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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 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 multi-component molecular systems and prepare input files. This study describes 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.

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.

RNA molecules both contribute to and are causative of many human diseases. One method to perturb RNA function is to target its structure with small molecules. However, discovering bioactive ligands for RNA targets is challenging. Here, we show that the bioactivity of a linear dimeric ligand that inactivates the RNA trinucleotide repeat expansion that causes myotonic dystrophy type 1 [DM1; r(CUG)(exp)] can be improved by macrocyclization. Indeed, the macrocyclic compound is ten times more potent than the linear compound for improving DM1‐associated defects in cells, including in patient‐derived myotubes (muscle cells). This enhancement in potency is due to the macrocycle’s increased affinity and selectively for the target, which inhibit r(CUG)(exp)’s toxic interaction with muscleblind‐like 1 (MBNL1), and its superior cell permeability. Macrocyclization could prove to be an effective way to enhance the bioactivity of modularly assembled ligands targeting RNA.

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.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G(4)C(2) RNA repeat [r(G(4)C(2))(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(G(4)C(2))(exp). The compound was able to selectively bind r(G(4)C(2))(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 patient–derived 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(G(4)C(2))(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(G(4)C(2))(exp) is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

We present a method for measuring the pore throat diameter of a simulated porous material. The pore throat diameter is the size of the narrowest pore that has both an entrance and an exit in a netw...

We present the results of discontinuous molecular dynamics (DMD) simulations aimed at understanding the formation of DNA-mediated hydrogels and assessing their drug loading ability. Poly(ethylene glycol) (PEG) precursors of four and six arms that are covalently functionalized on all ends with oligonucleotides are cross-linked by a single oligonucleotide whose sequence is complementary to the oligonucleotide conjugated to the precursor. We show that the precursors with large molecular weight and many arms are advantageous in forming a three-dimensional percolated network. Analysis of the percolated networks shows that the pore diameter distribution becomes narrower as the precursor concentration, the number of arms, and the molecular weight increase. The pore throat diameter, the size of the largest molecule that can travel through the hydrogel networks without being trapped, is determined. The percolated network slows the movement of molecules inside the pores. Molecules larger than the pore throat diamet...

[Image: see text] 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.

Significance The development of selective bioactive compounds that target RNA structures is difficult. Here we report an approach to identify low molecular weight fragments that bind to a cancer-causing RNA. By determining where the fragments bind within an RNA target, they can be assembled to provide a high-affinity and potent inhibitor. This approach could be a general way to develop bioactive small molecules that target RNA, which previously was considered “undruggable.” It was applied to provide a precision lead medicine against an RNA that contributes to cancer metastasis., 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.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r(G(4)C(2))(exp)] within the C9orf 72 gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r(G(4)C(2))(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(G(4)C(2))(exp) folds. Structure−activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r(G(4)C(2))(exp) that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r(G(4)C(2))(exp) that can be exploited for the development of lead medicines and chemical probes.

We describe the design of a small molecule that binds the structure of a r(CUG) repeat expansion [r(CUG)exp] and reverses molecular defects in two diseases mediated by the RNA - myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD). Thus, a single structure-specific ligand has potential therapeutic benefit for multiple diseases, in contrast to oligonucleotide-based modalities that are customized for each disease by nature of targeting the gene that harbors the repeat. Indeed, the small molecule binds the target with nanomolar affinity and >100-fold specificity vs. many other RNAs and DNA. 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 (UTR) of and mRNA, and the compound has no effect on the RNA’s abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule, by binding the repeat expansion, facilitates excision of the intron, which is then degraded by the exosome complex exonuclease, hRRP6. Thus, structure-specific, RNA-targeting small molecules can act disease-specifically to affect biology, either by disabling its gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD).Significance statementMany different diseases are caused by toxic structured RNAs. Herein, we designed a lead small molecule that binds a toxic structure and rescues disease biology. We show that a structure-specific small molecule can improve disease-associated defects in two diseases that share the common toxic RNA structure. In one disease, the toxic structure is harbored in an intron and causes its retention. The compound facilitates processing of a retained intron, enabling the disease-affected cell to remove the toxic RNA.

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.

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 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-wells and the MS-IBI scheme is well suited to the study of lipid bilayers across a range of temperatures with DMD simulations.

In this paper, we develop a coarse-grained nucleotide model for the purpose of simulating large-scale aptamer-based hydrogel network formation in future research. In the model, each nucleotide is represented by a single interaction site containing sugar, phosphate and base. Discontinuous molecular dynamics (DMD) simulations are performed to simulate formation and denaturation of oligonucleotide duplexes as a function of temperature. The simulated melting temperatures of oligonucleotide duplexes are calculated in simulations of systems with different sequences, lengths and concentrations of oligonucleotides, and compared to data from the OligoAnalyzer tool. The denaturation of oligonucleotide triplexes containing a hybridised structure of three different oligonucleotides is analysed using both simulations and experiments. The nucleotide model is found to be a good predictor of the oligonucleotide’s hybridised state for both duplexes and triplexes. This coarse-grained model has wide ranging applications in the development or optimisation of DNA-based technologies including DNA origami, DNA-enabled hydrogels and DNA-based biosensors.

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.

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-β-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.

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 mm. The size of microdroplets can be controlled through the changing continuous phase flow rate. Organo- clay-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.