Zoe Cournia

Structure-based virtual screening for drug discovery: principles, applications and recent advances.

Structure-based drug discovery (SBDD) is becoming an essential tool in assisting fast and cost-efficient lead discovery and optimization. The application of rational, structure-based drug design is proven to be more efficient than the traditional way of drug discovery since it aims to understand the molecular basis of a disease and utilizes the knowledge of the three-dimensional structure of the biological target in the process. In this review, we focus on the principles and applications of Virtual Screening (VS) within the context of SBDD and examine different procedures ranging from the initial stages of the process that include receptor and library pre-processing, to docking, scoring and post-processing of topscoring hits. Recent improvements in structure-based virtual screening (SBVS) efficiency through ensemble docking, induced fit and consensus docking are also discussed. The review highlights advances in the field within the framework of several success studies that have led to nM inhibition directly from VS and provides recent trends in library design as well as discusses limitations of the method. Applications of SBVS in the design of substrates for engineered proteins that enable the discovery of new metabolic and signal transduction pathways and the design of inhibitors of multifunctional proteins are also reviewed. Finally, we contribute two promising VS protocols recently developed by us that aim to increase inhibitor selectivity. In the first protocol, we describe the discovery of micromolar inhibitors through SBVS designed to inhibit the mutant H1047R PI3Kα kinase. Second, we discuss a strategy for the identification of selective binders for the RXRα nuclear receptor. In this protocol, a set of target structures is constructed for ensemble docking based on binding site shape characterization and clustering, aiming to enhance the hit rate of selective inhibitors for the desired protein target through the SBVS process.

Discovery of Human Macrophage Migration Inhibitory Factor (MIF)-CD74 Antagonists via Virtual Screening

Macrophage migration inhibitory factor (MIF) is a cytokine that is involved in the regulation of inflammation as well as cell proliferation and differentiation. Deactivation of MIF by antibodies or inhibition of MIF binding to its receptor, CD74, attenuates tumor growth and angiogenesis. To discover small-molecule inhibitors of MIFs biological activity, virtual screening was performed by docking 2.1 million compounds into the MIF tautomerase active site. After visual inspection of 1200 top-ranked MIF-ligand complexes, 26 possible inhibitors were selected and purchased and 23 of them were assayed. The in vitro binding assay for MIF with CD74 revealed that 11 of the compounds have inhibitory activity in the micromolar regime, including four compounds with IC(50) values below 5 microM. Inhibition of MIF tautomerase activity was also established for many of the compounds with IC(50) values as low as 0.5 microM; Michaelis-Menten analysis was performed for two cases and confirmed the competitive inhibition.

Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC(50) values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.

A molecular mechanics force field for biologically important sterols.

A parameterization has been performed of the biologically important sterols cholesterol, ergosterol, and lanosterol for the CHARMM27 all‐atom molecular mechanics force field. An automated parameterization method was used that involves fitting the potential to vibrational frequencies and eigenvectors derived from quantum‐chemical calculations. The partial charges were derived by fitting point charges to quantum‐chemically calculated electrostatic potentials. To model the dynamics of the hydroxyl groups of the sterols correctly, the parameter set was refined to reproduce the energy barrier for the rotation of the hydroxyl group around the carbon connected to the hydroxyl of each sterol. The frequency‐matching plots show good agreement between the CHARMM and quantum chemical normal modes. The parameters are tested in a molecular dynamics simulation of the cholesterol crystal structure. The experimental geometry and cell dimensions are well reproduced. The force field derived here is also useful for simulating other sterols such as the phytosterols sigmasterol, and campesterol, and a variety of steroids.

Therapeutic Effects of an Anti-Myc Drug on Mouse Pancreatic Cancer

BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is frequently driven by oncogenic KRAS(KRAS*) mutations. We developed a mouse model of KRAS*-induced PDA and, based on genetic results demonstrating that KRAS* tumorigenicity depends on Myc activity, we evaluated the therapeutic potential of an orally administered anti-Myc drug. METHODS We tested the efficacy of Mycro3, a small-molecule inhibitor of Myc-Max dimerization, in the treatment of mouse PDA (n = 9) and also of xenografts of human pancreatic cancer cell lines (NOD/SCID mice, n = 3-12). Tumor responses to the drug were evaluated by PET/CT imaging, and histological, immunohistochemical, molecular and microarray analyses. The Students t test was used for differences between groups. All statistical tests were two-sided. RESULTS Transgenic overexpression of KRAS* in the pancreas resulted in pancreatic intraepithelial neoplasia in two-week old mice, which developed invasive PDA a week later and became moribund at one month. However, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas. We then treated moribund, PDA-bearing mice daily with the Mycro3 Myc-inhibitor. The mice survived until killed at two months. PET/CT image analysis (n = 5) demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation (treated/untreated proliferation index ratio: 0.29, P < .001, n = 3, each group). Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice (n = 12) carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells (eg, mean tumor weight ± SD of treated heterotopic xenografts vs vehicle-treated controls: 15.2±5.8 mg vs 230.2±43.9 mg, P < .001). CONCLUSION These results provide strong justification for eventual clinical evaluation of anti-Myc drugs as potential chemotherapeutic agents for the treatment of PDA.

AFMM: A molecular mechanics force field vibrational parametrization program☆

AFMM (Automated Frequency Matching Method) is a program package for molecular mechanics force field parametrization. The method used fits the molecular mechanics potential function to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The program optimizes an initial parameter set (either pre-existing or using chemically-reasonable estimation) by iteratively changing them until the optimal fit with the reference set is obtained. By implementing a Monte Carlo-like algorithm to vary the parameters, the tedious task of manual parametrization is replaced by an efficient automated procedure. The program is best suited for optimization of small rigid molecules in a well-defined energy minimum, for which the harmonic approximation to the energy surface is appropriate for describing the intra-molecular degrees of freedom.

Membrane Protein Structure, Function and Dynamics: A Perspective from Experiments and Theory

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.

Free Energy Calculations Reveal the Origin of Binding Preference for Aminoadamantane Blockers of Influenza A/M2TM Pore.

Aminoadamantane derivatives, such as amantadine and rimantadine, have been reported to block the M2 membrane protein of influenza A virus (A/M2TM), but their use has been discontinued due to reported resistance in humans. Understanding the mechanism of action of amantadine derivatives could assist the development of novel potent inhibitors that overcome A/M2TM resistance. Here, we use Free Energy Perturbation calculations coupled with Molecular Dynamics simulations (FEP/MD) to rationalize the thermodynamic origin of binding preference of several aminoadamantane derivatives inside the A/M2TM pore. Our results demonstrate that apart from crucial protein-ligand intermolecular interactions, the flexibility of the protein, the water network around the ligand, and the desolvation free energy penalty to transfer the ligand from the aqueous environment to the transmembrane region are key elements for the binding preference of these compounds and thus for lead optimization. The high correlation of the FEP/MD results with available experimental data (R(2) = 0.85) demonstrates that this methodology holds predictive value and can be used to guide the optimization of drug candidates binding to membrane proteins.

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT1 receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartans pathway towards the AT1 receptor.