A mating mechanism to generate diversity for the Darwinian selection of DNA-encoded synthetic molecules.

DNA-encoded library technologies enable the screening of synthetic molecules but have thus far not tapped into the power of Darwinian selection with iterative cycles of selection, amplification and diversification. Here we report a simple strategy to rapidly assemble libraries of conformationally constrained peptides that are paired in a combinatorial fashion (suprabodies). We demonstrate that the pairing can be shuffled after each amplification cycle in a process similar to DNA shuffling or mating to regenerate diversity. Using simulations, we show the benefits of this recombination in yielding a more accurate correlation of selection fitness with affinity after multiple rounds of selection, particularly if the starting library is heterogeneous in the concentration of its members. The method was validated with selections against streptavidin and applied to the discovery of PD-L1 binders. We further demonstrate that the binding of self-assembled suprabodies can be recapitulated by smaller (∼7 kDa) synthetic products that maintain the conformational constraint of the peptides.

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs.

Potential RNA drug targets for small molecules are found throughout the human transcriptome, yet small molecules known to elicit a pharmacological response by directly targeting RNA are limited to antibacterials. Herein, we describe AbsorbArray, a small molecule microarray-based approach that allows for unmodified compounds, including FDA-approved drugs, to be probed for binding to RNA motif libraries in a massively parallel format. Several drug classes bind RNA including kinase and topoisomerase inhibitors. The latter avidly bound the motif found in the Dicer site of oncogenic microRNA (miR)-21 and inhibited its processing both in vitro and in cells. The most potent compound de-repressed a downstream protein target and inhibited a miR-21-mediated invasive phenotype. The compound's activity was ablated upon overexpression of pre-miR-21. Target validation via chemical crosslinking and isolation by pull-down showed direct engagement of pre-miR-21 by the small molecule in cells, demonstrating that RNAs should indeed be considered druggable.

A Massively Parallel Selection of Small Molecule-RNA Motif Binding Partners Informs Design of an Antiviral from Sequence.

Many RNAs cause disease; however, RNA is rarely exploited as a small-molecule drug target. Our programmatic focus is to define privileged RNA motif small-molecule interactions to enable the rational design of compounds that modulate RNA biology starting from only sequence. We completed a massive, library-versus-library screen that probed over 50 million binding events between RNA motifs and small molecules. The resulting data provide a rich encyclopedia of small-molecule RNA recognition patterns, defining chemotypes and RNA motifs that confer selective, avid binding. The resulting interaction maps were mined against the entire viral genome of hepatitis C virus (HCV). A small molecule was identified that avidly bound RNA motifs present in the HCV 30 UTR and inhibited viral replication while having no effect on host cells. Collectively, this study represents the first whole-genome pattern recognition between small molecules and RNA folds.

Small molecule chemical probes of microRNA function.

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA.

Photodynamic agents with anti-metastatic activities.

A new concept in multifunctional anticancer agents is demonstrated. Tetrakis-(diisopropyl-guanidino) zinc phthalocyanine (Zn-DIGP) exhibits excellent properties as a photodynamic therapy (PDT) agent, as well as potential anti-metastatic activities in vivo. Zn-DIGP exhibits good cellular uptake and low toxicity in the dark (EC50 > 80 µM) and is well tolerated upon its intravenous injection into mice at 8 mg/kg. Upon photoexcitation with red laser light (660 nm), Zn-DIGP exhibits a high quantum yield for singlet oxygen formation (Φ ≈ 0.51) that results in potent phototoxicity to cell cultures (EC50 ≈ 0.16 µM). Zn-DIGP is also capable of inhibiting the formation of tumor colonies in the lungs of C57BL/6 mice injected with B16F10 cells. Zn-DIGP therefore inhibits cancer growth by both light-dependent and light-independent pathways. The anti-metastatic activities of Zn-DIGP possibly result from its ability to interfere with the signaling between chemokine CXCL10 and the G protein-coupled receptor CXCR3. Zn-DIGP is a competitive inhibitor of CXCR3 activation (IC50 = 3.8 µM) and selectively inhibits downstream events such as CXCL10-activated cell migration. Consistent with the presence of feedback regulation between CXCR3 binding and CXCL10 expression, Zn-DIGP causes overexpression of CXCL10. Interestingly, Zn-DIGP binds to CXCR3 without activating the receptor yet is able to cause endocytosis and degradation of this GPCR. To the best of our knowledge, Zn-DIGP is the first PDT agent that can facilitate the photodynamic treatment of primary tumors while simultaneously inhibiting the formation of metastatic tumor colonies by a light-independent mode of action.

Fluorescent probes for G-quadruplex structures.

Mounting evidence supports the presence of biologically relevant G-quadruplexes in single-cell organisms, but the existence of endogenous G-quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure-function relationships of nucleic acids. In this review, we present progress towards the direct detection of G-quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell-permeable probes that selectively bind to G-quadruplex structures with high affinity, these same probes can induce G-quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as "internal" fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G-quadruplex, single-stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G-quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.