Ning Ma

Photophysical and Dynamical Properties of Doubly Linked Cy3 - DNA Constructs

Photophysical measurements are reported for Cy3-DNA constructs in which both Cy3 nitrogen atoms are attached to the DNA backbone by short linkers. While this linking was thought to rigidify the orientation of the dye and hinder cis-isomerization, the relatively low fluorescence quantum yield and the presence of a short component in the time-resolved fluorescence decay of the dye indicated that cis-isomerization remained possible. Fluorescence correlation spectroscopy and transient absorption experiments showed that photoisomerization occurred with high efficiency. Molecular dynamics simulations of the trans dye system indicated the presence of stacked and unstacked states, and free energy simulations showed that the barriers for stacking/unstacking were low. In addition, simulations showed that the ground cis state was feasible without DNA distortions. Based on these observations, a model is put forward in which the doubly linked dye can photoisomerize in the unstacked state.

Analysis of differential secondary effects of novel rexinoids: Select rexinoid X receptor ligands demonstrate differentiated side effect profiles

In order to determine the feasibility of utilizing novel rexinoids for chemotherapeutics and as potential treatments for neurological conditions, we undertook an assessment of the side effect profile of select rexinoid X receptor (RXR) analogs that we reported previously. We assessed pharmacokinetic profiles, lipid and thyroid‐stimulating hormone (TSH) levels in rats, and cell culture activity of rexinoids in sterol regulatory element‐binding protein (SREBP) induction and thyroid hormone inhibition assays. We also performed RNA sequencing of the brain tissues of rats that had been dosed with the compounds. We show here for the first time that potent rexinoid activity can be uncoupled from drastic lipid changes and thyroid axis variations, and we propose that rexinoids can be developed with improved side effect profiles than the parent compound, bexarotene (1).

Anisotropy of B-DNA Groove Bending

DNA bending is critical for DNA packaging, recognition, and repair, and occurs toward either the major or the minor groove. The anisotropy of B-DNA groove bending was quantified for eight DNA sequences by free energy simulations employing a novel reaction coordinate. The simulations show that bending toward the major groove is preferred for non-A-tracts while the A-tract has a high tendency of bending toward the minor groove. Persistence lengths were generally larger for bending toward the minor groove, which is thought to originate from differences in groove hydration. While this difference in stiffness is one of the factors determining the overall preference of bending direction, the dominant contribution is shown to be a free energy offset between major and minor groove bending. The data suggests that, for the A-tract, this offset is largely determined by inherent structural properties, while differences in groove hydration play a large role for non-A-tracts. By quantifying the energetics of DNA groove bending and rationalizing the origins of the anisotropy, the calculations provide important new insights into a key biological process.

Modeling, synthesis, and biological evaluation of potential retinoid X receptor (RXR) selective agonists: Novel analogues of 4-[1-(3,5,5,8,8- Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl) -4-hydroxyphenyl)acrylic acid (CD3254)

Three unreported analogues of 4-[1-(3,5,5,8,8-pentamethyl-5-6-7-8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), otherwise known as bexarotene, as well as four novel analogues of (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl)-4-hydroxyphenyl)acrylic acid (CD3254), are described and evaluated for their retinoid X receptor (RXR) selective agonism. Compound 1 has FDA approval as a treatment for cutaneous T-cell lymphoma (CTCL), although treatment with 1 can elicit side-effects by disrupting other RXR-heterodimer receptor pathways. Of the seven modeled novel compounds, all analogues stimulate RXR-regulated transcription in mammalian 2 hybrid and RXRE-mediated assays, possess comparable or elevated biological activity based on EC50 profiles, and retain similar or improved apoptotic activity in CTCL assays compared to 1. All novel compounds demonstrate selectivity for RXR and minimal crossover onto the retinoic acid receptor (RAR) compared to all-trans-retinoic acid, with select analogues also reducing inhibition of other RXR-dependent pathways (e.g., VDR-RXR). Our results demonstrate that further improvements in biological potency and selectivity of bexarotene can be achieved through rational drug design.

Right-Handed Helical Foldamers Consisting of De Novo d-AApeptides

New types of foldamer scaffolds are formidably challenging to design and synthesize, yet highly desirable as structural mimics of peptides/proteins with a wide repertoire of functions. In particular, the development of peptidomimetic helical foldamers holds promise for new biomaterials, catalysts, and drug molecules. Unnatural l-sulfono-γ-AApeptides were recently developed and shown to have potential applications in both biomedical and material sciences. However, d-sulfono-γ-AApeptides, the enantiomers of l-sulfono-γ-AApeptides, have never been studied due to the lack of high-resolution three-dimensional structures to guide structure-based design. Herein, we report the first synthesis and X-ray crystal structures of a series of 2:1 l-amino acid/d-sulfono-γ-AApeptide hybrid foldamers, and elucidate their folded conformation at the atomic level. Single-crystal X-ray crystallography indicates that this class of oligomers folds into well-defined right-handed helices with unique helical parameters. The helical structures were consistent with data obtained from solution 2D NMR, CD studies, and molecular dynamics simulations. Our findings are expected to inspire the structure-based design of this type of unique folding biopolymers for biomaterials and biomedical applications.

Free energy simulation of helical transitions

An umbrella sampling method for the calculation of free energies for helical transitions is presented. The method biases structures toward helices of a desired radius and pitch. Although computationally complex, the method has negligible overhead in actual applications. To illustrate the method, calculations of the helical free energy landscape of several peptides are presented for both the CHARMM and the AMBER force fields.

Computer Simulations of the Retinoid X Receptor: Conformational Dynamics and Allosteric Networks

As the heterodimerization partner for a large number of nuclear receptors, the retinoid X receptor (RXR) is important for a large and diverse set of biochemical pathways. Activation and regulation of RXR heterodimers is achieved by complex allosteric mechanisms, which involve the binding of ligands, DNA, coactivators and corepressors, and entail large and subtle conformational motions. Complementing experiments, computer simulations have provided detailed insights into the origins of the allostery by investigating the changes in structure, motion, and interactions upon dimerization, ligand and cofactor binding. This review will summarize a number of simulation studies that have furthered the understanding of the conformational dynamics and the allosteric activation and control of RXR complexes. While the review focuses on the RXR and RXR heterodimers, relevant simulation studies of other nuclear receptors will be discussed as well.

A novel gene expression analytics-based approach to structure aided design of rexinoids for development as next-generation cancer therapeutics

HIGHLIGHTSCTCL cells reveal differential gene expression when treated with chemically distinct rexinoids.Rexinoids can be partitioned into discrete classes employing gene expression profiles.Minor variations in rexinoid structure lead to unique gene expression signatures.Divergence scoring using expression profiles can lead to valuable structural and decision making insights in rexinoid design.Five of the twelve studied bexarotene analogs show promise for further analysis/drug development. ABSTRACT Rexinoids are powerful ligands that bind to retinoid‐X‐receptors (RXRs) and show great promise as therapeutics for a wide range of diseases, including cancer. However, only one rexinoid, bexarotene (Targretin TM) has been successfully transitioned from the bench to the clinic and used to treat cutaneous T‐cell lymphoma (CTCL). Our goal is to develop novel potent rexinoids with a less untoward side effect profile than bexarotene. To this end, we have synthesized a wide array of rexinoids with EC50 values and biological activity similar to bexarotene. In order to determine their suitability for additional downstream analysis, and to identify potential candidate analogs for clinical translation, we treated human CTCL cells in culture and employed microarray technology to assess gene expression profiles. We analyzed twelve rexinoids and found they could be stratified into three distinct categories based on their gene expression: similar to bexarotene, moderately different from bexarotene, and substantially different from bexarotene. Surprisingly, small changes in the structure of the bexarotene parent compound led to marked differences in gene expression profiles. Furthermore, specific analogs diverged markedly from our hypothesis in expression of genes expected to be important for therapeutic promise. However, promoter analysis of genes whose expression was analyzed indicates general regulatory trends along structural frameworks. Our results suggest that certain structural motifs, particularly the basic frameworks found in analog 4 and analog 9, represent important starting points to exploit in generating additional rexinoids for future study and therapeutic applications.

Free Energy Coupling between DNA Bending and Base Flipping

Free energy simulations are presented to probe the energetic coupling between DNA bending and the flipping of a central thymine in double stranded DNA 13mers. The energetics are shown to depend on the neighboring base pairs, and upstream C or T or downstream C tended to make flipping more costly. Flipping to the major groove side was generally preferred. Bending aids flipping, by pushing the system up in free energy, but for small and intermediate bending angles the two were uncorrelated. At higher bending angles, bending and flipping became correlated, and bending primed the system for base flipping toward the major groove. Flipping of the 6-4 pyrimidine-pyrimidone and pyrimidine dimer photoproducts is shown to be more facile than for undamaged DNA. For the damages, major groove flipping was preferred, and DNA bending was much facilitated in the 6-4 pyrimidine-pyrimidone damaged system. Aspects of the calculations were verified by structural analyses of protein-DNA complexes with flipped bases.

[KCl] Dependence of B-DNA Groove Bending Anisotropy

The energetics of B-DNA bending toward the major and minor grooves were quantified by free energy simulations at four different KCl concentrations. Increased [KCl] led to more flexible DNA, with persistence lengths that agreed well with experimental values. At all salt concentrations, major groove bending was preferred, although preferences for major and minor groove bending were similar for the A-tract containing sequence. Since the phosphate repulsions and DNA internal energy favored minor groove bending, the preference for major groove bending was thought to originate from differences in solvation. Water in the minor groove was tighter bound than water in the major groove, and harder to displace than major groove water, which favored the compression of the major groove upon bending. Higher [KCl] decreased the persistence length for both major and minor groove bending but did not greatly affect the free energy spacing between the minor and major groove bending curves. For sequences without A-tracts, salt affected major and minor bending to nearly the same degree, and did not change the preference for major groove bending. For the A-tract containing sequence, an increase in salt concentration decreased the already small energetic difference between major and minor groove bending. Since salts did not significantly affect the relative differences in bending energetics and hydration, it is likely that the increased bending flexibilities upon salt increase are simply due to screening.