Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold-small molecule interactions.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif-small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between 'undruggable' biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Progress toward the development of the small molecule equivalent of small interfering RNA.

Given that many small molecules could bind to structured regions at sites that will not affect function, approaches that trigger degradation of RNA could provide a general way to affect biology. Indeed, targeted RNA degradation is an effective strategy to selectively and potently modulate biology. We describe several approaches to endow small molecules with the power to cleave RNAs. Central to these strategies is Inforna, which designs small molecules targeting RNA from human genome sequence. Inforna deduces the uniqueness of a druggable pocket, enables generation of hypotheses about functionality of the pocket, and defines on- and off-targets to drive compound optimization. RNA-binding compounds are then converted into cleavers that degrade the target directly or recruit an endogenous nuclease to do so. Cleaving compounds have significantly contributed to understanding and manipulating biological functions. Yet, there is much to be learned about how to affect human RNA biology with small molecules.

Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy.

There are many RNA targets in the transcriptome to which small molecule chemical probes and lead therapeutics are desired. However, identifying compounds that bind and modulate RNA function in cellulo is difficult. Although rational design approaches have been developed, they are still in their infancies and leave many RNAs "undruggable". In an effort to develop a small molecule library that is biased for binding RNA, we computationally identified "drug-like" compounds from screening collections that have favorable properties for binding RNA and for suitability as lead drugs. As proof-of-concept, this collection was screened for binding to and modulating the cellular dysfunction of the expanded repeating RNA (r(CUG)(exp)) that causes myotonic dystrophy type 1. Hit compounds bind the target in cellulo, as determined by the target identification approach Competitive Chemical Cross-Linking and Isolation by Pull-down (C-ChemCLIP), and selectively improve several disease-associated defects. The best compounds identified from our 320-member library are more potent in cellulo than compounds identified by high-throughput screening (HTS) campaigns against this RNA. Furthermore, the compound collection has a higher hit rate (9% compared to 0.01-3%), and the bioactive compounds identified are not charged; thus, RNA can be "drugged" with compounds that have favorable pharmacological properties. Finally, this RNA-focused small molecule library may serve as a useful starting point to identify lead "drug-like" chemical probes that affect the biological (dys)function of other RNA targets by direct target engagement.

Small Molecule Inhibition of miR-544 Biogenesis Disrupts Adaptive Responses to Hypoxia by Modulating ATM-mTOR Signaling.

Hypoxia induces a complex circuit of gene expression that drives tumor progression and increases drug resistance. Defining these changes allows for an understanding of how hypoxia alters tumor biology and informs design of lead therapeutics. We probed the role of microRNA-544 (miR-544), which silences mammalian target of rapamycin (mTOR), in a hypoxic breast cancer model by using a small molecule (1) that selectively impedes the microRNA's biogenesis. Application of 1 to hypoxic tumor cells selectively inhibited production of the mature microRNA, sensitized cells to 5-fluorouracil, and derepressed mRNAs affected by miR-544 in cellulo and in vivo, including boosting mTOR expression. Thus, small molecule inhibition of miR-544 reverses a tumor cell's physiological response to hypoxia. Importantly, 1 sensitized tumor cells to hypoxia-associated apoptosis at a 25-fold lower concentration than a 2'-O-methyl RNA antagomir and was as selective. Further, the apoptotic effect of 1 was suppressed by treatment of cell with rapamycin, a well-known inhibitor of the mTOR signaling pathway, illustrating the selectivity of the compound. Thus, RNA-directed chemical probes, which could also serve as lead therapeutics, enable interrogation of complex cellular networks in cells and animals.

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis.

RNA is an important therapeutic target; however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096 member 3 × 3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, druglike benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

Two-dimensional combinatorial screening identifies specific 6'-acylated kanamycin A- and 6'-acylated neamine-RNA hairpin interactions.

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6'-acylated kanamycin A (1) and 6'-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K(d)'s ranging from 235 to 1035 nM, with an average K(d) of 618 nM. For 2, the selected hairpins bind with K(d)'s ranging from 135 to 2300 nM, with an average K(d) of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5'GC and 5'CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections.

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.

Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris, Candida albicans, and Candida dubliniensis.

Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC50) of 30 microM in 2 or 4 mM Mg2+, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s ranged from 1 to 9 microM for strains with group I introns and were 12 and 32 microM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.