glutamates of GUSs, resulting in the most potent inhibition (Ki ≥ 11 nM). C6-propyl analogue of 2 (6) displayed 700-fold selectivity for opportunistic bacterial GUSs (Ki = 74 nM for E. coli GUS and 51.8 µM for RgGUS). In comparison with 2, there was 200-fold enhancement in the selectivity, which was attributed to differential interactions between the propyl group and loop 5 residues of the GUSs. The results provide useful insights to develop potent and selective inhibitors for undesired GUSs.

Synthesis of ( R)- and ( S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity.

A robust synthetic route has been developed for preparing optically pure, Fmoc-protected diethylene glycol-containing ( R)- and ( S)-γPNA monomers. The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of epimerization in the O-alkylation and reductive amination steps. The optical purities of the final monomers were determined to be greater than 99.5% ee, as assessed by 19F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer production, with most synthetic steps providing excellent chemical yields without the need for chromatographic purification other than a simple workup and precipitation.

Orthogonal γPNA Dimerization Domains Empower DNA Binders with Cooperativity and Versatility Mimicking that of Transcription Factor Pairs.

Synthetic molecules capable of DNA binding and mimicking cooperation of transcription factor (TF) pairs have long been considered a promising tool for manipulating gene expression. Our previously reported Pip-HoGu system, a programmable DNA binder pyrrole-imidazole polyamides (PIPs) conjugated to host-guest moiety, defined a general framework for mimicking cooperative TF pair-DNA interactions. Here, we supplanted the cooperation modules with left-handed (LH) γPNA modules: i.e., PIPs conjugated with nucleic acid-based cooperation system (Pip-NaCo). LH γPNA was chosen because of its bioorthogonality, sequence-specific interaction, and high binding affinity toward the partner strand. From the results of the Pip-NaCo system, cooperativity is highly comparable to the natural TF pair-DNA system, with a minimum energetics of cooperation of -3.27 kcal mol-1 . Moreover, through changing the linker conjugation site, binding mode, and the length of γPNAs sequence, the cooperative energetics of Pip-NaCo can be tuned independently and rationally. The current Pip-NaCo platform might also have the potential for precise manipulation of biological processes through the construction of triple to multiple heterobinding systems.

In utero nanoparticle delivery for site-specific genome editing.

Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington's Disease.

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington's disease and a number of other related neuromuscular and neurodegenerative disorders.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

RNA-Templated Concatenation of Triplet Nucleic-Acid Probe.

Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.

Anti-tumor Activity of miniPEG-γ-Modified PNAs to Inhibit MicroRNA-210 for Cancer Therapy.

MicroRNAs (miRs) are frequently overexpressed in human cancers. In particular, miR-210 is induced in hypoxic cells and acts to orchestrate the adaptation of tumor cells to hypoxia. Silencing oncogenic miRs such as miR-210 may therefore offer a promising approach to anticancer therapy. We have developed a miR-210 inhibition strategy based on a new class of conformationally preorganized antisense γ peptide nucleic acids (γPNAs) that possess vastly superior RNA-binding affinity, improved solubility, and favorable biocompatibility. For cellular delivery, we encapsulated the γPNAs in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Our results show that γPNAs targeting miR-210 cause significant delay in growth of a human tumor xenograft in mice compared to conventional PNAs. Further, histopathological analyses show considerable necrosis, fibrosis, and reduced cell proliferation in γPNA-treated tumors compared to controls. Overall, our work provides a chemical framework for a novel anti-miR therapeutic approach using γPNAs that should facilitate rational design of agents to potently inhibit oncogenic microRNAs.

Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly.

Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

Development of oseltamivir phosphonate congeners as anti-influenza agents.

Oseltamivir phosphonic acid (tamiphosphor, 3a), its monoethyl ester (3c), guanidino-tamiphosphor (4a), and its monoethyl ester (4c) are potent inhibitors of influenza neuraminidases. They inhibit the replication of influenza viruses, including the oseltamivir-resistant H275Y strain, at low nanomolar to picomolar levels, and significantly protect mice from infection with lethal doses of influenza viruses when orally administered with 1 mg/kg or higher doses. These compounds are stable in simulated gastric fluid, liver microsomes, and human blood and are largely free from binding to plasma proteins. Pharmacokinetic properties of these inhibitors are thoroughly studied in dogs, rats, and mice. The absolute oral bioavailability of these compounds was lower than 12%. No conversion of monoester 4c to phosphonic acid 4a was observed in rats after intravenous administration, but partial conversion of 4c was observed with oral administration. Advanced formulation may be investigated to develop these new anti-influenza agents for better therapeutic use.