Trent E. Balius

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/.

Docking Validation Resources: Protein Family and Ligand Flexibility Experiments

A database consisting of 780 ligand-receptor complexes, termed SB2010, has been derived from the Protein Databank to evaluate the accuracy of docking protocols for regenerating bound ligand conformations. The goal is to provide easily accessible community resources for development of improved procedures to aid virtual screening for ligands with a wide range of flexibilities. Three core experiments using the program DOCK, which employ rigid (RGD), fixed anchor (FAD), and flexible (FLX) protocols, were used to gauge performance by several different metrics: (1) global results, (2) ligand flexibility, (3) protein family, and (4) cross-docking. Global spectrum plots of successes and failures vs rmsd reveal well-defined inflection regions, which suggest the commonly used 2 Å criteria is a reasonable choice for defining success. Across all 780 systems, success tracks with the relative difficulty of the calculations: RGD (82.3%) > FAD (78.1%) > FLX (63.8%). In general, failures due to scoring strongly outweigh those due to sampling. Subsets of SB2010 grouped by ligand flexibility (7-or-less, 8-to-15, and 15-plus rotatable bonds) reveal that success degrades linearly for FAD and FLX protocols, in contrast to RGD, which remains constant. Despite the challenges associated with FLX anchor orientation and on-the-fly flexible growth, success rates for the 7-or-less (74.5%) and, in particular, the 8-to-15 (55.2%) subset are encouraging. Poorer results for the very flexible 15-plus set (39.3%) indicate substantial room for improvement. Family-based success appears largely independent of ligand flexibility, suggesting a strong dependence on the binding site environment. For example, zinc-containing proteins are generally problematic, despite moderately flexible ligands. Finally, representative cross-docking examples, for carbonic anhydrase, thermolysin, and neuraminidase families, show the utility of family-based analysis for rapid identification of particularly good or bad docking trends, and the type of failures involved (scoring/sampling), which will likely be of interest to researchers making specific receptor choices for virtual screening. SB2010 is available for download at http://rizzolab.org .

Targeting fatty acid binding protein (FABP) anandamide transporters - a novel strategy for development of anti-inflammatory and anti-nociceptive drugs.

Fatty acid binding proteins (FABPs), in particular FABP5 and FABP7, have recently been identified by us as intracellular transporters for the endocannabinoid anandamide (AEA). Furthermore, animal studies by others have shown that elevated levels of endocannabinoids resulted in beneficial pharmacological effects on stress, pain and inflammation and also ameliorate the effects of drug withdrawal. Based on these observations, we hypothesized that FABP5 and FABP7 would provide excellent pharmacological targets. Thus, we performed a virtual screening of over one million compounds using DOCK and employed a novel footprint similarity scoring function to identify lead compounds with binding profiles similar to oleic acid, a natural FABP substrate. Forty-eight compounds were purchased based on their footprint similarity scores (FPS) and assayed for biological activity against purified human FABP5 employing a fluorescent displacement-binding assay. Four compounds were found to exhibit approximately 50% inhibition or greater at 10 µM, as good as or better inhibitors of FABP5 than BMS309403, a commercially available inhibitor. The most potent inhibitor, γ-truxillic acid 1-naphthyl ester (ChemDiv 8009-2334), was determined to have Ki value of 1.19±0.01 µM. Accordingly a novel α-truxillic acid 1-naphthyl mono-ester (SB-FI-26) was synthesized and assayed for its inhibitory activity against FABP5, wherein SB-FI-26 exhibited strong binding (Ki 0.93±0.08 µM). Additionally, we found SB-FI-26 to act as a potent anti-nociceptive agent with mild anti-inflammatory activity in mice, which strongly supports our hypothesis that the inhibition of FABPs and subsequent elevation of anandamide is a promising new approach to drug discovery. Truxillic acids and their derivatives were also shown by others to have anti-inflammatory and anti-nociceptive effects in mice and to be the active component of Chinese a herbal medicine (Incarvillea sinensis) used to treat rheumatism and pain in humans. Our results provide a likely mechanism by which these compounds exert their effects.

Vorinostat increases carboplatin and paclitaxel activity in non-small cell lung cancer cells

We observed a 53% response rate in non‐small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 μM) inhibited growth by: 17% ± 7% in A549, 28% ± 6% in 128‐88T, 39% ± 8% in Calu1 and 41% ± 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 μM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128‐88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 μM vorinostat, 83% ± 10%; 5 μM carboplatin, 41% ± 11%; carboplatin/vorinostat, 8% ± 4%; 2 nM paclitaxel, 53% ± 11%; paclitaxel/vorinostat, 46% ± 21%. In A549 and 128‐88T, vorinostat potentiated carboplatin induction of gamma‐H2AX (a DNA damage marker) and increased α‐tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in α‐tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat‐mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

Quantitative Prediction of Fold Resistance for Inhibitors of EGFR

Clinical use of ATP-competitive inhibitors of the epidermal growth factor receptor (EGFR) kinase domain can lead to an acquired drug resistant mutant L858R&T790M which dramatically reduces binding affinity relative to a prevalent cancer causing mutation L858R. In this study, we have used molecular dynamics (MD) computer simulations, free energy calculations (MM-GBSA method), and per-residue footprint analysis to characterize binding of three inhibitors (erlotinib, gefitinib, and AEE788) with wildtype EGFR and three mutants. The goal is to characterize how variation in structure and energy correlate with changes in experimental activities and to deduce origins of drug resistance. For seven fold resistance values, each computed from the difference of two independent computer simulations, excellent agreement was obtained with available experimental data (r2 = 0.84). Importantly, the results correctly predict that affinity will increase as a result of L858R and decrease due to L858R&T790M. Per-residue analysis shows an increase in favorable packing at the site of the methionine mutation reaffirming that a steric clash hypothesis is unlikely; however, large losses in van der Waals, Coulombic, and H-bond interactions strongly suggest that resistance is not due solely to changes in affinity for the native substrate ATP as recently proposed. Instead, the present results indicate that drug resistance more likely involves disruption of favorable interactions, including a water-mediated H-bond network between the ligands and residues T854, T790, and Q791, which could have important implication for guiding rational design of inhibitors with improved resistance profiles.

Implementation and evaluation of a docking‐rescoring method using molecular footprint comparisons

A docking‐rescoring method, based on per‐residue van der Waals (VDW), electrostatic (ES), or hydrogen bond (HB) energies has been developed to aid discovery of ligands that have interaction signatures with a target (footprints) similar to that of a reference. Biologically useful references could include known drugs, inhibitors, substrates, transition states, or side‐chains that mediate protein‐protein interactions. Termed footprint similarity (FPS) score, the method, as implemented in the program DOCK, was validated and characterized using: (1) pose identification, (2) crossdocking, (3) enrichment, and (4) virtual screening. Improvements in pose identification (6–12%) were obtained using footprint‐based (FPSVDW+ES) vs. standard DOCK (DCEVDW+ES) scoring as evaluated on three large datasets (680–775 systems) from the SB2010 database. Enhanced pose identification was also observed using FPS (45.4% or 70.9%) compared with DCE (17.8%) methods to rank challenging crossdocking ensembles from carbonic anhydrase. Enrichment tests, for three representative systems, revealed FPSVDW+ES scoring yields significant early fold enrichment in the top 10% of ranked databases. For EGFR, top FPS poses are nicely accommodated in the molecular envelope defined by the reference in comparison with DCE, which yields distinct molecular weight bias toward larger molecules. Results from a representative virtual screen of ca. 1 million compounds additionally illustrate how ligands with footprints similar to a known inhibitor can readily be identified from within large commercially available databases. By providing an alternative way to rank ligand poses in a simple yet directed manner we anticipate that FPS scoring will be a useful tool for docking and structure‐based design.

Origins of Resistance to the HIVgp41 Viral Entry Inhibitor T20

Peptide T20, which targets the HIV protein gp41, represents the first approved member of the class of HIV drugs known as membrane fusion inhibitors. However, mechanisms which lead to resistance through clinical use of T20 are not well-understood because the structure of the bound complex remains undetermined. In this report, an atomic-level model of a T20-gp41 complex embedded in an explicit DOPC membrane was constructed, and molecular dynamics simulations, followed by binding energy analysis (MM-GBSA method), were performed to delineate structural and energetic features that contribute to drug resistance. Per-residue binding footprints for T20 with wild-type gp41 reveal strong intermolecular van der Waals, Coulombic, and H-bond interactions in striking agreement with clinically observed resistance patterns. In addition, seven deleterious gp41 point mutations (L33Q, L33S, G36V, I37K, V38E, Q40H, and Q40K) were simulated, and all correctly exhibited decreases in the level of binding, including the fact that L33Q and Q40K are most detrimental. Six of the seven simulations yield good quantitative agreement (r(2) = 0.72; N = 6) with available experimental fold resistance data. Results from energy decomposition, heat map analysis, and differential (mutant minus wild-type) footprinting indicate the following. (1) Mutations disrupt intermolecular H-bonding and reduce the level of favorable contact with gp41 at M19. (2) Charged mutations (I37K, Q40K, and V38E) lead to significant Coulombic changes that weaken favorable van der Waals interactions. (3) Q40K is more detrimental than I37K because of interaction differences with a polar/charged patch on T20 in the initial (wild-type) state. (4) Resistance for L33S versus L33Q likely involves side chain packing differences in the final (mutated) state. A valuable finding of the work involves identification of favorable interactions among the C-terminal end of T20 (WNWF motif), residues on gp41 (including the fusion peptide), and headgroups in the adjacent membrane. The results suggest a complete T20 binding site would contribute to a stable complex, which could help to explain why prior studies, which employed truncated gp41 constructs, reported that C-terminal T20 residues may not interact with gp41. A hypothesis resulting from this study is that peptides could be designed to increase the level of favorable contact with both the membrane and gp41 which would lead to enhanced activity.

Homologous ligands accommodated by discrete conformations of a buried cavity

Significance Many medicinal chemistry programs change ligands incrementally to explore protein binding and to optimize binding affinity. How a protein accommodates such a growing ligand series has received remarkably little structural attention. Here we investigate eight congeneric ligands that grow by single-methylene additions, determining their protein-bound structures by X-ray crystallography, to investigate how a protein accommodates these changes. Rather than changing conformation smoothly to complement the ever-larger ligands, the protein site adopts a few discrete conformations as it expands. Inspection of the few other homologous series in the Protein Data Bank suggests that such discrete conformational adaptations to ligand binding are common, and may be an important consideration in ligand design. Conformational change in protein–ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

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 identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation into the cytosols of motor neurons. In this work, high-throughput in silico screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogs of the lead compounds were synthesized and their potency examined. One of these analogs showed an enhanced activity than the lead compounds.