Diane Joseph-McCarthy

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.

Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of 4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Triad of polar residues implicated in pH specificity of acidic mammalian chitinase

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X‐ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the β‐anomer of the substrate. A triad of polar residues in the second‐shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.

Use of the multiple copy simultaneous search (MCSS) method to design a new class of picornavirus capsid binding drugs

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analyzed and selected minima are connected with (CH2) n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented. Proteins 29:32–58, 1997.

Automated generation of MCSS‐derived pharmacophoric DOCK site points for searching multiconformation databases

All docking methods employ some sort of heuristic to orient the ligand molecules into the binding site of the target structure. An automated method, MCSS2SPTS, for generating chemically labeled site points for docking is presented. MCSS2SPTS employs the program Multiple Copy Simultaneous Search (MCSS) to determine target‐based theoretical pharmacophores. More specifically, chemically labeled site points are automatically extracted from selected low‐energy functional‐group minima and clustered together. These pharmacophoric site points can then be directly matched to the pharmacophoric features of database molecules with the use of either DOCK or PhDOCK to place the small molecules into the binding site. Several examples of the ability of MCSS2SPTS to reproduce the three‐dimensional pharmacophoric features of ligands from known ligand–protein complex structures are discussed. In addition, a site‐point set calculated for one human immunodeficiency virus 1 (HIV1) protease structure is used with PhDOCK to dock a set of HIV1 protease ligands; the docked poses are compared to the corresponding complex structures of the ligands. Finally, the use of an MCSS2SPTS‐derived site‐point set for acyl carrier protein synthase is compared to the use of atomic positions from a bound ligand as site points for a large‐scale DOCK search. In general, MCSS2SPTS‐generated site points focus the search on the more relevant areas and thereby allow for more effective sampling of the target site. Proteins 2003;51:189–202.

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.

Ensemble-based docking using biased molecular dynamics.

Proteins are dynamic molecules, and understanding their movements, especially as they relate to molecular recognition and protein-ligand interactions, poses a significant challenge to structure-based drug discovery. In most instances, protein flexibility is underrepresented in computer-aided drug design due to uncertainties on how it should be accurately modeled as well as the computational cost associated with attempting to incorporate flexibility in the calculations. One approach that aims to address these issues is ensemble-based docking. With this technique, ligands are docked to an ensemble of rigid protein conformations. Molecular dynamics (MD) simulations can be used to generate the ensemble of protein conformations for the subsequent docking. Here we present a novel approach that uses biased-MD simulations to generate the docking ensemble. The MD simulations are biased toward an initial protein-ligand X-ray complex structure. The biasing maintains some of the original crystallographic pocket-ligand information and thereby enhances sampling of the more relevant conformational space of the protein. Resulting trajectories are clustered to select a representative set of protein conformations, and ligands are docked to that reduced set of conformations. Cross-docking to this ensemble and then selecting the lowest scoring pose enables reliable identification of the correct binding mode. Various levels of biasing are investigated, and the method is validated for cyclin-dependent kinase 2 and factor Xa.

In Vivo Validation of Thymidylate Kinase (TMK) with a Rationally Designed, Selective Antibacterial Compound

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.

Identification and Characterization of Acidic Mammalian Chitinase Inhibitors

Acidic mammalian chitinase (AMCase) is a member of the glycosyl hydrolase 18 family (EC 3.2.1.14) that has been implicated in the pathophysiology of allergic airway disease such as asthma. Small molecule inhibitors of AMCase were identified using a combination of high-throughput screening, fragment screening, and virtual screening techniques and characterized by enzyme inhibition and NMR and Biacore binding experiments. X-ray structures of the inhibitors in complex with AMCase revealed that the larger more potent HTS hits, e.g. 5-(4-(2-(4-bromophenoxy)ethyl)piperazine-1-yl)-1H-1,2,4-triazol-3-amine 1, spanned from the active site pocket to a hydrophobic pocket. Smaller fragments identified by FBS occupy both these pockets independently and suggest potential strategies for linking fragments. Compound 1 is a 200 nM AMCase inhibitor which reduced AMCase enzymatic activity in the bronchoalveolar lavage fluid in allergen-challenged mice after oral dosing.