Alexei Pushechnikov

Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease: Application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3

Herein, we describe the design of high affinity ligands that bind expanded rCUG and rCAG repeat RNAs expressed in myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 3. These ligands also inhibit, with nanomolar IC(50) values, the formation of RNA-protein complexes that are implicated in both disorders. The expanded rCUG and rCAG repeats form stable RNA hairpins with regularly repeating internal loops in the stem and have deleterious effects on cell function. The ligands that bind the repeats display a derivative of the bisbenzimidazole Hoechst 33258, which was identified by searching known RNA-ligand interactions for ligands that bind the internal loop displayed in these hairpins. A series of 13 modularly assembled ligands with defined valencies and distances between ligand modules was synthesized to target multiple motifs in these RNAs simultaneously. The most avid binder, a pentamer, binds the rCUG repeat hairpin with a K(d) of 13 nM. When compared to a series of related RNAs, the pentamer binds to rCUG repeats with 4.4- to >200-fold specificity. Furthermore, the affinity of binding to rCUG repeats shows incremental gains with increasing valency, while the background binding to genomic DNA is correspondingly reduced. Then, it was determined whether the modularly assembled ligands inhibit the recognition of RNA repeats by Muscleblind-like 1 (MBNL1) protein, the expanded-rCUG binding protein whose sequestration leads to splicing defects in DM1. Among several compounds with nanomolar IC(50) values, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes. Comparison of the binding data for the designed synthetic ligands and MBNL1 to repeating RNAs shows that the synthetic ligand is 23-fold higher affinity and more specific to DM1 RNAs than MBNL1. Further studies show that the designed ligands are cell permeable to mouse myoblasts. Thus, cell permeable ligands that bind repetitive RNAs have been designed that exhibit higher affinity and specificity for binding RNA than natural proteins. These studies suggest a general approach to targeting RNA, including those that cause RNA dominant disease.

Two-Dimensional Combinatorial Screening Identifies Specific Aminoglycoside−RNA Internal Loop Partners

Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.

The Role of Flexibility in the Rational Design of Modularly Assembled Ligands Targeting the RNAs that Cause the Myotonic Dystrophies

Modularly assembled ligands were designed to target the RNAs that cause two currently untreatable neuromuscular disorders, myotonic dystrophy types 1 (DM1) and 2 (DM2). DM1 is caused by an expanded repeating sequence of CUG, and DM2 is caused by expanded CCUG repeats. Both are present in noncoding regions and fold into hairpins with either repeating 1×1 nucleotide UU (DM1) or 2×2 nucleotide 5′‐CU/3′‐UC (DM2) internal loops separated by two GC pairs. The repeats are toxic because they sequester the RNA splicing regulator muscleblind‐like 1 protein (MBNL1). Rational design of ligands targeting these RNAs was enabled by a database of RNA motif–ligand partners compiled by using two‐dimensional combinatorial screening (2DCS). One 2DCS study found that the 6′′‐azido‐kanamycin A module binds internal loops similar to those found in DM1 and DM2. In order to further enhance affinity and specificity, the ligand was assembled on a peptoid backbone to precisely control valency and the distance between ligand modules. Designed compounds are more potent and specific binders to the toxic RNAs than MBNL1 and inhibit the formation of the RNA–protein complexes with nanomolar IC50 values. This study shows that three important factors govern potent inhibition: 1) the surface area sequestered by the assembled ligands; 2) the spacing between ligand modules since a longer distance is required to target DM2 RNAs than DM1 RNAs; and 3) flexibility in the modular assembly scaffold used to display the RNA‐binding module. These results have impacts on the general design of assembled ligands targeting RNAs present in genomic sequence.

Probing a 2-Aminobenzimidazole Library for Binding to RNA Internal Loops via Two-Dimensional Combinatorial Screening

There are many potential RNA drug targets in bacterial, viral, and human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition.

A Chemoenzymatic Route to Diversify Aminolgycosides Enables a Microarray‐Based Method to Probe Acetyltransferase Activity

Specific modification of functional groups in aminoglycosides poses a significant synthetic challenge. In this report, a chemoenzymatic route for modification of aminoglycosides is disclosed. The critical feature of this approach is the discovery that the aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli [1] accepts azido acetyl coenzyme A (AzAcCoA) as a substrate similarly as the natural substrate, acetyl coenzyme A (AcCoA). After enzymatic delivery of an azido acetyl group, it can be chemically modified via a Huisgen dipolar cycloaddition reaction (HDCR)[2] enabling further diversification. Thus, this method accelerates access to modified compounds with diversity beyond that which can be installed directly via AAC(3) and a modified CoA thioester. The approach was further developed to study modification of aminoglycosides by AAC(3), which causes broad-scale aminoglycoside inactivation, using a fluorescence-based microarray platform. This platform is a useful analytical tool for the facile identification of both protein and carbohydrate substrates for acetyltransferases, which play critical roles in a multitude of cellular processes.[3]