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Computational Research in Molecular Chemistry

Abstracts of Articles in 2014

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  1. Trapping the dynamic acyl carrier protein in fatty acid biosynthesis
  2. Exploring the role of receptor flexibility in structure-based drug discovery.
  3. Free Energy Landscape of G-Protein Coupled Receptors
  4. The Marine Cyanobacterial Metabolite Gallinamide A is a Potent and Selective Inhibitor of Human Cathepsin
  5. Discovery of Novel Inhibitors of HIV-1 Reverse Transcriptase through Virtual Screening of Experimental and Theoretical Ensembles.
  6. Drug screening strategy for human membrane proteins: From NMR protein backbone structure to in silica- and NMR-screened hits
  7. Structural and computational studies of the Staphylococcus aureus SrtB-substrate complex reveal a substrate-stabilized oxyanion hole
  8. Identification and Validation of Modulators of Epac Activity: Structure-function Implications for Epac Activation and Inhibition.
  9. Variational Implicit Solvation with Poisson–Boltzmann Theory.
  10. A model study of sequential enzyme reactions and electrostatic channeling
  11. Dipeptide aggregation in aqueous solution from fixed point-charge force fields.
  12. Predicting the influence of long-range molecular interactions on macroscopic-scale diffusion by homogenization of the Smoluchowski equation
  13. Unconventional Coordination Chemistry by Metal Chelating Fragments in a Metalloprotein Active Site
  14. Accelerated Adaptive Integration Method
  15. Molecular dynamics simulation study of conformational changes of Transcript Factor TFIIS during RNA Polymerase II transcriptional arrest and reactivation
  16. Improved Reweighting of Accelerated Molecular Dynamics Simulations for Free Energy Calculation
  17. Undecaprenyl Diphosphate Synthase Inhibitors: Antibacterial Drug Leads
  18. Taxodione and Arenarone Inhibit Farnesyl Diphosphate Synthase by Binding to the Isopentenyl Diphosphate Site
  19. PKA phosphorylation of cardiac troponin I modulates activation and relaxation kinetics of ventricular myofibrils.
  20. Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics.
  21. Trypsinogen activation as observed in accelerated molecular dynamics simulations.
  22. Protocol utilizing constant pH molecular dynamics simulations to compute pH-dependent binding free energies.
  23. Exploring the Influence of the Protein Environment on Metal-Binding Pharmacophores.
  24. Celastrol inhibits Plasmodium falciparum enoyl-acyl carrier protein reductase.
  25. Allosteric Inhibition of Epac: Computational Modeling and Experimental Validation to Identify Allosteric Sites and Inhibitors.
  26. Use of Broken-Symmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition.
  27. Electrostatic channeling in P.falciparum DHFR-TS: Brownian dynamics and continuum modeling.
Trapping the dynamic acyl carrier protein in fatty acid biosynthesisNguyen, C., R.W. Haushalter, D.J. Lee, P.R.L. Markwick, J. Bruegger, G. Caldara-Festin, K. Finzel, D.R. Jackson, F. Ishikawa, B.A. O'Dowd, J.A. McCammon, S.J. Opella, S.C. Tsai, M.D. Burkart.Nature, 505(7483):427-31 (2014)    [PubMed 24362570]
Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway. The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 Å crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.
Exploring the role of receptor flexibility in structure-based drug discovery.Feixas, F., S. Lindert, W. Sinko, J.A. McCammon.Biophys. Chem. 186, 31-45 (2014).    [PubMed 24332165]
The proper understanding of biomolecular recognitionmechanisms that take place in a drug target is of paramount importance to improve the efficiency of drug discovery and development. The intrinsic dynamic character of proteins has a strong influence on biomolecular recognition mechanisms andmodels such as conformational selection have beenwidely used to account for this dynamic association process. However, conformational changes occurring in the receptor prior and upon associationwith other molecules are diverse and not obvious to predictwhen only a few structures of the receptor are available. In view of the prominent role of protein flexibility in ligand binding and its implications for drug discovery, it is of great interest to identify receptor conformations that play a major role in biomolecular recognition before starting rational drug design efforts. In this review, we discuss a number of recent advances in computer-aided drug discovery techniques that have been proposed to incorporate receptor flexibility into structure-based drug design. The allowance for receptor flexibility provided by computational techniques such as molecular dynamics simulations or enhanced sampling techniques helps to improve the accuracy of methods used to estimate binding affinities and, thus, such methods can contribute to the discovery of novel drug leads.
Free Energy Landscape of G-Protein Coupled ReceptorsMiao, Y., S.E. Nichols, J.A. McCammon.Explored by Accelerated Molecular Dynamics. Phys. Chem. Chem. Phys. 2014; 16(14):6398-406.    [PubMed 24445284]
G-protein coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. They are known to adopt multiple conformational states (e.g., inactive, intermediate and active) during their modulation of various cell signaling pathways. Here, the free energy landscape of GPCRs is explored using accelerated molecular dynamics (aMD) simulations as demonstrated on the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes. Free energy profiles of important structural motifs that undergo conformational transitions upon GPCR activation and allosteric signaling are analyzed in detail, including the Arg(3.50)-Glu(6.30) ionic lock, the Trp(6.48) toggle switch and the hydrogen interactions between Tyr(5.58)-Tyr(7.53).
The Marine Cyanobacterial Metabolite Gallinamide A is a Potent and Selective Inhibitor of Human CathepsinMiller, B., A.J. Friedman, H. Choi, J.A. McCammon, V. Hook, W.H. Gerwick.L. J. Nat. Prod. 2014, 77 (1), pp 92–99    
A number of marine natural products are potent inhibitors of proteases, an important drug target class in human diseases. Hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Herein, we have shown that gallinamide A potently and selectively inhibits the human cysteine protease cathepsin L. With 30 min of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9000 ± 260 Mi–1 s–1. Preincubation–dilution and activity-probe experiments revealed an irreversible mode of inhibition, and comparative IC50 values display a 28- to 320-fold greater selectivity toward cathepsin L than closely related human cysteine cathepsin V or B. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enamide of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.
Discovery of Novel Inhibitors of HIV-1 Reverse Transcriptase through Virtual Screening of Experimental and Theoretical Ensembles.Ivetac, A., S.E. Swift, P.L. Boyer, A. Diaz, J. Naughton, J.A.T. Young, S.H. Hughes, J.A. McCammon.Chem. Biol. Drug Disc., Vol. 83,(5) pp. 521-531(2014).    [PubMed 24405985]
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are potent anti-HIV chemotherapeutics. Although there are FDA-approved NNRTIs, challenges such as the development of resistance have limited their utility. Here, we describe the identification of novel NNRTIs through a combination of computational and experimental approaches. Based on the known plasticity of the NNRTI binding pocket (NNIBP), we adopted an ensemble-based virtual screening strategy: coupling receptor conformations from 10 X-ray crystal structures with 120 snapshots from a total of 480 ns of molecular dynamics (MD) trajectories. A screening library of 2864 National Cancer Institute (NCI) compounds was built and docked against the ensembles in a hierarchical fashion. Sixteen diverse compounds were tested for their ability to block HIV infection in human tissue cultures using a luciferase-based reporter assay. Three promising compounds were further characterized, using a HIV-1 RT-based polymerase assay, to determine the specific mechanism of inhibition. We found that 2 of the three compounds inhibited the polymerase activity of RT (with potency similar to the positive control, the FDA-approved drug nevirapine). Through a computational approach, we were able to discover two compounds which inhibit HIV replication and block the activity of RT, thus offering the potential for optimization into mature inhibitors.
Drug screening strategy for human membrane proteins: From NMR protein backbone structure to in silica- and NMR-screened hitsLindert, S., I. Maslennikov, E.J. Chiu, L.C Pierce, J.A. McCammon, S. ChoeBiochem Biophys Res Commun, 445(4):724-733 (2014)    [PubMed 24525125]
About 8000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such “omics” approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B
Structural and computational studies of the Staphylococcus aureus SrtB-substrate complex reveal a substrate-stabilized oxyanion holeJacobitz, A.W., J. Wereszczynski, S.W. Yi, B.R. Amer, G.L. Huang, A.V. Nguyen, M.R. Sawaya, M. Jung, J.A. McCammon, R.T. Clubb.J. Biol. Chem. 289, 8891-8902 (2014)    [PubMed 24519933]
Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria which frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C-terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus Sortase B enzyme (SrtB) in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics (MD) simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form an oxyanion hole in SrtB that stabilizes high-energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. MD simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.
Identification and Validation of Modulators of Epac Activity: Structure-function Implications for Epac Activation and Inhibition.Loren M. Brown, Kathleen E. Rogers, J. Andrew McCammon and Paul A. InselJ. Biol. Chem. 289, 8217-8230 (2014)    [PubMed 24497631]
The signaling molecule cAMP primarily mediates its effects by activating PKA and/or Epac. Epac has been implicated in many responses in cells but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a BRET-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-CPT-cAMP), and super-agonists (8-CPT-2′-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g., Rp-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.
Variational Implicit Solvation with Poisson–Boltzmann Theory.Zhou, S., L.-T. Cheng, J. Dzubiella, B. Li, J.A. McCammonJ. Chem. Theory Comput. 10, 1454-1467 (2014)    
We incorporate the Poisson–Boltzmann (PB) theory of electrostatics into our variational implicit-solvent model (VISM) for the solvation of charged molecules in an aqueous solvent. In order to numerically relax the VISM free-energy functional by our level-set method, we develop highly accurate methods for solving the dielectric PB equation and for computing the dielectric boundary force. We also apply our VISM-PB theory to analyze the solvent potentials of mean force and the effect of charges on the hydrophobic hydration for some selected molecular systems. These include some single ions, two charged particles, two charged plates, and the host–guest system Cucurbit[7]uril and Bicyclo[2.2.2]octane. Our computational results show that VISM with PB theory can capture well the sensitive response of capillary evaporation to the charge in hydrophobic confinement and the polymodal hydration behavior and can provide accurate estimates of binding affinity of the host–guest system. We finally discuss several issues for further improvement of VISM.
A model study of sequential enzyme reactions and electrostatic channelingEun, C., P.M. Kekenes-Huskey, V.T. Metzger, J.A. McCammon.J. Chem. Phys. 140, 105101 (2014);    [PubMed 24628210]
We study models of two sequential enzyme-catalyzed reactions as a basic functional building block for coupled biochemical networks. We investigate the influence of enzyme distributions and long-range molecular interactions on reaction kinetics, which have been exploited in biological systems to maximize metabolic efficiency and signaling effects. Specifically, we examine how the maximal rate of product generation in a series of sequential reactions is dependent on the enzyme distribution and the electrostatic composition of its participant enzymes and substrates. We find that close proximity between enzymes does not guarantee optimal reaction rates, as the benefit of decreasing enzyme separation is countered by the volume excluded by adjacent enzymes. We further quantify the extent to which the electrostatic potential increases the efficiency of transferring substrate between enzymes, which supports the existence of electrostatic channeling in nature. Here, a major finding is that the role of attractive electrostatic interactions in confining intermediate substrates in the vicinity of the enzymes can contribute more to net reactive throughput than the directional properties of the electrostatic fields. These findings shed light on the interplay of long-range interactions and enzyme distributions in coupled enzyme-catalyzed reactions, and their influence on signaling in biological systems.
Dipeptide aggregation in aqueous solution from fixed point-charge force fields.Goetz, A., D. Bucher, S. Lindert, J.A. McCammon.J. Chem. Theory Comput., 10 (4), pp 1631-1637 (2014)    
The description of aggregation processes with molecular dynamics simulations is a playground for testing biomolecular force fields, including a new generation of force fields that explicitly describe electronic polarization. In this work we study a system consisting of 50 glycyl-L-alanine (Gly-Ala) dipeptides in solution with 1,001 water molecules. Neutron diffraction experiments have shown that at this concentration Gly-Ala aggregates into large clusters. However, general-purpose force fields in combination with established water models can fail to correctly describe this aggregation process, highlighting important deficiencies in how solute-solute and solute-solvent interactions are parameterized in these force fields. We found that even for the fully polarizable AMOEBA force field, the degree of association is considerably underestimated. Instead, a fixed point-charge approach based on the newly developed IPolQ scheme [Cerutti et al., J. Phys. Chem. 117 (2013) 2328] allows for the correct modeling of the dipeptide aggregation in aqueous solution. This result should stimulate interest in novel fitting schemes that aim to improve the description of the solvent polarization effect within both explicitly polarizable and fixed point-charge frameworks.
Predicting the influence of long-range molecular interactions on macroscopic-scale diffusion by homogenization of the Smoluchowski equationKekenes-Huskey, P.M., A.K. Gillette, J.A. McCammon.J. Chem. Phys. 140 (7) 174106 (2014).    
The macroscopic diffusion constant for a charged diffuser is in part dependent on (1) the volume excluded by solute "obstacles" and (2) long-range interactions between those obstacles and the diffuser. Increasing excluded volume reduces transport of the diffuser, while long-range interactions can either increase or decrease diffusivity, depending on the nature of the potential. We previously demonstrated [P. M. Kekenes-Huskey et al., Biophys. J. 105, 2130 (2013)] using homogenization theory that the configuration of molecular-scale obstacles can both hinder diffusion and induce diffusional anisotropy for small ions. As the density of molecular obstacles increases, van der Waals (vdW) and electrostatic interactions between obstacle and a diffuser become significant and can strongly influence the latter's diffusivity, which was neglected in our original model. Here, we extend this methodology to include a fixed (time-independent) potential of mean force, through homogenization of the Smoluchowski equation. We consider the diffusion of ions in crowded, hydrophilic environments at physiological ionic strengths and find that electrostatic and vdW interactions can enhance or depress effective diffusion rates for attractive or repulsive forces, respectively. Additionally, we show that the observed diffusion rate may be reduced independent of non-specific electrostatic and vdW interactions by treating obstacles that exhibit specific binding interactions as "buffers" that absorb free diffusers. Finally, we demonstrate that effective diffusion rates are sensitive to distribution of surface charge on a globular protein, Troponin C, suggesting that the use of molecular structures with atomistic-scale resolution can account for electrostatic influences on substrate transport. This approach offers new insight into the influence of molecular-scale, long-range interactions on transport of charged species, particularly for diffusion-influenced signaling events occurring in crowded cellular environments.
Unconventional Coordination Chemistry by Metal Chelating Fragments in a Metalloprotein Active SiteMartin D.P., Blachly P.G., Marts A.R., Woodruff T.M., de Oliveira C.A., McCammon J.A., Tierney D.L., Cohen S.M.J Am Chem Soc. 136 (14), pp 5400-5406 (2014)    [PubMed 24635441]
The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn2+ ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.
Accelerated Adaptive Integration MethodKaus, J.W., M. Arrar, J.A. McCammonJ. Phys. Chem. B,118 (19), pp 5109-5118 (2014)    
Conformational changes that occur upon ligand binding may be too slow to observe on the time scales routinely accessible using molecular dynamics simulations. The adaptive integration method (AIM) leverages the notion that when a ligand is either fully coupled or decoupled, according to ?, barrier heights may change, making some conformational transitions more accessible at certain ? values. AIM adaptively changes the value of ? in a single simulation so that conformations sampled at one value of ? seed the conformational space sampled at another ? value. Adapting the value of ? throughout a simulation, however, does not resolve issues in sampling when barriers remain high regardless of the ? value. In this work, we introduce a new method, called Accelerated AIM (AcclAIM), in which the potential energy function is flattened at intermediate values of ?, promoting the exploration of conformational space as the ligand is decoupled from its receptor. We show, with both a simple model system (Bromocyclohexane) and the more complex biomolecule Thrombin, that AcclAIM is a promising approach to overcome high barriers in the calculation of free energies, without the need for any statistical reweighting or additional processors.
Molecular dynamics simulation study of conformational changes of Transcript Factor TFIIS during RNA Polymerase II transcriptional arrest and reactivationEun, C., J.M. Ortiz-Sánchez, L. Da, D. Wang, J.A. McCammonPLoS ONE, Volume 9, Issue 5, e97975 (2014)    
Transcription factor IIS (TFIIS) is a protein known for catalyzing the cleavage reaction of the 3?-end of backtracked RNA transcript, allowing RNA polymerase II (Pol II) to reactivate the transcription process from the arrested state. Recent structural studies have provided a molecular basis of protein-protein interaction between TFIIS and Pol II. However, the detailed dynamic conformational changes of TFIIS upon binding to Pol II and the related thermodynamic information are largely unknown. Here we use computational approaches to investigate the conformational space of TFIIS in the Pol II-bound and Pol II-free (unbound) states. Our results reveal two distinct conformations of TFIIS: the closed and the open forms. The closed form is dominant in the Pol II-free (unbound) state of TFIIS, whereas the open form is favorable in the Pol II-bound state. Furthermore, we discuss the free energy difference involved in the conformational changes between the two forms in the presence or absence of Pol II. Additionally, our analysis indicates that hydrophobic interactions and the protein-protein interactions between TFIIS and Pol II are crucial for inducing the conformational changes of TFIIS. Our results provide novel insights into the functional interplay between Pol II and TFIIS as well as mechanism of reactivation of Pol II transcription by TFIIS.
Improved Reweighting of Accelerated Molecular Dynamics Simulations for Free Energy CalculationMiao, Y., W. Sinko, L. Pierce, D. Bucher, R.C. Walker, J.A. McCammonJ. Med. Chem. 10 (7), pp 2677-2689 (2014)    
Accelerated molecular dynamics (aMD) simulations greatly improve the efficiency of conventional molecular dynamics (cMD) for sampling biomolecular conformations, but they require proper reweighting for free energy calculation. In this work, we systematically compare the accuracy of different reweighting algorithms including the exponential average, Maclaurin series, and cumulant expansion on three model systems: alanine dipeptide, chignolin, and Trp-cage. Exponential average reweighting can recover the original free energy profiles easily only when the distribution of the boost potential is narrow (e.g., the range ?20kBT) as found in dihedral-boost aMD simulation of alanine dipeptide. In dual-boost aMD simulations of the studied systems, exponential average generally leads to high energetic fluctuations, largely due to the fact that the Boltzmann reweighting factors are dominated by a very few high boost potential frames. In comparison, reweighting based on Maclaurin series expansion (equivalent to cumulant expansion on the first order) greatly suppresses the energetic noise but often gives incorrect energy minimum positions and significant errors at the energy barriers 2`3kBT). Finally, reweighting using cumulant expansion to the second order is able to recover the most accurate free energy profiles within statistical errors of kBT, particularly when the distribution of the boost potential exhibits low anharmonicity (i.e., near-Gaussian distribution), and should be of wide applicability. A toolkit of Python scripts for aMD reweighting [PyReweighting] is distributed free of charge at
Undecaprenyl Diphosphate Synthase Inhibitors: Antibacterial Drug LeadsSinko, W., Y. Wang, W. Zhu, Y. Zhang, F. Feixas, C. Cox, D. Mitchell, E. Oldfield, J.A. McCammonJ. Med. Chem. 57(13), pp 5693-5701 (2014)    
There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ~2 ?M, Ki ~300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis and a vancomycin-resistant Enterococcus spp. with MIC or IC50 values in the 0.25-4 ?g/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug Epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.
Taxodione and Arenarone Inhibit Farnesyl Diphosphate Synthase by Binding to the Isopentenyl Diphosphate SiteLiu, Y.L., S. Lindert, W. Zhu, K. Wang, J.A. McCammon, E. OldfieldProc. Natl. Acad. USA published online 2014 June 24; 111 (25): E2530-E2539 (2014)    
We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC50 versus TbFPPS ca. 20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC50 values in the range of ca. 1-3 µM (as compared with ca. 0.5 µM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg(2+) to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (Tm), quite different to the large increases in δTm seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads.
PKA phosphorylation of cardiac troponin I modulates activation and relaxation kinetics of ventricular myofibrils.Rao, V., Y. Cheng, S. Lindert, P. Kekenes-Huskey, D. Wang, L. Oxenford, A.D. McCulloch, J.A. McCammon, P. Arzberger, M. Regnier.Biophys. J. Vol. 107, Issue 5, 1196-1204 (2014)    
Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca(2+) levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca(2+) concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca(2+) activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca(2+), and reduced the Ca(2+) sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca(2+)-cTn binding affinity and C-I interaction were monitored. Ca(2+) binding to cTn (ΔpCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca(2+). These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.
Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics.Cheng, Y., S. Lindert, P. Kekenes-Huskey, V. Rao, R. Solaro, P.R Rosevear, R.E. Amaro, A. McCulloch, J.A. McCammon, M. Regnier.Biophys. J. 107, 1675-1685 (2014).    [PubMed 25296321]
During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1-40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.
Trypsinogen activation as observed in accelerated molecular dynamics simulations.Boechi, L., L. Pierce, E.A. Komives. J.A. McCammon.Protein Sci., Volume 23, Issue 11, 1550-1558 (2014)    
Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N-terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N-terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.
Protocol utilizing constant pH molecular dynamics simulations to compute pH-dependent binding free energies.Kim, M.O., P.G. Blachly, J. Kaus, J.A. McCammon.J. Phys. Chem. B 119 (3), pp 861-872 (2014). (Bill Jorgensen Special Issue)    
In protein-ligand binding, the electrostatic environments of the two binding partners may vary significantly in bound and unbound states, which may lead to protonation changes upon binding. In cases where ligand binding results in a net uptake or release of protons, the free energy of binding is pH-dependent. Nevertheless, conventional free energy calculations and molecular docking protocols typically do not rigorously account for changes in protonation that may occur upon ligand binding. To address these shortcomings, we present a simple methodology based on Wyman's binding polynomial formalism to account for the pH dependence of binding free energies and demonstrate its use on cucurbit[7]uril (CB[7]) host-guest systems. Using constant pH molecular dynamics and a reference binding free energy that is taken either from experiment or from thermodynamic integration computations, the pH-dependent binding free energy is determined. This computational protocol accurately captures the large pKa shifts observed experimentally upon CB[7]:guest association and reproduces experimental binding free energies at different levels of pH. We show that incorrect assignment of fixed protonation states in free energy computations can give errors of > 2 kcal/mol in these host-guest systems. Use of the methods presented here avoids such errors, thus suggesting their utility in computing proton-linked binding free energies for protein-ligand complexes.
Exploring the Influence of the Protein Environment on Metal-Binding Pharmacophores.Martin, D.P, P.G. Blachly, J.A. McCammon, S.M. Cohen.J. Med. Chem. 57, 7126-7135 (2014).    
The binding of a series of metal-binding pharmacophores (MBPs) related to the ligand 1-hydroxypyridine-2-(1H)-thione (1,2-HOPTO) in the active site of human carbonic anhydrase II (hCAII) has been investigated. The presence and/or position of a single methyl substituent drastically alters inhibitor potency and can result in coordination modes not observed in small-molecule model complexes. It is shown that this unexpected binding mode is the result of a steric clash between the methyl group and a highly ordered water network in the active site that is further stabilized by the formation of a hydrogen bond and favorable hydrophobic contacts. The affinity of MBPs is dependent on a large number of factors including donor atom identity, orientation, electrostatics, and van der Waals interactions. These results suggest that metal coordination by metalloenzyme inhibitors is a malleable interaction and that it is thus more appropriate to consider the metal-binding motif of these inhibitors as a pharmacophore rather than a "chelator". The rational design of inhibitors targeting metalloenzymes will benefit greatly from a deeper understanding of the interplay between the variety of forces governing the binding of MBPs to active site metal ions.
Celastrol inhibits Plasmodium falciparum enoyl-acyl carrier protein reductase.Tallorin, L.C., J.D. Durrant, Q.G. Nguyen, J.A. McCammon, M.D. Burkart.Bioorg. Med. Chem. 22(21), 6053-6061 (2014).    
Enoyl-acyl carrier protein reductase (ENR), a critical enzyme in type II fatty acid biosynthesis, is a promising target for drug discovery against hepatocyte-stage Plasmodium falciparum. In order to identify PfENR-specific inhibitors, we docked 70 FDA-approved, bioactive, and/or natural product small molecules known to inhibit the growth of whole-cell blood-stage P. falciparum into several PfENR crystallographic structures. Subsequent in vitro activity assays identified a noncompetitive low-micromolar PfENR inhibitor, celastrol, from this set of compounds.
Allosteric Inhibition of Epac: Computational Modeling and Experimental Validation to Identify Allosteric Sites and Inhibitors.Brown, L.M., K.E. Rogers, N. Aroonsakool, J.A. McCammon, P.A. Insel.J. Biol. Chem. 289: 29148-29157 (2014).    
Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. We found that the conserved hinge region of the cyclic nucleotide binding domain (CNBD) of Epac1 is a potentially druggable region of the protein. Using a BRET-based assay (CAMYEL), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the compound's ability to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that binds to the hinge region of the CNBD of Epac1 and Epac2 to prevent their activation by cAMP.
Use of Broken-Symmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition.Blachly, P.G., G.M. Sandala, D.A. Giammona, T. Liu, D. Bashford, J.A. McCammon, L. Noodleman.J. Chem. Theory Comp. 10, 3871-3884 (2014).    [PubMed 25221444]
With current therapies becoming less efficacious due to increased drug resistance, new inhibitors of both bacterial and malarial targets are desperately needed. The recently discovered methylerythritol phosphate (MEP) pathway for isoprenoid synthesis provides novel targets for the development of such drugs. Particular attention has focused on the IspH protein, the final enzyme in the MEP pathway, which uses its [4Fe-4S] cluster to catalyze the formation of the isoprenoid precursors IPP and DMAPP from HMBPP. IspH catalysis is achieved via a 2e -/2H+ reductive dehydroxylation of HMBPP; the mechanism by which catalysis is achieved, however, is highly controversial. The work presented herein provides the first step in assessing different routes to catalysis by using computational methods. By performing broken-symmetry density functional theory (BS-DFT) calculations that employ both the conductor-like screening solvation model (DFT/COSMO) and a finite-difference Poisson-Boltzmann self-consistent reaction field methodology (DFT/SCRF), we evaluate geometries, energies, and Mössbauer signatures of the different protonation states that may exist in the oxidized state of the IspH catalytic cycle. From DFT/SCRF computations performed on the oxidized state, we find a state where the substrate, HMBPP, coordinates the apical iron in the [4Fe-4S] cluster as an alcohol group (ROH) to be one of two, isoenergetic, lowest-energy states. In this state, the HMBPP pyrophosphate moiety and an adjacent glutamate residue (E126) are both fully deprotonated, making the active site highly anionic. Our findings that this low-energy state also matches the experimental geometry of the active site and that its computed isomer shifts agree with experiment validate the use of the DFT/SCRF method to assess relative energies along the IspH reaction pathway. Additional studies of IspH catalytic intermediates are currently being pursued.
Electrostatic channeling in P.falciparum DHFR-TS: Brownian dynamics and continuum modeling.Metzger, V.T., C. Eun, P.M. Kekenes-Huskey3, G. Huber, J.A. McCammon.Biophys. J. Volume 107 (10):2394-402 (2014).    
We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (?15-25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.
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