Demetri T. Moustakas

Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase

The hydrophobic effect—a rationalization of the insolubility of nonpolar molecules in water—is centrally important to biomolecular recognition. Despite extensive research devoted to the hydrophobic effect, its molecular mechanisms remain controversial, and there are still no reliably predictive models for its role in protein–ligand binding. Here we describe a particularly well-defined system of protein and ligands—carbonic anhydrase and a series of structurally homologous heterocyclic aromatic sulfonamides—that we use to characterize hydrophobic interactions thermodynamically and structurally. In binding to this structurally rigid protein, a set of ligands (also defined to be structurally rigid) shows the expected gain in binding free energy as hydrophobic surface area is added. Isothermal titration calorimetry demonstrates that enthalpy determines these increases in binding affinity, and that changes in the heat capacity of binding are negative. X-ray crystallography and molecular dynamics simulations are compatible with the proposal that the differences in binding between the homologous ligands stem from changes in the number and organization of water molecules localized in the active site in the bound complexes, rather than (or perhaps in addition to) release of structured water from the apposed hydrophobic surfaces. These results support the hypothesis that structured water molecules—including both the molecules of water displaced by the ligands and those reorganized upon ligand binding—determine the thermodynamics of binding of these ligands at the active site of the protein. Hydrophobic effects in various contexts have different structural and thermodynamic origins, although all may be manifestations of the differences in characteristics of bulk water and water close to hydrophobic surfaces.

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/.

Pharmacophore‐based molecular docking to account for ligand flexibility

Rapid computational mining of large 3D molecular databases is central to generating new drug leads. Accurate virtual screening of large 3D molecular databases requires consideration of the conformational flexibility of the ligand molecules. Ligand flexibility can be included without prohibitively increasing the search time by docking ensembles of precomputed conformers from a conformationally expanded database. A pharmacophore‐based docking method whereby conformers of the same or different molecules are overlaid by their largest 3D pharmacophore and simultaneously docked by partial matches to that pharmacophore is presented. The method is implemented in DOCK 4.0. Proteins 2003;51:172–188.

Fragment-based lead discovery and design.

Fragment-based lead discovery and design has and continues to show increasing promise in drug discovery. In this article, the current state of the art in terms of hot-spot characterization, fragment screening techniques, and fragment-based design is discussed. Three overall fragment-based lead generation strategies are explored and involve the chemical biology characterization of biological targets via fragment screening, fragment screening as a complementary approach to high-throughput screening of drug-like compounds, and direct fragment-based drug discovery, respectively. The evolution and development of fragment libraries is described. With an emphasis on computational approaches and the strategies applied at AstraZeneca, the review illustrates how integration of data from one regime can inform the design of experiments in the other, ultimately leading to the discovery of high quality chemical matter.

Dependence of Avidity on Linker Length for a Bivalent Ligand–Bivalent Receptor Model System

This paper describes a synthetic dimer of carbonic anhydrase, and a series of bivalent sulfonamide ligands with different lengths (25 to 69 Å between the ends of the fully extended ligands), as a model system to use in examining the binding of bivalent antibodies to antigens. Assays based on analytical ultracentrifugation and fluorescence binding indicate that this system forms cyclic, noncovalent complexes with a stoichiometry of one bivalent ligand to one dimer. This dimer binds the series of bivalent ligands with low picomolar avidities (K(d)(avidity) = 3-40 pM). A structurally analogous monovalent ligand binds to one active site of the dimer with K(d)(mono) = 16 nM. The bivalent association is thus significantly stronger (K(d)(mono)/K(d)(avidity) ranging from ~500 to 5000 unitless) than the monovalent association. We infer from these results, and by comparison of these results to previous studies, that bivalency in antibodies can lead to associations much tighter than monovalent associations (although the observed bivalent association is much weaker than predicted from the simplest level of theory: predicted K(d)(avidity) of ~0.002 pM and K(d)(mono)/K(d)(avidity) ~ 8 × 10(6) unitless).

Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase

The hydrophobic effect, the free-energetically favorable association of nonpolar solutes in water, makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different but structurally similar hydrophobic groups, aliphatic hydrocarbons and aliphatic fluorocarbons, and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H(2)NSO(2)C(6)H(4)-CONHCH(2)(CX(2))(n)CX(3), n = 0-4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of nonoptimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and entropic contributions to the hydrophobic effect in this representative system of protein and ligand: hydrophobic interactions, here, seem to comprise approximately equal contributions from enthalpy (plausibly from strengthening networks of hydrogen bonds among molecules of water) and entropy (from release of water from configurationally restricted positions).

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Preclinical pharmacokinetic/pharmacodynamic models to predict synergistic effects of co-administered anti-cancer agents

PurposePharmacokinetic/pharmacodynamic (PK/PD) models have been shown to be useful in predicting tumor growth rates in mouse xenografts. We applied novel PK/PD models to the published anticancer combination therapies of tumor growth inhibition to simulate synergistic changes in tumor growth rates. The parameters from the PK/PD model were further used to estimate clinical doses of the combination.MethodsA PK/PD model was built that linked the dosing regimen of a compound to the inhibition of tumor growth in mouse xenograft models. Two subsequent PK/PD models were developed to simulate the published tumor growth profiles of combination treatments. Model I predicts the tumor growth curve assuming that the effect of two anticancer drugs, AZD7762 and irinotecan, is synergistic when given in combination. Model II predicts the tumor growth curve assuming that the effect of co-administering flavopiridol and irinotecan is maximally synergistic when dosed at an optimal interval.ResultsModel I was able to account for the synergistic effects of AZD7762 following the administration of irinotecan. When Model II was applied to the antitumor activity of irinotecan and flavopiridol combination therapy, the modeling was able to reproduce the optimal dosing interval between administrations of the compounds. Furthermore, Model II was able to estimate the biologically active dose of flavopiridol recommended for phase II studies.ConclusionsThe timing of clinical combination therapy doses is often selected empirically. PK/PD models provide a theoretical structure useful in the design of the optimal clinical dose, frequency of administration and the optimal timing of administration between anticancer agents to maximize tumor suppression.

Evaluating Free Energies of Binding and Conservation of Crystallographic Waters Using SZMAP.

The SZMAP method computes binding free energies and the corresponding thermodynamic components for water molecules in the binding site of a protein structure [ SZMAP, 1.0.0 ; OpenEye Scientific Software Inc. : Santa Fe, NM, USA , 2011 ]. In this work, the ability of SZMAP to predict water structure and thermodynamic stability is examined for the X-ray crystal structures of a series of protein-ligand complexes. SZMAP results correlate with higher-level replica exchange thermodynamic integration double decoupling calculations of the absolute free energy of bound waters in the test set complexes. In addition, SZMAP calculations show good agreement with experimental data in terms of water conservation (across multiple crystal structures) and B-factors over a subset of the test set. In particular, the SZMAP neutral entropy difference term calculated at crystallographic water positions within each of the complex structures correlates well with whether that crystallographic water is conserved or displaceable. Furthermore, the calculated entropy of the water probe relative to the continuum shows a significant degree of correlation with the B-factors associated with the oxygen atoms of the water molecules. Taken together, these results indicate that SZMAP is capable of quantitatively predicting water positions and their energetics and is potentially a useful tool for determining which waters to attempt to displace, maintain, or build in through water-mediated interactions when evolving a lead series during a drug discovery program.

Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site.

The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 μM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.