Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences.

Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference.

Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable.

Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides.

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype.

Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.

A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers.

99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer.

Unlabelled: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel (99m)Tc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans.

Methods: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with (99m)Tc and evaluated in vitro and in vivo.

Synthesis and SAR of ⁹⁹mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates.

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel (99m)Tc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells.

Synthesis and SAR of novel Re/99mTc-labeled benzenesulfonamide carbonic anhydrase IX inhibitors for molecular imaging of tumor hypoxia.

Carbonic anhydrase IX (CA-IX) is upregulated in cancer in response to the hypoxic tumor microenvironment, making it an attractive molecular target for the detection of hypoxic solid tumors. A series of small molecule benzenesulfonamide based CA-IX inhibitors containing novel tridentate chelates complexed with the M(CO)(3) core (M = Re or (99m)Tc) were designed and synthesized. The in vitro binding affinity of the benzenesulfonamide rhenium complexes yielded IC(50) values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa cells.

Universal peptidomimetics.

This paper concerns peptidomimetic scaffolds that can present side chains in conformations resembling those of amino acids in secondary structures without incurring excessive entropic or enthalpic penalties. Compounds of this type are referred to here as minimalist mimics. The core hypothesis of this paper is that small sets of such scaffolds can be designed to analogue local pairs of amino acids (including noncontiguous ones) in any secondary structure; i.

Novel polar single amino acid chelates for technetium-99m tricarbonyl-based radiopharmaceuticals with enhanced renal clearance: application to octreotide.

Single amino acid chelate (SAAC) systems for the incorporation of the M(CO)(3) moiety (M = Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation Tc(CO)(3)-dipyridyl complexes has resulted in substantial hepatobiliary uptake when either examined as lysine derivatives or integrated into biologically active small molecules and peptides. Here we designed, synthesized, and evaluated novel SAAC systems that have been chemically modified to promote overall Tc(CO)(3)L(3) complex hydrophilicity with the intent of enhancing renal clearance.

A diversity oriented synthesis of 3'-O-modified nucleoside triphosphates for DNA 'sequencing by synthesis'.

The nucleoside triphosphates 1, containing a photochemically cleavable group, and 2, having one that may be cleaved via palladium catalysis, were prepared as a prelude to investigating sequencing of DNA via sequencing by synthesis.

An iterative route to "decorated" ethylene glycol-based linkers.

Iterative copper-catalyzed cycloadditions of azides to alkynes were used to join functionalized triethylene glycol molecules to give "linkers" of defined lengths equipped with several different end-group functionalities.

Polymer-supported palladacycles: efficient reagents for synthesis of benzopyrans with palladium recovery. Relationship among resin loading, Pd:P ratio, and reactivity of immobilized palladacycles.

[reaction: see text] Oxapalladacycles were immobilized on polystyrene-divinylbenzene supports and treated with 3-aryl-2-propynoates or 1-alkyl-1,2-propadienes to afford 2H-1-benzopyrans in yields superior to those for solution-phase experiments. Isolation of benzopyrans was facilitated, and 71-80% of the palladium was recovered. Effects of resin loading with phosphorus and palladium were studied, and the optimum immobilized palladacycles featuring a medium loading with P (1.

Regio- and diastereoselective insertion of allenes into stable oxapalladacycles with a metal-bonded stereogenic carbon. preparation of contiguously substituted 3,4-dihydro-2H-1-benzopyrans.

Insertion of monosubstituted allenes into stable oxapalladacycle I was studied. The aim of this work was to define steric and electronic parameters of allenes that would allow for a regio- and diastereoselective synthesis of 2,3-disubstituted 3,4-dihydro-2H-1-benzopyrans, which could not be prepared via related catalytic protocols. Allenes with electron-donating alkyl substituents R sterically unencumbered at the C-3 and C-4 carbons reacted with palladacycles I to afford benzopyrans IV in good yields (45-81%), exclusively as cis diastereomers.

Mechanism of stereoinduction in asymmetric synthesis of highly functionalized 1,2-dihydroquinolines and 2H-1-benzopyrans via nonracemic palladacycles with a metal-bonded stereogenic carbon.

To establish the synthetic utility of palladacycles, a stable racemic benzannulated azapalladacycle featuring a palladium-bonded sp(3)-hybridized stereogenic carbon was prepared and converted into a series of racemic 2,3,4-trisubstituted 1,2-dihydroquinolines via a regioselective insertion of activated alkynes (RCCCOOEt). Analogous diastereomerically enriched azapalladacyle (92% de) and oxapalladacycle (64% de) were synthesized from arylpalladium(II) iodo complexes possessing a nonracemic spectator ligand ((1R,2R)-N,N,N',N'-tetramethyl-1,2-diaminocyclohexane) via an intramolecular displacement of the iodide by an ester enolate. Absolute configurations of the metal-bonded stereocenters in the diastereomerically enriched palladacycles were unequivocally assigned, and the efficiency of stereoinduction was systematically studied.