Targeting RNA Structure to Inhibit Editing in Trypanosomes.

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.

Mutated KLF4(K409Q) in meningioma binds STRs and activates FGF3 gene expression.

Krüppel-like factor 4 (KLF4) is a transcription factor that has been proven necessary for both induction and maintenance of pluripotency and self-renewal. Whole-genome sequencing defined a unique mutation in KLF4 (KLF4K409Q) in human meningiomas. However, the molecular mechanism of this tumor-specific KLF4 mutation is unknown. Using genome-wide high-throughput and focused quantitative transcriptional approaches in human cell lines, primary meningeal cells, and meningioma tumor tissue, we found that a change in the evolutionarily conserved DNA-binding domain of KLF4 alters its DNA recognition preference, resulting in a shift in downstream transcriptional activity. In the KLF4K409Q-specific targets, the normally silent fibroblast growth factor 3 (FGF3) is activated. We demonstrated a neomorphic function of KLF4K409Q in stimulating FGF3 transcription through binding to its promoter and in using short tandem repeats (STRs) located within the locus as enhancers.

Coupling of acceptor-substituted diazo compounds and tertiary thioamides: synthesis of enamino carbonyl compounds and their pharmacological evaluation.

This paper describes the synthesis of enamino carbonyl compounds by the copper(i)-catalyzed coupling of acceptor-substituted diazo compounds and tertiary thioamides. We plan to use this method to synthesize indolizidine (-)-237D analogs to find α6-selective antismoking agents. Therefore, we also performed in silico α6-nAchRs binding studies of selected products. Compounds with low root-mean-square deviation values showed more favorable binding free energies. We also report preliminary pharmacokinetic data on indolizidine (-)-237D and found it to have weak activity at CYP3A4. In addition, as enamino carbonyl compounds are also known for antimicrobial properties, we screened previously reported and new enamino carbonyl compounds for antibacterial, antimicrobial, and antifungal properties. Eleven compounds showed significant antimicrobial activities.

Engineering Crystal Packing in RNA Structures I: Past and Future Strategies for Engineering RNA Packing in Crystals.

X-ray crystallography remains a powerful method to gain atomistic insights into the catalytic and regulatory functions of RNA molecules. However, the technique requires the preparation of diffraction-quality crystals. This is often a resource- and time-consuming venture because RNA crystallization is hindered by the conformational heterogeneity of RNA, as well as the limited opportunities for stereospecific intermolecular interactions between RNA molecules. The limited success at crystallization explains in part the smaller number of RNA-only structures in the Protein Data Bank. Several approaches have been developed to aid the formation of well-ordered RNA crystals. The majority of these are construct-engineering techniques that aim to introduce crystal contacts to favor the formation of well-diffracting crystals. A typical example is the insertion of tetraloop-tetraloop receptor pairs into non-essential RNA segments to promote intermolecular association. Other methods of promoting crystallization involve chaperones and crystallization-friendly molecules that increase RNA stability and improve crystal packing. In this review, we discuss the various techniques that have been successfully used to facilitate crystal packing of RNA molecules, recent advances in construct engineering, and directions for future research in this vital aspect of RNA crystallography.

Simulations of Promising Indolizidine-α6-ß2 Nicotinic Acetylcholine Receptor Complexes.

Smoking-cessation drugs bind many off-target nicotinic acetylcholine receptors (nAChRs) and cause severe side effects if they are based on nicotine. New drugs that bind only those receptors, such as α6ß2* nAChR, implicated in nicotine addiction would avoid the off-target binding. Indolizidine (-)-237D (IND (-)-237D), a bicyclic alkaloid, has been shown to block α6ß2* containing nAChRs and functionally inhibit the nicotine-evoked dopamine release. To improve the affinity of indolizidine (-)-237D for α6ß2*, we built a library of 2226 analogs. We screened virtually the library against a homology model of α6ß2 nAChR that we derived from the recent crystal structure of α4ß2 nAChR. We also screened the crystal structure of α4ß2 nAChR as a control on specificity. We ranked the compounds based on their predicted free energy of binding. We selected the top eight compounds bound in their best pose and subjected the complexes to 100 ns molecular dynamics simulations to assess the stability of the complexes. All eight analogs formed stable complexes for the duration of the simulations. The results from this work highlight nine distinct analogs of IND (-)-237D with high affinity towards α6ß2* nAChR. These leads can be synthesized and tested in in vitro and in vivo studies as lead candidates for drugs to treat nicotine addiction.