Dynamic docking of small molecules targeting RNA CUG repeats causing myotonic dystrophy type 1.

Expansion of RNA CUG repeats causes myotonic dystrophy type 1 (DM1). Once transcribed, the expanded CUG repeats strongly attract muscleblind-like 1 (MBNL1) proteins and disturb their functions in cells. Because of its unique structural form, expanded RNA CUG repeats are prospective drug targets, where small molecules can be utilized to target RNA CUG repeats to inhibit MBNL1 binding and ameliorate DM1-associated defects. In this contribution, we developed two physics-based dynamic docking approaches (DynaD and DynaD/Auto) and applied them to nine small molecules known to specifically target RNA CUG repeats. While DynaD uses a distance-based reaction coordinate to study the binding phenomenon, DynaD/Auto combines results of umbrella sampling calculations performed on 1 × 1 UU internal loops and AutoDock calculations to efficiently sample the energy landscape of binding. Predictions are compared with experimental data, displaying a positive correlation with correlation coefficient (R) values of 0.70 and 0.81 for DynaD and DynaD/Auto, respectively. Furthermore, we found that the best correlation was achieved with MM/3D-RISM calculations, highlighting the importance of solvation in binding calculations. Moreover, we detected that DynaD/Auto performed better than DynaD because of the use of prior knowledge about the binding site arising from umbrella sampling calculations. Finally, we developed dendrograms to present how bound states are connected to each other in a binding process. Results are exciting, as DynaD and DynaD/Auto will allow researchers to utilize two novel physics-based and computer-aided drug-design methodologies to perform in silico calculations on drug-like molecules aiming to target complex RNA loops.

CHARMM-GUI Implicit Solvent Modeler for Various Generalized Born Models in Different Simulation Programs.

Implicit solvent models are widely used because they are advantageous to speed up simulations by drastically decreasing the number of solvent degrees of freedom, which allows one to achieve long simulation time scales for large system sizes. CHARMM-GUI, a web-based platform, has been developed to support the setup of complex multicomponent molecular systems and prepare input files. This study describes an Implicit Solvent Modeler (ISM) in CHARMM-GUI for various generalized Born (GB) implicit solvent simulations in different molecular dynamics programs such as AMBER, CHARMM, GENESIS, NAMD, OpenMM, and Tinker. The GB models available in ISM include GB-HCT, GB-OBC, GB-neck, GBMV, and GBSW with the CHARMM and Amber force fields for protein, DNA, RNA, glycan, and ligand systems. Using the system and input files generated by ISM, implicit solvent simulations of protein, DNA, and RNA systems produce similar results for different simulation packages with the same input information. Protein-ligand systems are also considered to further validate the systems and input files generated by ISM. Simple ligand root-mean-square deviation (RMSD) and molecular mechanics generalized Born surface area (MM/GBSA) calculations show that the performance of implicit simulations is better than docking and can be used for early stage ligand screening. These reasonable results indicate that ISM is a useful and reliable tool to provide various implicit solvent simulation applications.

Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patient­derived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

Massively Parallel Optimization of the Linker Domain in Small Molecule Dimers Targeting a Toxic r(CUG) Repeat Expansion.

RNA contributes to disease pathobiology and is an important therapeutic target. The downstream biology of disease-causing RNAs can be short-circuited with small molecules that recognize structured regions. The discovery and optimization of small molecules interacting with RNA is, however, challenging. Herein, we demonstrate a massively parallel one-bead-one-compound methodology, employed to optimize the linker region of a dimeric compound that binds the toxic r(CUG) repeat expansion [r(CUG)exp] causative of myotonic dystrophy type 1 (DM1). Indeed, affinity selection on a 331,776-member library allowed the discovery of a compound with enhanced potency both in vitro (10-fold) and in DM1-patient-derived myotubes (5-fold). Molecular dynamics simulations revealed additional interactions between the optimized linker and the RNA, resulting in ca. 10 kcal/mol lower binding free energy. The compound was conjugated to a cleavage module, which directly cleaved the transcript harboring the r(CUG)exp and alleviated disease-associated defects.

A Small Molecule that Binds an RNA Repeat Expansion Stimulates Its Decay via the Exosome Complex.

Many diseases are caused by toxic RNA repeats. Herein, we designed a lead small molecule that binds the structure of the r(CUG) repeat expansion [r(CUG)exp] that causes myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD) and rescues disease biology in patient-derived cells and in vivo. Interestingly, the compound's downstream effects are different in the two diseases, owing to the location of the repeat expansion. In DM1, r(CUG)exp is harbored in the 3' untranslated region, and the compound has no effect on the mRNA's abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule facilitates excision of the intron, which is then degraded by the RNA exosome complex. Thus, structure-specific, RNA-targeting small molecules can act disease specifically to affect biology, either by disabling the gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD).

A general fragment-based approach to identify and optimize bioactive ligands targeting RNA.

RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21-associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.

Structural Features of Small Molecules Targeting the RNA Repeat Expansion That Causes Genetically Defined ALS/FTD.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r(G4C2)exp] within the C9orf72 gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r(G4C2)exp RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1 × 1 GG internal loop structure, 5'CGG/3'GGC, that is formed when r(G4C2)exp folds. Structure-activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r(G4C2)exp that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r(G4C2)exp that can be exploited for the development of lead medicines and chemical probes.

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.

Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer.

As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non-oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre-miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre-miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.

Development of a coarse-grained lipid model, LIME 2.0, for DSPE using multistate iterative Boltzmann inversion and discontinuous molecular dynamics simulations.

We suggest an improved version of the intermediate resolution implicit solvent model for lipids, LIME, that was previously developed for use with discontinuous molecular dynamics (DMD) simulations. LIME gets its geometrical and the energy parameters between bonded and nonbonded pairs of coarse-grained (CG) sites from atomistic simulations. The improved model, LIME 2.0, uses multiple square wells rather than the single square well used in original LIME to obtain intermolecular interactions that more faithfully mimic those from atomistic simulations. The multi-state iterative Boltzmann inversion (MS-IBI) scheme is used to determine the interaction parameters. This means that a single set of interaction parameters between coarse-grained sites can be used to represent the lipid bilayers at different temperatures. The physical properties of CG DSPE lipid bilayer are calculated using CG simulations and compared to atomistic simulations results to verify the improved model. The phase transition temperature of the lipid bilayer is measured accurately and the lipid translocation phenomenon, " flip-flop" is observed through CG simulation. These results suggest that CG parameterization using multiple square-well and the MS-IBI scheme is well suited to the study of lipid bilayers cross a range of temperatures with DMD simulations.

In vitro biosynthesis of metal nanoparticles in microdroplets.

We report the use of a hydrogel polymer, recombinant Escherichia coli cell extracts, and a microdroplet-based microfluidic device to fabricate artificial cellular bioreactors which act as reactors to synthesize diverse metal nanoparticles (NPs). The combination of cell extracts, microdroplet-based microfluidic device, and hydrogel was able to produce a mass amount of artificial cellular bioreactors with uniform size and shape. For the first time, we report the alternating generation of microdroplets through one orifice for the fabrication of the artificial cellular reactors using the cell extract as inner cellular components and hydrogel as an artificial cellular membrane. Notably, the hydrogels were able to protect the encapsulated cell extracts from the surrounding environment and maintain the functionality of cellular component for the further cellular bioreactor applications. Furthermore, the successful applications of the fabricated artificial cellular bioreactors to synthesize various NPs including quantum dots, iron, and gold was demonstrated. By employing this microfluidic technique, the artificial cellular bioreactors could be applicable for the synthesis of diverse metal NPs through simple dipping of the reactors to the metal precursor solutions. Thus, the different size of NPs can be synthesized through controlling the concentration of metal precursors. This artificial cellular bioreactors offer promising abilities to biofriendly ways to synthesis diverse NPs and can be applicable in chemical, biomedical, and bioengineering applications.

Organoclay-assisted interfacial polymerization for microfluidic production of monodisperse PEG-microdroplets and in situ encapsulation of E. coli.

We developed a novel one-pot synthetic strategy for preparing monodisperse polyethylene glycol diacrylate (PEGDA) microdroplets via organoclay-assisted interfacial polymerization approach for Escherichia coli encapsulation. Based on the mechanism of spontaneous and rapid polymerization of PEGDA precursor solution with Mg-organoclay, the prepared PEGDA microdroplets have uniform size and fine round shape, with size range of 74-118 µm. The size of microdroplets can be controlled through the changing continuous phase flow rate. Organoclay-assisted polymerization method provides a unique environment to produce non-toxic ways of fabricating microorganism encapsulated microdroplets and to prohibit microdroplets merge during the processes. Furthermore, we successfully carried out to entrap E. coli inside of the PEGDA microdroplets. E. coli expressing a green fluorescent protein shows a good viability inside the PEGDA microdroplets. The in situ microfluidic synthetic method provides a novel approach for the preparation of monodisperse PEGDA microdroplets via a one-pot route.

Ultrasound-aided formation of gold nanoparticles on multi-walled carbon nanotubes functionalized with mercaptobenzene moieties.

A hybrid of multi-walled carbon nanotube (MWCNT) and gold nanoparticle (Au NP) was prepared under ultrasound irradiation. The approach starts with the functionalization of the walls of MWCNTs with mercaptobenzene moieties for the subsequent immobilization of Au NPs. From the Raman spectra, mercaptobenzene was proven to exist on the MWCNTs. Gold ions were added to the aqueous dispersion of functionalized MWCNTs (f-MWCNTs), and were reduced with the aid of ultrasound and ammonium hydroxide. The reduced gold nanoparticles were examined from the TEM images. Au NPs adhered specifically on the thiol groups of mercaptobenzene to be deposited uniformly on the outer walls of the f-MWCNTs. The application of ultrasound led to a high yield of MWCNT-Au nanocomposites and to the dense distribution of the Au NPs. Moreover, the synthesis reaction rate of the hybrid was considerably enhanced relative to synthesis with mechanical agitation. Through an adsorption test using gold-binding-peptide-(GBP)-modified biomolecules, the hybrid's potential for biological diagnosis was verified.

Synthesis and utilization of E. coli-encapsulated PEG-based microdroplet using a microfluidic chip for biological application.

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single-step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non-toxic ways of fabricating microorganism-encapsulated hydrogel microbeads. Due to these unique properties of micro-sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl-ß-D-thiogalactopyranoside induction, suggesting that there is no low-molecular weight substrate transfer limitation inside of microbeads. Furthermore, non-toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells.