Idealized Models of Protofilaments of Human Islet Amyloid Polypeptide

Fibrils formed by assembly of human islet amyloid polypeptide (hIAPP) are found in most patients with type II diabetes. Structurally, these fibrils are composed of multiple protofilaments and are characterized by extended beta sheets, variable helical twists, and different morphologies. We have previously derived models for the hIAPP protofilament using simulations constrained by data from EPR spectroscopy. In the current work, these models were used as a basis for generating idealized hIAPP protofilaments with symmetrical geometrical properties using a new algorithm, MFIBRIL. We show good agreement of the idealized protofilaments with experimental data for amino acid side chain orientations and geometrical features including the inter-β sheet distance and the protofilament radius. These idealized protofilaments can be used in MFIBRIL to generate fibril models that may be experimentally testable at the molecular level. MFIBRIL can also be used for building structures of any repetitive molecular assembly starting with a single building block obtained from any source

ESI-MS/MS analysis of N³dC-mech-N³dC.

<p><b>A:</b> ESI-MS/MS spectrum of N³dC-mech-N³dC. <b>B:</b> Fragmentation pattern of N³dC-mech-N³dC. The IUPAC name for N³dC-mech-N³dC is [4-amino-3-[2-[2-[6-amino-3-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-2-oxo-pyrimidin-1-ium-1-yl]ethyl-methyl-amino]ethyl]-1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-3-ium-2-one].</p

MALDI-TOF-MS spectrum of the mechlorethamine-crosslinked DNA duplex.

<p>MALDI-TOF-MS spectrum of the mechlorethamine-crosslinked DNA duplex.</p

Structures of possible mechlorethamine crosslinks at a cytosine-cytosine mismatch pair.

<p><b>A:</b> A mechlorethamine crosslink through O² of cytosine. <b>B:</b> A mechlorethamine crosslink through N³ of cytosine. <b>C:</b> A mechlorethamine crosslink through N⁴ of cytosine. Note that the O² and N⁴ crosslinks are neutral species that form with loss of 2H⁺ from the nucleosides, whereas the N³ crosslink has a double positive charge.</p

HPLC-UV chromatograms of DNA and the mechlorethamine-crosslinked DNA duplex.

<p><b>A:</b> Top-strand DNA. <b>B:</b> Bottom-strand DNA. <b>C:</b> DNA duplex (denatured on the column). <b>D:</b> The mechlorethamine-crosslinked DNA duplex.</p

Physiologically Based Pharmacokinetic Modeling in Lead Optimization. 1. Evaluation and Adaptation of GastroPlus To Predict Bioavailability of Medchem Series

When medicinal chemists need to improve bioavailability (%F) within a chemical series during lead optimization, they synthesize new series members with systematically modified properties mainly by following experience and general rules of thumb. More quantitative models that predict %F of proposed compounds from chemical structure alone have proven elusive. Global empirical %F quantitative structure–property (QSPR) models perform poorly, and projects have too little data to train local %F QSPR models. Mechanistic oral absorption and physiologically based pharmacokinetic (PBPK) models simulate the dissolution, absorption, systemic distribution, and clearance of a drug in preclinical species and humans. Attempts to build global PBPK models based purely on calculated inputs have not achieved the <2-fold average error needed to guide lead optimization. In this work, local GastroPlus PBPK models are instead customized for individual medchem series. The key innovation was building a local QSPR for a numerically fitted effective intrinsic clearance (CL_loc). All inputs are subsequently computed from structure alone, so the models can be applied in advance of synthesis. Training CL_loc on the first 15–18 rat %F measurements gave adequate predictions, with clear improvements up to about 30 measurements, and incremental improvements beyond that

Physiologically Based Pharmacokinetic Modeling in Lead Optimization. 2. Rational Bioavailability Design by Global Sensitivity Analysis To Identify Properties Affecting Bioavailability

When medicinal chemists need to improve oral bioavailability (%F) during lead optimization, they systematically modify compound properties mainly based on their own experience and general rules of thumb. However, at least a dozen properties can influence %F, and the difficulty of multiparameter optimization for such complex nonlinear processes grows combinatorially with the number of variables. Furthermore, strategies can be in conflict. For example, adding a polar or charged group will generally increase solubility but decrease permeability. Identifying the 2 or 3 properties that most influence %F for a given compound series would make %F optimization much more efficient. We previously reported an adaptation of physiologically based pharmacokinetic (PBPK) simulations to predict %F for lead series from purely computational inputs within a 2-fold average error. Here, we run thousands of such simulations to generate a comprehensive “bioavailability landscape” for each series. A key innovation was recognition that the large and variable number of p<i>K</i>_a’s in drug molecules could be replaced by just the two straddling the isoelectric point. Another was use of the ZINC database to cull out chemically inaccessible regions of property space. A quadratic partial least squares regression (PLS) accurately fits a continuous surface to these thousands of bioavailability predictions. The PLS coefficients indicate the globally sensitive compound properties. The PLS surface also displays the %F landscape in these sensitive properties locally around compounds of particular interest. Finally, being quick to calculate, the PLS equation can be combined with models for activity and other properties for multiobjective lead optimization

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron–Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H_T and H_L) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H_T and H_L stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial

Nitroxide Sensing of a DNA Microenvironment: Mechanistic Insights from EPR Spectroscopy and Molecular Dynamics Simulations

The behavior of the nitroxide spin labels 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a) and 1-oxyl-2,2,5,5-tetramethylpyrroline (R5) attached at a phosphorothioate-substituted site in a DNA duplex is modulated by the DNA in a site- and stereospecific manner. A better understanding of the mechanisms of R5a/R5 sensing of the DNA microenvironment will enhance our capability to relate information from nitroxide spectra to sequence-dependent properties of DNA. Toward this goal, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate R5 and R5a attached as R_<i>p</i> and S_<i>p</i> diastereomers at phosphorothioate _pSC₇ of d­(CTACTG_pSC₇Y₈TTAG). d­(CTAAAGCAGTAG) (Y = T or U). X-band continuous-wave EPR spectra revealed that the dT₈ to dU₈ change alters nanosecond rotational motions of R_<i>p</i>-R5a but produces no detectable differences for S_<i>p</i>-R5a, R_<i>p</i>-R5, and S_<i>p</i>-R5. MD simulations were able to qualitatively account for these spectral variations and provide a plausible physical basis for the R5/R5a behavior. The simulations also revealed a correlation between DNA backbone B_I/B_II conformations and R5/R5a rotational diffusion, thus suggesting a direct connection between DNA local backbone dynamics and EPR-detectable R5/R5a motion. These results advance our understanding of how a DNA microenvironment influences nitroxide motion and the observed EPR spectra. This may enable use of R5/R5a for a quantitative description of the sequence-dependent properties of large biologically relevant DNA molecules

A curcumin-diglutaric acid conjugated prodrug with improved water solubility and antinociceptive properties compared to curcumin

<p>In this work, a curcumin-diglutaric acid (CurDG) prodrug was synthesized by conjugation of curcumin with glutaric acid via an ester linkage. The water solubility, partition coefficient, release characteristics, and antinociceptive activity of CurDG were compared to those of curcumin. The aqueous solubility of CurDG (7.48 μg/mL) is significantly greater than that of curcumin (0.068 μg/mL). A study in human plasma showed that the CurDG completely releases curcumin within 2 h, suggesting the ability of CurDG to serve as a prodrug of curcumin. A hot plate test in mice showed the highest antinociceptive effect dose of curcumin at 200 mg/kg p.o., whereas CurDG showed the same effect at an effective dose of 100 mg/kg p.o., indicating that CurDG significantly enhanced the antinociceptive effect compared to curcumin. The enhanced antinociceptive effect of CurDG may be due to improved water solubility and increased oral bioavailability compared to curcumin.</p