Towards a templated reaction that translates RNA in cells into a proaptotic peptide-PNA conjugate.

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10 nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.

Toward conditional control of Smac mimetic activity by RNA-templated reduction of azidopeptides on PNA or 2'-OMe-RNA.

Oligonucleotide templated reactions can be used to control the activity of functional molecules based on the presence of a specific trigger sequence. We report an RNA-controlled reaction system to conditionally restore the N-terminal amino group and thus binding affinity of azide-modified Smac mimetic compounds (SMCs) for their target protein X-linked Inhibitor of Apoptosis Protein (XIAP). Two templated reactions were compared: Staudinger reduction with phosphines and a photocatalytic reaction with Ru(bpy)2 (mcbpy). The latter proved faster and more efficient, especially for the activation of a bivalent SMC, which requires two consecutive reduction steps. The templated reaction proceeds with turnover when 2'-OMe-RNA probes are used, but is significantly more efficient with PNA, catalyzing a reaction in the presence of low, substoichiometric amounts (1%-3%, 10 nM) of target RNA.

Simultaneous Targeting of Two Master Regulators of Apoptosis with Dual-Action PNA- and DNA-Peptide Conjugates.

Conjugation of peptides with oligonucleotides offers opportunities for combining the strengths of both biopolymer classes. Herein, we show that the combination of a peptide-based module with an antisense oligonucleotide module provides for enhancements of potency and a widened scope of cell delivery options. The peptide unit comprises a Smac mimetic compound (SMCs) which antagonizes the action of inhibitor of apoptosis proteins (IAPs) frequently overexpressed in cancer cells. To counteract SMC resistance, the antisense module downregulates the cellular FLICE-like protein (c-FLIP), a master regulator of the extrinsic apoptosis pathway. We compared c-FLIP antisense units based on oligophosphorothioate (PSO) and peptide nucleic acid (PNA) architectures. Owing to the ease of synthesis, PNA conjugates combined with a cell penetrating peptide (CPP) offer a seemingly ideal solution for cell delivery of dual activity agents. However, our investigations revealed that such congeners have to be handled with care to avoid off-target effects. By contrast, PSO conjugates provided a more robust and specific activity for inducing death of SMC-resistant A549 cells due to a simultaneous activation of caspases and c-FLIP knockdown. We show that lipofection is a convenient approach for delivery of peptide-PSO conjugates into cells. The results highlight that the combination of the peptide and the DNA world confers properties inaccessible by the unconjugated components.