The Scripps Research Institute

The State University of New York

Massachusetts Institute of Technology

University of Florida Research Foundation Inc

Expansion Therapeutics, Inc.

Matthew D Disney

Suzanne Rzuczek

Wang-Yong Yang

Biao Liu

Jessica L Childs-Disney

Lirui Guan

Sai Velagapudi

Timothy Manning Swager

Joseph P Vacca

Peter H Seeberger

Timothy Allen Blizzard

Andy Jennings

Alexei Pushechnikov

John Ndungu

Juan Zheng

Methods and computer systems are described herein for identifying small molecules that bind to selected RNA structural features (e.g., to RNA secondary structures). Also described are compounds and compositions that modulate RNA function and/or activity.

Transcriptome-wide design of selective, bioactive small molecules targeting RNA

Provided herein are compounds and methods for modulating abnormal repeat expansions of gene sequences. More particularly, provided are inhibitors of RNA and the uses of such inhibitors in regulating nucleotide repeat expansions, e.g., to treat Myotonic Dystrophy Type 1 (DM1), Myotonic Dystrophy Type 2 (DM2), Fuchs dystrophy, Huntington Disease, Amyotrophic Lateral Sclerosis, or Frontotemporal Dementia.

Bis-benzimidazole compounds and methods of using the same

Potent modulators of RNA function can be assembled in cellulo by using the cell as a reaction vessel and a disease-causing RNA as a catalyst. When designing small molecule effectors of function, a balance between permeability and potency must be struck. Low molecular weight compounds are more permeable while higher molecular weight compounds are more potent. The advantages of both types of compounds could be synergized if low molecular weight molecules could be transformed into potent, multivalent ligands via a reaction catalyzed by binding to a target in cells expressing a genetic defect. We demonstrate that this approach is indeed viable in cellulo. Small molecule modules with precisely positioned alkyne and azide moieties bind adjacent internal loops in r(CCUG), the causative agent of myotonic dystrophy type 2 (DM2), and are transformed into oligomeric, potent inhibitors of DM2 RNA dysfunction via a 1,3 Huisgen dipolar cycloaddition reaction, a variant of click chemistry. Additionally, we show that this approach is applicable to the r(CUG) repeating RNA that causes myotonic dystrophy type 1 (DM1). The click chemistry approach also allows for FRET sensors to be synthesized on-site by using r(CUG) repeats as a catalyst. Furthermore it is shown that small molecule binding sites in patient-derived cells can be identified by using reactive approaches termed Chem-CLIP and Chem-CLIP-Map. Lastly, it is shown that small molecules that target r(CUG) expansions can be designed to cleave this RNA by appending a small molecule with a nucleic acid cleaving module.

Toxic RNA inhibitors self-assembled in situ

A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing 'c9RAN proteins'. Since neutralizing r(GGGGCC)could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC). Chemical and enzymatic probing of r(GGGGCC)indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC).

Treatment of C9FTD/ALS by targeting RNA expanded repeat sequences

The invention provides a series of bioactive small molecules that target expanded r(CGG) repeats, termed r(CGG), that causes Fragile X-associated Tremor Ataxia Syndrome (FXTAS). The compound was identified by using information on the chemotypes and RNA motifs that interact. Specifically, 9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium, binds the 5'CG/3&#x2032;GC motifs in r(CGG)and disrupts a toxic r(CGG)-protein complex. Specifically, dimeric compounds incorporating two 9-hydroxyellipticine analog structures can even more potently bind the 5&#x2032;CG/3&#x2032;GC motifs in r(CGG)and disrupts a toxic r(CGG)-protein complex. Structure-activity relationships (SAR) studies determined that the alkylated pyridyl and phenolic side chains are important chemotypes that drive molecular recognition of r(CGG) repeats, such as r(CGG). Importantly, the compound is efficacious in FXTAS model cellular systems as evidenced by its ability to improve FXTAS-associated pre-mRNA splicing defects and to reduce the size and number of r(CGG)-protein aggregates.

Small molecules targeting repeat r(CGG) sequences

The present invention provides small molecule compounds that can form covalent adducts with specific sequences of RNA, such as the hairpin loop r(CUG)sequence which is a cause of myotonic dystrophy type 1 (DM1), or the r(CGG)sequence which is a cause of fragile X-associated tremor/ataxia syndrome (FXTAS); to methods of making the small molecule compounds; and to methods of using the small molecular compounds in the treatment of DM1 or of FXTAS in patients afflicted therewith. The invention further provides a method for identifying an RNA target of a small molecule drug in vivo, using a small molecule drug conjugated to an RNA-reactive crosslinker group and a reporter group, contacting a cell or nucleic acid extract with the small molecule drug conjugate, then separating RNA targets crosslinked to the small molecule drug conjugate by interaction of the affinity group with a complementary affinity group.

Specific targeting of RNA expanded repeat sequences

To study RNA function using small molecules, we designed bioactive, modularly assembled small molecules that target the noncoding expanded RNA repeat that causes myotonic dystrophy type 1 (Dm1), r(CUG)exp. Different modular assembly scaffolds were investigated including polyamines, alpha-peptides, beta-peptides, and peptide tertiary amides (PT As). Based on activity as assessed by improvement of DM1 -associated defects, stability against proteases, cellular permeability, and toxicity, we discovered that constrained backbones, namely PT As, are optimal.

Modularly assembled small molecules for the treatment of myotonic dystrophy type 1

Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

Methods for identifying ligands that target nucleic acid molecules and nucleic acid structural motifs

Disclosed are RNA targeting compounds, methods for using the subject RNA targeting compounds to treat myotonic dystrophy and other diseases are also disclosed.

RNA targeting compounds and methods for making and using same

Disclosed are RNA targeting compounds having the formula: wherein j is an integer from 1 to 100; each i is the same or different and is zero or an integer from 1 to 100; each Zrepresents the same or different linking moiety; each Ris the same or different and represents an alkyl group or an aryl group; each Qrepresents the same or different RNA binding ligand; Qis an alkyl group; Qis a halogen, an alkyl group, an aryl group, or an amine. Also disclosed are RNA targeting compounds that include a polymer backbone and two or more pendant RNA binding ligands that are bound to the polymer backbone. Methods for using the subject RNA targeting compounds to treat myotonic dystrophy and other diseases are also disclosed, as are compounds that can be used to prepare the subject RNA targeting compounds.

The present invention generally relates to organic polymers able to participate in an analyte-recognition process, where an analyte facilitates an energy transfer between an energy donor and an energy acceptor. Certain embodiments of the invention make use of fluorescent conjugated polymers, such as poly(phenylene ethynylene)s and other polymers comprising pi-conjugated backbones. For example, one aspect of the invention provides a fluorescent conjugated polymer and an indicator that can interact with each other in the presence of an analyte to produce an emissive signal. In some cases, the interaction may include energy exchange mechanisms, such as Dexter energy transfer or the strong coupling effect. The interaction of the conjugated polymer and the indicator, in some instances, may be facilitated through specific interactions, such as a protein/carbohydrate interaction, a ligand/receptor interaction, etc. Another aspect of the invention provides for the detection of biological entities, for example, pathogenic bacteria such as, or viruses such as influenza virus. In some cases, biological recognition elements may be used to determine the biological entity, for instance, carbohydrates that can be used to specifically interact with at least part of the biological entity, such as a protein in the cell membrane of a bacterium. Still other aspects of the invention involve articles, devices, and kits using any of the above-described systems.

Growing community of inventors

Williamsville, NY, United States of America

Matthew D Disney