Dendritic closomers: novel spherical hybrid dendrimers.

We report construction of monodisperse PAMAM-type dendrimers based on a dodecahydroxy closo-dodecaborane scaffold. The ideal sphericity and high functional group density of the icosahedral core permits ready access to hybrid dendrimers (dendritic closomers) having rigid, uniformly shaped exterior surfaces.

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.

Approach to the library of fused pyridine-4-carboxylic acids by Combes-type reaction of acyl pyruvates and electron-rich amino heterocycles.

A library of fused pyridine-4-carboxylic acids (including pyrazolo[3,4-b]pyridines, isoxazolo[5,4-b]pyridines, furo[2,3-b]pyridines, thieno[2,3-b]pyridines, and pyrido[2,3-d]pyrimidines) was generated by Combes-type reaction of acyl pyruvates and electron-rich amino heterocycles followed by hydrolysis of the ester. The library members were also demonstrated to undergo the standard combinatorial transformations including amide coupling and esterification, as well as less common heterocyclizations to 1,2,4-triazoles and 1,2,4-oxadiazoles.

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 1x1 nucleotide UU (DM1) or 2x2 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 IC(50) 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.

Controlling the specificity of modularly assembled small molecules for RNA via ligand module spacing: targeting the RNAs that cause myotonic muscular dystrophy.

Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in noncoding regions of RNA. In DM1, the expansion is an rCUG triplet repeat, whereas the DM2 expansion is an rCCUG quadruplet repeat. Both RNAs fold into hairpin structures with periodically repeating internal loops separated by two 5'GC/3'CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 x 1 nucleotide UU loops while the DM2 repeat forms 2 x 2 nucleotide 5'CU/3'UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6'-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 x 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective for the DM2 RNA over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by a shorter distance. Indeed, here the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with K(d)'s of 20 nM (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective) and inhibit the RNA-protein interaction with IC(50)'s of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- x 3-fold). The trimer and tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.

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.

Rational and modular design of potent ligands targeting the RNA that causes myotonic dystrophy 2.

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6'-N-5-hexynoate kanamycin A (1) prefers to bind 2x2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops are found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC(50) of 25 nM. This ligand has higher affinity and is more specific for binding the DM2 RNA than MBNL-1. It binds the DM2 RNA at least 30 times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6'-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNA-protein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable.

Small molecule microarrays of RNA-focused peptoids help identify inhibitors of a pathogenic group I intron.

Peptoids that inhibit the group I intron RNA from Candida albicans, an opportunistic pathogen that kills immunocompromised hosts, have been identified using microarrays. The arrayed peptoid library was constructed using submonomers with moieties similar to ones found in small molecules known to bind RNA. Library members that passed quality control analysis were spotted onto a microarray and screened for binding to the C. albicans group I intron ribozyme. Each ligand binder identified from microarray-based screening inhibited self-splicing in the presence of 1 mM nucleotide concentration of bulk yeast tRNA with IC(50)'s between 150 and 2200 microM. The binding signals and the corresponding IC(50)'s were used to identify features in the peptoids that predispose them for RNA binding. After statistical analysis of the peptoids' structures that bind, a second generation of inhibitors was constructed using these important features; all second generation inhibitors have improved potencies with IC(50)'s of <100 microM. The most potent inhibitor is composed of one phenylguanidine and three tryptamine submonomers and has an IC(50) of 31 microM. This compound is 6-fold more potent than pentamidine, a clinically used drug that inhibits self-splicing. These results show that (i) modulators of RNA function can be identified by designing RNA-focused chemical libraries and screening them via microarray; (ii) statistical analysis of ligand binders can identify features in leads that predispose them for binding to their targets; and (iii) features can then be programmed into second generation inhibitors to design ligands with improved potencies.

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray.

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2') from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2'). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2') is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.

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.

A small molecule microarray platform to select RNA internal loop-ligand interactions.

Herein, we report the development of a microarray platform to select RNA motif-ligand interactions that allows simultaneous screening of both RNA and chemical space. We used this platform to identify the RNA internal loops that bind 6'- N-5-hexynoate kanamycin A ( 1). Selected internal loops that bind 1 were studied in detail and commonly display an adenine across from a cytosine independent of the size of the loop. Additional preferences are also observed. For 3 x 3 nucleotide loops, there is a preference for purines, and for 2 x 2 nucleotide loops there is a preference for pyrimidines neighbored by an adenine across from a cytosine. This technique has several advantageous features for selecting RNA motif-ligand interactions: (1) higher affinity RNA motif-ligand interactions are identified by harvesting bound RNAs from lower ligand loadings; (2) bound RNAs are harvested from the array via gel extraction, mitigating kinetic biases in selections; and (3) multiple selections are completed on a single array surface. To further demonstrate that multiple selections can be completed in parallel on the same array surface, we selected the RNA internal loops from a 4096-member RNA internal loop library that bound a four-member aminoglycoside library. These experiments probed 16,384 (4 aminoglycoside x 4096-member RNA library) interactions in a single experiment. These studies allow for parallel screening of both chemical and RNA space to improve our understanding of RNA-ligand interactions. This information may facilitate the rational and modular design of small molecules targeting RNA.

Synthesis of a highly reactive heterocyclic reactant and its unusual photochemistry; mechanistic and exploratory organic photochemistry.

[reaction: see text] A unique new set of reactions has been observed in heterocyclic photochemistry. 2-Methyl-4,4-diphenyl-3,4-dihydropyrimidin-1(2H)-one has been synthesized and its photochemistry investigated. This compound has been found to lead to a rearranged, dimeric product arising from a unique bond-scission process.