The rotavirus inner capsid particle, known as the "double-layered particle" (DLP), is the "payload" delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of VP2 and VP6, respectively, package the eleven segments of double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ~700 Å) in the asymmetric unit of the P2 1 2 1 2 1 unit cell, of dimensions a= 740 Å, b= 1198 Å, c= 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular-replacement model for initial phase determination to about 18.5 Å resolution and the sixty-fold redundancy of the icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outerlayer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus two per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin, principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two "subdomain" boundaries) allow VP2A and B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the fivefold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoreoviruses, VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a "fivefold hub" that projects into the DLP interior along the fivefold axis; residues 81-100 link the ten polypeptide chains emerging from a fivefold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The fivefold hub appears to have several distinct functions. One is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of (+)-strand RNA, as a decamer of VP2 assembles. A second is to serve as Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript a shaft around which can coil a segment of dsRNA. A third is to guide nascent mRNA, synthesized in the DLP int...

Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense, mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner, double-layered particle (DLP) into the cyotosol. DLP structures determined by x-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a “fivefold hub” of more structured density projecting inward from the VP2 shell of the DLP along each of the 12 fivefold axes. Analysis of the x-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the x-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the fivefold axis. The template strand enters from within the particle, and the dsRNA product of the initial, replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

Small molecule protein kinase inhibitors are widely employed as biological reagents and as leads in the design of drugs for a variety of diseases. We investigated the phenomenon of kinase-likeness, i.e., the propensity of ligands to inhibit protein kinases, in the context of kinase-specific substructural fragments. The frequency of occurrence of multiple structural fragments in kinase inhibitor libraries relative to nonkinase compounds has been analyzed. A combination of structural fragment counts, termed the "2-0" kinase-likeness rule, provides approximately 5-fold enrichment in kinase active compounds. This rule has been validated using in-house kinase counterscreening data and applied prospectively to uncover kinase activities in marketed drugs. In addition, the role of discriminating fragments in kinase recognition was interrogated using available structural data, providing an insight into their effect on inhibitor potency and selectivity. One of these fragments, bisarylaniline, has been characterized as a kinase-privileged fragment with specific binding preferences and a link to increased activity within kinases.

The solubility of organic molecules is of critical importance to the pharmaceutical industry; however, robust computational methods to predict this quantity from first-principles are lacking. Solubility can be computed from a thermodynamic cycle that decomposes standard state solubility into the sum of solid–vapor sublimation and vapor–liquid solvation free energies ΔG solubility ° = ΔG sub ° + ΔG solv °. Over the past few decades, alchemical simulation methods to compute solvation free energy using classical force fields have become widely used. However, analogous methods for determining the free energy of the sublimation/deposition phase transition are currently limited by the necessity of a priori knowledge of the atomic coordinates of the crystal. Here, we describe progress toward an alternative scheme based on growth of the asymmetric unit into a crystal via alchemy (GAUCHE). GAUCHE computes deposition free energy ΔG dep ° = −ΔG sub ° = −k B T ln(V c/V g) + ΔG AU + ΔG AU→UC as the sum of an entropic term to account for compressing a vapor at 1 M standard state (V g) into the molar volume of the crystal (V c), where k B is Boltzmann’s constant and T is temperature in degrees Kelvin, plus two simulation steps. In the first simulation step, the deposition free energy ΔG AU for a system composed of only N AU asymmetric unit (AU) molecule(s) is computed beginning from an arbitrary conformation in vacuum. In the second simulation step, the change in free energy ΔG AU→UC to expand the asymmetric unit degrees of freedom into a unit cell (UC) composed of N UC independent molecules is computed. This latter step accounts for the favorable free energy of removing the constraint that every symmetry mate of the asymmetric unit has an identical conformation and intermolecular interactions. The current work is based on NVT simulations, which requires knowledge of the crystal space group and unit cell parameters from experiment, but not a priori knowledge of crystalline atomic coordinates. GAUCHE was applied to 5 organic molecules whose sublimation free energy has been measured experimentally, based on the polarizable AMOEBA force field and more than a microsecond of sampling per compound in the program Force Field X. The mean unsigned and RMS errors were only 1.6 and 1.7 kcal/mol, respectively, which indicates that GAUCHE is capable of accurate prediction of absolute sublimation thermodynamics.

As compounds are optimized for greater potency during pharmaceutical discovery, their aqueous solubility often decreases, making them less viable as orally-administered drugs. To investigate whether potency and insolubility share a common origin, we examined the structural and thermodynamic properties of telaprevir, a sparingly soluble inhibitor of hepatitis C virus protease. Comparison of the hydrogen bond motifs in crystalline telaprevir with those present in the protease-telaprevir complex revealed striking similarities. Additionally, the thermodynamics of telaprevir dissolution closely resembles those of protein-ligand dissociation. Together, these findings point to a common origin of potency and insolubility rooted in particular amide-amide hydrogen bond patterns. The insolubility of telaprevir is shown by computational analysis to be caused by interactions in the crystal, not unfavorable hydrophobic hydration. Accordingly, competing out the particular amide-amide hydrogen bond motifs in crystalline telaprevir with 4-hydroxybenzoic acid yielded a co-crystalline solid with excellent aqueous dissolution and oral absorption. The analysis suggests a generalizable approach for identifying drug candidate compounds that either can or cannot be rendered orally bioavailable by alteration of their crystalline solid phases, in an approach that provides a pragmatic way to attain substantial enhancements in the success rate of drug discovery and development.