William J. Allen

GridMAT-MD: a grid-based membrane analysis tool for use with molecular dynamics.

GridMAT‐MD is a new program developed to aid in the analysis of lipid bilayers from molecular dynamics simulations. It reads a GROMACS coordinate file and generates two types of data: a two‐dimensional contour plot depicting membrane thickness, and a polygon‐based tessellation of the individual lipid headgroups. GridMAT‐MD can also account for proteins or small molecules within the headgroups of the lipids, closely approximating their occupied lateral area. The program requires no installation, is fast, and is freely available.

DOCK 6: Impact of new features and current docking performance

This manuscript presents the latest algorithmic and methodological developments to the structure‐based design program DOCK 6.7 focused on an updated internal energy function, new anchor selection control, enhanced minimization options, a footprint similarity scoring function, a symmetry‐corrected root‐mean‐square deviation algorithm, a database filter, and docking forensic tools. An important strategy during development involved use of three orthogonal metrics for assessment and validation: pose reproduction over a large database of 1043 protein‐ligand complexes (SB2012 test set), cross‐docking to 24 drug‐target protein families, and database enrichment using large active and decoy datasets (Directory of Useful Decoys [DUD]‐E test set) for five important proteins including HIV protease and IGF‐1R. Relative to earlier versions, a key outcome of the work is a significant increase in pose reproduction success in going from DOCK 4.0.2 (51.4%) → 5.4 (65.2%) → 6.7 (73.3%) as a result of significant decreases in failure arising from both sampling 24.1% → 13.6% → 9.1% and scoring 24.4% → 21.1% → 17.5%. Companion cross‐docking and enrichment studies with the new version highlight other strengths and remaining areas for improvement, especially for systems containing metal ions. The source code for DOCK 6.7 is available for download and free for academic users at http://dock.compbio.ucsf.edu/.

Implementation of the Hungarian algorithm to account for ligand symmetry and similarity in structure-based design.

False negative docking outcomes for highly symmetric molecules are a barrier to the accurate evaluation of docking programs, scoring functions, and protocols. This work describes an implementation of a symmetry-corrected root-mean-square deviation (RMSD) method into the program DOCK based on the Hungarian algorithm for solving the minimum assignment problem, which dynamically assigns atom correspondence in molecules with symmetry. The algorithm adds only a trivial amount of computation time to the RMSD calculations and is shown to increase the reported overall docking success rate by approximately 5% when tested over 1043 receptor–ligand systems. For some families of protein systems the results are even more dramatic, with success rate increases up to 16.7%. Several additional applications of the method are also presented including as a pairwise similarity metric to compare molecules during de novo design, as a scoring function to rank-order virtual screening results, and for the analysis of trajectories from molecular dynamics simulation. The new method, including source code, is available to registered users of DOCK6 (http://dock.compbio.ucsf.edu).

Structural Basis for Ceramide Recognition and Hydrolysis by Human Neutral Ceramidase

Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. As an enzyme regulating the balance of ceramide and sphingosine-1-phosphate, nCDase is emerging as a therapeutic target for cancer. Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Utilizing flexible ligand docking, we predict a likely binding mode for ceramide that superimposes closely with the crystallographically observed transition state analog phosphate. Our results suggest that nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide. Together, these data provide a foundation to aid drug development and establish common themes for how proteins recognize the bioactive lipid ceramide.

Modeling the Relationship between the p53 C-Terminal Domain and Its Binding Partners Using Molecular Dynamics

Fifty percent of all cancer cases result from mutations of the TP53 gene, which encodes the tumor suppressor p53, and it is hypothesized that the p53-mediated checkpoint pathway is compromised in most of the remaining cases. The p53 C-terminal domain (CTD) is an important site of p53 regulation but by nature is difficult to study, as it is intrinsically disordered. In this study, we performed molecular dynamics simulations on the p53 CTD and five known regulatory binding partners. We identified distinct trends in fluctuation within and around the p53 CTD binding site on each partner demonstrating a behavior that facilitates association. Further, we present evidence that the size of the hydrophobic pocket in each p53 CTD binding site governs the secondary structure of the p53 CTD when in the bound state. This information will be useful for predicting new binding partners for the p53 CTD, identifying interacting regions within other known partners, and discovering inhibitors that provide additional points of control over p53 activity.

Steered Molecular Dynamics Simulations Reveal Important Mechanisms in Reversible Monoamine Oxidase B Inhibition

The monotopic membrane protein monoamine oxidase B (MAO B) is an important drug target for Parkinsons disease. In order to design more specific, and thereby more effective, inhibitors for this enzyme, it is necessary to determine what factors govern inhibitor specificity and the inhibitor binding process, including the roles of the lipid bilayer, the active site loop, and several key residues within the binding pocket. Atomistic molecular dynamics simulations of MAO B either embedded in a lipid bilayer or free in solution have been performed. The simulations suggest that the bilayer controls the availability of the active site cavity by regulating the degree of fluctuation in two key loops that form the greater part of the active site entrance (residues 85-110 and 155-165). In turn, the enzyme itself causes local thinning and a decrease in area per lipid of the surrounding bilayer environment. Additional MD simulations of MAO B in complex with seven different reversible inhibitors followed by nonequilibrium steered MD simulations of the inhibitor unbinding have also been performed. The simulations demonstrate that the average energy of interaction between inhibitor and MAO B residues during inhibitor egress is an effective indicator of inhibitor strength and is also useful for identifying key residues that govern inhibitor specificity. These data provide researchers with valuable tools for designing effective MAO B inhibitors as well as outline a method that can be translated to the study of other enzyme-inhibitor complexes.

Small molecule inhibitors of HIVgp41 N-heptad repeat trimer formation

Identification of mechanistically novel anti-HIV fusion inhibitors was accomplished using a computer-aided structure-based design approach with the goal of blocking the formation of the N-heptad repeat (NHR) trimer of the viral protein gp41. A virtual screening strategy that included per-residue interaction patterns (footprints) was employed to identify small molecules compatible with putative binding pockets at the internal interface of the NHR helices at the core native viral six-helix bundle. From a screen of ∼2.8 million compounds using the DOCK program, 120 with favorable energetic and footprint overlap characteristics were purchased and experimentally tested leading to two compounds with favorable cell-cell fusion (IC50) and cytotoxicity profiles. Importantly, both hits were identified on the basis of scores containing footprint overlap terms and would not have been identified using the standard DOCK energy function alone. To our knowledge, these compounds represent the first reported small molecules that inhibit viral entry via the proposed NHR-trimer obstruction mechanism.

Grid-based molecular footprint comparison method for docking and de novo design: Application to HIVgp41

Scoring functions are a critically important component of computer‐aided screening methods for the identification of lead compounds during early stages of drug discovery. Here, we present a new multigrid implementation of the footprint similarity (FPS) scoring function that was recently developed in our laboratory which has proven useful for identification of compounds which bind to a protein on a per‐residue basis in a way that resembles a known reference. The grid‐based FPS method is much faster than its Cartesian‐space counterpart, which makes it computationally tractable for on‐the‐fly docking, virtual screening, or de novo design. In this work, we establish that: (i) relatively few grids can be used to accurately approximate Cartesian space footprint similarity, (ii) the method yields improved success over the standard DOCK energy function for pose identification across a large test set of experimental co‐crystal structures, for crossdocking, and for database enrichment, and (iii) grid‐based FPS scoring can be used to tailor construction of new molecules to have specific properties, as demonstrated in a series of test cases targeting the viral protein HIVgp41. The method is available in the program DOCK6.

Computer-Aided Approaches for Targeting HIVgp41

Virus-cell fusion is the primary means by which the human immunodeficiency virus-1 (HIV) delivers its genetic material into the human T-cell host. Fusion is mediated in large part by the viral glycoprotein 41 (gp41) which advances through four distinct conformational states: (i) native, (ii) pre-hairpin intermediate, (iii) fusion active (fusogenic), and (iv) post-fusion. The pre-hairpin intermediate is a particularly attractive step for therapeutic intervention given that gp41 N-terminal heptad repeat (NHR) and C‑terminal heptad repeat (CHR) domains are transiently exposed prior to the formation of a six-helix bundle required for fusion. Most peptide-based inhibitors, including the FDA‑approved drug T20, target the intermediate and there are significant efforts to develop small molecule alternatives. Here, we review current approaches to studying interactions of inhibitors with gp41 with an emphasis on atomic-level computer modeling methods including molecular dynamics, free energy analysis, and docking. Atomistic modeling yields a unique level of structural and energetic detail, complementary to experimental approaches, which will be important for the design of improved next generation anti-HIV drugs.

Steered molecular dynamics identifies critical residues of the Nodamura virus B2 suppressor of RNAi

Nearly all RNA viruses produce double-stranded RNA (dsRNA) during their replication cycles—an important pathogen-associated molecular pattern recognized by the RNA interference (RNAi) pathway in invertebrates and plants. Nodamura virus (NoV) encodes a suppressor of RNA silencing termed B2, which binds to dsRNA and prevents the initiation of RNAi as well as the loading of silencing complexes. Using the published crystal structure of NoV-B2, we performed a series of molecular dynamics (MD) simulations to determine the relative electrostatic and van der Waals contributions of various residues in binding dsRNA, identifying four novel potential interactors: R56, E48, P68 and R69. Additionally, steered MD was used to simulate the binding affinity of NoV-B2 sequences bearing substitutions at positions F49, R56 or R59 to dsRNA, with F49S and R56L/R59L substitutions found to have a significant negative impact on the ability of NoV-B2 to bind dsRNA. NoV RNA1 variants were tested for self-directed replication in both vertebrate (RNAi−) and invertebrate (RNAi+) cultured cells. Consistent with a role in dsRNA binding, NoV replication in F49C and F49S variant constructs was affected negatively only in RNAi+ cells. Thus, we used a combination of MD simulations and experimental mutagenesis to further characterize residues important for NoV-dsRNA interactions.