Rapid, Accurate, Ranking of Protein-Ligand Binding Affinities with VM2, the Second-Generation Mining Minima Method.

The structure-based technologies most widely used to rank the affinities of candidate small molecule drugs for proteins range from faster but less reliable docking methods to slower but more accurate explicit solvent free energy methods. In recent years, we have advanced another technology, which is called mining minima because it "mines" out the main contributions to the chemical potentials of the free and bound molecular species by identifying and characterizing their main local energy minima. The present study provides systematic benchmarks of the accuracy and computational speed of mining minima, as implemented in the VeraChem Mining Minima Generation 2 (VM2) code, across two well-regarded protein-ligand benchmark data sets, for which there are already benchmark data for docking, free energy, and other computational methods. A core result is that VM2's accuracy approaches that of explicit solvent free energy methods at a far lower computational cost. In finer-grained analyses, we also examine the influence of various run settings, such as the treatment of crystallographic water molecules, on the accuracy, and define the costs in time and dollars of representative runs on Amazon Web Services (AWS) compute instances with various CPU and GPU combinations. We also use the benchmark data to determine the importance of VM2's correction from generalized Born to finite-difference Poisson-Boltzmann results for each energy well and find that this correction affords a remarkably consistent improvement in accuracy at a modest computational cost. The present results establish VM2 as a distinctive technology for early-stage drug discovery, which provides a strong combination of efficiency and predictivity.

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase.

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory calculations indicate that both superoxo and hydroperoxo active site states are stable. The hydroperoxo formed is potentially an early LPMO catalytic reaction intermediate or the first step in the mechanism of hydrogen peroxide formation in the absence of substrate. We observe that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by molecular dynamics and mining minima free energy calculations we establish that the conserved second-shell His161 in MtPMO3* displays conformational flexibility in solution and that this flexibility is also observed, though to a lesser extent, in His157 of NcLPMO9D. The imidazolate form of His157 observed in our structure following oxygen intermediate protonation can be attributed to abolished His157 flexibility due steric hindrance in the crystal as well as the solvent-occluded active site environment due to crystal packing. A neutron crystal structure of NcLPMO9D at low pH further supports occlusion of the active site since His157 remains singly protonated even at acidic conditions.

Computation of host-guest binding free energies with a new quantum mechanics based mining minima algorithm.

A new method called QM-VM2 is presented that efficiently combines statistical mechanics with quantum mechanical (QM) energy potentials in order to calculate noncovalent binding free energies of host-guest systems. QM-VM2 efficiently couples the use of semi-empirical QM (SEQM) energies and geometry optimizations with an underlying molecular mechanics (MM) based conformational search, to find low SEQM energy minima, and allows for processing of these minima at higher levels of ab initio QM theory. A progressive geometry optimization scheme is introduced as a means to increase conformational sampling efficiency. The newly implemented QM-VM2 is used to compute the binding free energies of the host molecule cucurbit[7]uril and a set of 15 guest molecules. The results are presented along with comparisons to experimentally determined binding affinities. For the full set of 15 host-guest complexes, which have a range of formal charges from +1 to +3, SEQM-VM2 based binding free energies show poor correlation with experiment, whereas for the ten +1 complexes only, a significant correlation (R2 = 0.8) is achieved. SEQM-VM2 generation of conformers followed by single-point ab initio QM calculations at the dispersion corrected restricted Hartree-Fock-D3(BJ) and TPSS-D3(BJ) levels of theory, as post-processing corrections, yields a reasonable correlation with experiment for the full set of host-guest complexes (R2 = 0.6 and R2 = 0.7, respectively) and an excellent correlation for the +1 formal charge set (R2 = 1.0 and R2 = 0.9, respectively), as long as a sufficiently large basis set (triple-zeta quality) is employed. The importance of the inclusion of configurational entropy, even at the MM level, for the achievement of good correlation with experiment was demonstrated by comparing the calculated ΔE values with experiment and finding a considerably poorer correlation with experiment than for the calculated free energy ΔE - TΔS. For the complete set of host-guest systems with the range of formal charges, it was observed that the deviation of the predicted binding free energy from experiment correlates somewhat with the net charge of the systems. This observation leads to a simple empirical interpolation scheme to improve the linear regression of the full set.

Solvent-induced frequency shifts: configuration interaction singles combined with the effective fragment potential method.

The simplest variational method for treating electronic excited states, configuration interaction with single excitations (CIS), has been interfaced with the effective fragment potential (EFP) method to provide an effective and computationally efficient approach for studying the qualitative effects of solvents on the electronic spectra of molecules. Three different approaches for interfacing a non-self-consistent field (SCF) excited-state quantum mechanics (QM) method and the EFP method are discussed. The most sophisticated and complex approach (termed fully self consistent) calculates the excited-state electron density with fully self-consistent accounting for the polarization (induction) energy of effective fragments. The simplest approach (method 1) includes a strategy that indirectly adds the EFP perturbation to the CIS wave function and energy via modified Hartree-Fock molecular orbitals, so that there is no direct EFP interaction with the excited-state density. An intermediate approach (method 2) accomplishes the latter in a noniterative perturbative manner. Theoretical descriptions of the three approaches are presented, and test results of solvent-induced shifts using methods 1 and 2 are compared with fully ab initio values. These comparisons illustrate that, at least for the test cases examined here, modification of the ground-state Hartree-Fock orbitals is the largest and most important factor in the calculated solvent-induced shifts. Method 1 is then employed to study the aqueous solvation of coumarin 151 and compared with experimental measurements.

Modeling Protein-Ligand Binding by Mining Minima.

We present the first application of the mining minima algorithm to protein-small molecule binding. This end-point approach use an empirical force field and implicit solvent models, treats the protein binding-site as fully flexible and estimates free energies as sums over local energy wells. The calculations are found to yield encouraging agreement with experiment for three sets of HIV-1protease inhibitors and a set of phosphodiesterase 10a inhibitors. The contributions of various aspects of the model to its accuracy are examined, and the Poisson-Boltzmann correction is found to be the most critical. Interestingly, the computed changes in configurational entropy upon binding fall roughly along the same entropy-energy correlation previously observed for smaller host-guest systems. Strengths and weaknesses of the method are discussed, as are the prospects for enhancing accuracy and speed.

Proteomic analysis identifies oxidative stress induction by adaphostin.

PURPOSE: Activities distinct from inhibition of Bcr/abl have led to adaphostin (NSC 680410) being described as "a drug in search of a mechanism." In this study, proteomic analysis of adaphostin-treated myeloid leukemia cell lines was used to further elucidate a mechanism of action. EXPERIMENTAL DESIGN: HL60 and K562 cells treated with adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. The contribution of the redox-active hydroquinone group in adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H(2)O(2). RESULTS: Analysis of adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1, PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1 as an adaphostin resistance gene. Subsequent analysis of HL60 cells treated with 1,4-dihydroxybenzene or H(2)O(2) showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with adaphostin resistance. The role of SOD as a second adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H(2)O(2) replicated adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon. CONCLUSION: Adaphostin should be classified as a redox-active-substituted dihydroquinone.

Determinants of the relative reduction potentials of type-1 copper sites in proteins.

The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.