Design, synthesis, and computational studies of inhibitors of Bcl-XL

One of the primary objectives in the design of protein inhibitors is to shape the three-dimensional structures of small molecules to be complementary to the binding site of a target protein. In the course of our efforts to discover potent inhibitors of Bcl-2 family proteins, we found a unique folded conformation adopted by tethered aromatic groups in the ligand that significantly enhanced binding affinity to Bcl-XL. This finding led us to design compounds that were biased by nonbonding interactions present in a urea tether to adopt this bioactive, folded motif. To characterize the key interactions that induce the desired conformational bias, a series of substituted N,N'-diarylureas were prepared and analyzed using X-ray crystallography and quantum mechanical calculations. Stabilizing pi-stacking interactions and destabilizing steric interactions were predicted to work in concert in two of the substitution patterns to promote the bioactive conformation as a global energy minimum and result in a high target binding affinity. Conversely, intramolecular hydrogen bonding present in the third substitution motif promotes a less active, extended conformer as the energetically favored geometry. These findings were corroborated when the inhibition constant of binding to Bcl-XL was determined for fully elaborated analogues bearing these structural motifs. Finally, we obtained the NMR solution structure of the disubstituted N,N'-diarylurea bound to Bcl-XL demonstrating the folded conformation of the urea motif engaged in extensive pi-interactions with the protein.     

Temperature artifacts in protein structures bias ligand-binding predictions

X-ray crystallography is the gold standard to resolve conformational ensembles that are significant for protein function, ligand discovery, and computational methods development. However, relevant conformational states may be missed at common cryogenic (cryo) data-collection temperatures but can be populated at room temperature. To assess the impact of temperature on making structural and computational discoveries, we systematically investigated protein conformational changes in response to temperature and ligand binding in a structural and computational workhorse, the T4 lysozyme L99A cavity. Despite decades of work on this protein, shifting to RT reveals new global and local structural changes. These include uncovering an apo helix conformation that is hidden at cryo but relevant for ligand binding, and altered side chain and ligand conformations. To evaluate the impact of temperature-induced protein and ligand changes on the utility of structural information in computation, we evaluated how temperature can mislead computational methods that employ cryo structures for validation. We find that when comparing simulated structures just to experimental cryo structures, hidden successes and failures often go unnoticed. When using structural information in ligand binding predictions, both coarse docking and rigorous binding free energy calculations are influenced by temperature effects. The trend that cryo artifacts limit the utility of structures for computation holds across five distinct protein classes. Our results suggest caution when consulting cryogenic structural data alone, as temperature artifacts can conceal errors and prevent successful computational predictions, which can mislead the development and application of computational methods in discovering bioactive molecules.

Temperature artifacts in protein structures impact the utility of structural information in computation by misleading validation and application of computational methods in discovering bioactive molecules.     

Toward accurate relative energy predictions of the bioactive conformation of drugs

Quantifying the relative energy of a ligand in its target-bound state (i.e. the bioactive conformation) is essential to understand the process of molecular recognition, to optimize the potency of bioactive molecules and to increase the accuracy of structure-based drug design methods. This is, nevertheless, seriously hampered by two interrelated issues, namely the difficulty in carrying out an exhaustive sampling of the conformational space and the shortcomings of the energy functions, usually based on parametric methods of limited accuracy. Matters are further complicated by the experimental uncertainty on the atomic coordinates, which precludes a univocal definition of the bioactive conformation. In this article we investigate the relative energy of bioactive conformations introducing two major improvements over previous studies: the use sophisticated QM-based methods to take into account both the internal energy of the ligand and the solvation effect, and the application of physically meaningful constraints to refine the bioactive conformation. On a set of 99 drug-like molecules, we find that, contrary to previous observations, two thirds of bioactive conformations lie within 0.5 kcal mol(-1) of a local minimum, with penalties above 2.0 kcal mol(-1) being generally attributable to structural determination inaccuracies. The methodology herein described opens the door to obtain quantitative estimates of the energy of bioactive conformations and can be used both as an aid in refining crystallographic structures and as a tool in drug discovery.     

Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: Comparison of predicted and experimental conformations of inhibitors of three enzymes

Summary A computer procedure TFIT, which uses a molecular superposition force field to flexibly match test compounds to a 3D pharmacophore, was evaluated to find out whether it could reliably predict the bioactive conformations of flexible ligands. The program superposition force field optimizes the overlap of those atoms of the test ligand and template that are of similar chemical type, by applying an attractive force between atoms of the test ligand and template which are close together and of similar type (hydrogen bonding, charge, hydrophobicity). A procedure involving Monte Carlo torsion perturbations, followed by torsional energy minimization, is used to find conformations of the test ligand which cominimize the internal energy of the ligand and the superposition energy of ligand and template. The procedure was tested by applying it to a series of flexible ligands for which the bioactive conformation was known experimentally. The 15 molecules tested were inhibitors of thermolysin, HIV-1 protease or endothiapepsin for which X-ray structures of the bioactive conformation were available. For each enzyme, one of the molecules served as a template and the others, after being conformationally randomized, were fitted. The fitted conformation was then compared to the known binding geometry. The matching procedure was successful in predicting the bioactive conformations of many of the structures tested. Significant deviation from experimental results was found only for parts of molecules where it was readily apparent that the template did not contain sufficient information to accurately determine the bioactive conformation.     

A proposed bioactive conformation of Peptide T

The conformational profiles of Peptide T, (5–8)Peptide T, [Abu⁵](4–8)Peptide T and (4–8)Peptide T were computed independently to assess the geometrical characteristics of the bioactive conformation of Peptide T. The conformational profiles of the peptides were computed within the molecular mechanics framework using an effective dielectric constant of 80. The conformational space was thoroughly sampled using an iterative simulated annealing protocol. The bioactive conformation was assessed by pairwise cross comparisons of each of the unique low energy conformations found for each of the different analogs studied. After a putative bioactive conformation was selected, in order to further validate our hypothesis the conformational profile of the potent compound cyclo(Thr-Thr-Asn-Tyr-Thr-Asp) was computed and the putative bioactive conformation was found. The conformation exhibits a pseudo -turn involving the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷ forming a C12 ring.     

Exploring the use of conformationally locked aminoglycosides as a new strategy to overcome bacterial resistance

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.     

Conformational analysis of kainate in aqueous solution in relation to its binding to AMPA and kainic acid receptors

Conformational analyses for kainate in aqueous solution have been performed by using the MM3*, AMBER* and MMFF94 force fields in conjunction with the Generalized Born Solvent Accessible Surface (GB/SA) hydration model. A comparison of calculated results with experimentally determined conformational data indicates that MM3*-GB/SA strongly overestimates the stability of a hydrogen bonded ion-pair in aqueous solution in comparison with the separated and solvated ions. This results in an incorrect prediction by MM3* of the most stable conformer of kainate in aqueous solution, whereas AMBER* and MMFF94 correctly predict the lowest energy conformer. Calculated conformational energy penalties for binding of kainate to the AMPA iGluR2 receptor indicate that the lower affinity of kainate for AMPA receptors compared to its affinity for kainic acid (KA) receptors is not due to a higher energy bioactive conformation of kainate at AMPA receptors. This conclusion is strongly supported by an analysis of a recently reported nonselective AMPA/KA ligand and a comparison of the conformational and structural properties of this ligand with iGluR2-bound kainate. This comparison strongly suggests that kainate binds to AMPA and KA receptors in closely the same conformation.     

Conformational energy penalties of protein-bound ligands

The conformational energies required for ligands to adopt their bioactive conformations were calculated for 33 ligand–protein complexes including 28 different ligands. In order to monitor the force field dependence of the results, two force fields, MM3 and AMBER, were employed for the calculations. Conformational analyses were performed in vacuo and in aqueous solution by using the generalized Born/solvent accessible surface (GB/SA) solvation model. The protein-bound conformations were relaxed by using flat-bottomed Cartesian constraints. For about 70% of the ligand–protein complexes studied, the conformational energies of the bioactive conformations were calculated to be 3 kcal/mol. It is demonstrated that the aqueous conformational ensemble for the unbound ligand must be used as a reference state in this type of calculations. The calculations for the ligand–protein complexes with conformational energy penalties of the ligand calculated to be larger than 3 kcal/mol suffer from uncertainties in the interpretation of the experimental data or limitations of the computational methods. For example, in the case of long-chain flexible ligands (e.g. fatty acids), it is demonstrated that several conformations may be found which are very similar to the conformation determined by X-ray crystallography and which display significantly lower conformational energy penalties for binding than obtained by using the experimental conformation. For strongly polar molecules, e.g. amino acids, the results indicate that further developments of the force fields and of the dielectric continuum solvation model are required for reliable calculations on the conformational properties of this type of compounds.     

Exploring the energy landscape of a molecular engineered analog of a tumor-homing peptide

Recently a new non-coded amino acid was designed as a replacement for Arg, to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This constrained Arg analog, denoted c(5)Arg, was engineered to also promote the stability of the CREKA bioactive conformation. The conformational profile of the CREKA analog obtained by replacing Arg by c(5)Arg has been extensively investigated in this work. Two molecular dynamics simulations-based strategies have been employed: a modified simulated annealing and replica exchange. Results obtained using both techniques show that the conformational features of the new analog fulfill the purpose of its design. The new CREKA analog not only preserves the main structural attributes found for the bioactive conformation of the parent peptide but also shows lower flexibility. Moreover, the conformational profile of the mutated peptide narrows towards the most stable structures previously observed for the parent CREKA peptide.     

A CoMFA analysis with conformational propensity: An attempt to analyze the SAR of a set of molecules with different conformational flexibility using a 3D-QSAR method

CoMFA analysis, a widely used 3D-QSAR method, has limitations to handle a set of SAR data containing diverse conformational flexibility since it does not explicitly include the conformational entropic effects into the analysis. Here, we present an attempt to incorporate the conformational entropy effects of a molecule into a 3D-QSAR analysis. Our attempt is based on the assumption that the conformational entropic loss of a ligand upon making a ligand-receptor complex is small if the ligand in an unbound state has a conformational propensity to adopt an active conformation in a complex state. For a QSAR analysis, this assumption was interpreted as follows: a potent ligand should have a higher conformational propensity to adopt an `active-conformation'-like structure in an unbound state than an inactive one. The conformational propensity value was defined as the populational ratio, N<sub>active</sub>/N<sub>stable</sub>, of the number of energetically stable conformers, N<sub>stable</sub>, to the number of `active-conformation'-like structures, N_active. The latter number was calculated by counting the number of conformers that satisfied the structural parameters deduced from the active conformation. A set of SAR data of imidazoleglycerol phosphate dehydratase inhibitors containing 20 molecules with different conformational flexibility was used as a training set for developing a 3D structure-activity relationship by a CoMFA analysis with the conformational propensity value. This resulted in a cross-validated squared correlation coefficient of the CoMFA model with the conformational propensity value (R ² _cross = 0.640) higher than that of the standard CoMFA model (R ² _cross = 0.431). Then we evaluated the quality of the CoMFA models by predicting the inhibitory activity for a new molecule.     

The conformation of cyclo(-D-Pro-Ala4-) as a model for cyclic pentapeptides of the DL4 type

The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.     

T-Taxol and the electron crystallographic density in beta-tubulin

[chemical structure: see text]. T-Taxol has been proposed as the bioactive conformation on beta-tubulin and subsequently utilized in the design of a series of highly active bridged taxane analogues. A modified T-form with a reversed C-13 side chain orientation has recently been proposed as an equally plausible bioactive shape. A comparison of the two spatial alternatives within the tubulin binding site electron crystallographic density suggests strongly that T-Taxol is the bioactive conformation.     

Binding to an RNA aptamer changes the charge distribution and conformation of malachite green

RNA plays a central role in many biological processes and is therefore an important target for drug development. In recent years an increasing wealth of structural and functional information about RNA-ligand complexes has been obtained using in vitro selected RNAs (aptamers). However, all those studies focused on structure and changes of the nucleic acid and mostly considered the ligand as a rigid target. To develop a detailed picture of ligand structure and dynamics in RNA-small molecule complexes, the malachite green binding aptamer was studied. Isotopically labeled ligand in complex with RNA was analyzed by NMR spectroscopy in solution. The surprisingly asymmetric changes in the (13)C chemical shift of the ligand methyl groups indicate that the dye undergoes changes in its conformation and charge distribution upon binding. The role of the RNA electrostatic field in this interaction was explored using ab initio calculations of the ligand structure and charge distribution. The results indicate that the uneven charge distribution in the RNA binding pocket provides a major contribution to the driving force of the ligand structural changes. The observation that not only the RNA adapts to the ligand, in what is called adaptive binding, but that the ligand itself also undergoes conformational changes ("induced fit") is crucial for the rational design of RNA ligands and for understanding the properties of RNA-ligand complexes.     

蛋白质分子与配体结合过程构象变化的研究

蛋白质分子与配体的作用模式主要有直接的环区结合及铰链式结合两种方式。针对这两种不同的作用方式,我们提出采用不同的策略进行结合过程的构象研究。对于直接的环区结合模式,通过建立环区主链构象库,来实现蛋白质环区与配体的准柔性对接,并以链霉抗生物素蛋白体系为例对构象库建立的可行性进行了验证计算。对铰链结合方式,采用分步对接的方法进行计算,并具体应用于HIV蛋白酶与其小分子配体的结合过程。计算结果表明,这两种处理方法分别能较好地模拟不同类型的蛋白质与配体结合的的构象变化。     

GalaxyDock3: Protein–ligand docking that considers the full ligand conformational flexibility

Predicting conformational changes of both the protein and the ligand is a major challenge when a protein–ligand complex structure is predicted from the unbound protein and ligand structures. Herein, we introduce a new protein–ligand docking program called GalaxyDock3 that considers the full ligand conformational flexibility by explicitly sampling the ligand ring conformation and allowing the relaxation of the full ligand degrees of freedom, including bond angles and lengths. This method is based on the previous version (GalaxyDock2) which performs the global optimization of a designed score function. Ligand ring conformation is sampled from a ring conformation library constructed from structure databases. The GalaxyDock3 score function was trained with an additional bonded energy term for the ligand on a large set of complex structures. The performance of GalaxyDock3 was improved compared to GalaxyDock2 when predicted ligand conformation was used as the input for docking, especially when the input ligand conformation differs significantly from the crystal conformation. GalaxyDock3 also compared favorably with other available docking programs on two benchmark tests that contained diverse ligand rings. The program is freely available at http://galaxy.seoklab.org/softwares/galaxydock.html . © 2019 Wiley Periodicals, Inc.     

Design, synthesis, and biological evaluation of HSP90 inhibitors based on conformational analysis of radicicol and its analogues

The molecular chaperone HSP90 is an attractive target for chemotherapy because its activity is required for the functional maturation of a number of oncogenes. Among the known inhibitors, radicicol, a 14-member macrolide, stands out as the most potent. A molecular dynamics/minimization of radicicol showed that there were three low energy conformers of the macrocycle. The lowest of these is the bioactive conformation observed in the cocrystal structure of radicicol with HSP90. Corresponding conformational analyses of several known analogues gave a good correlation between the bioactivity and the energy of the bioactive conformer, relative to other conformers. Based on this observation, a number of proposed analogues were analyzed for their propensity to adopt the bioactive conformation prior to synthesis. This led to the identification of pochonin D, a recently isolated secondary metabolite of Pochonia chlamydosporia, as a potential inhibitor of HSP90. Pochonin D was synthesized using polymer-bound reagents and shown to be nearly as potent an HSP90 inhibitor as radicicol.     

The Confine-and-Release Method: Obtaining Correct Binding Free Energies in the Presence of Protein Conformational Change

Free energy calculations are increasingly being used to estimate absolute and relative binding free energies of ligands to proteins. However, computed free energies often appear to depend on the initial protein conformation, indicating incomplete sampling. This is especially true when proteins can change conformation on ligand binding, as free energies associated with these conformational changes are either ignored or assumed to be included by virtue of the sampling performed in the calculation. Here, we show that, in a model protein system (a designed binding site in T4 Lysozyme), conformational changes can make a difference of several kcal/mol in computed binding free energies, and that they are neglected in computed binding free energies if the system remains kinetically trapped in a particular metastable state on simulation timescales. We introduce a general "confine-and-release" framework for free energy calculations that accounts for these free energies of conformational change. We illustrate its use in this model system by demonstrating that an umbrella sampling protocol can obtain converged binding free energies that are independent of the starting protein structure and include these conformational change free energies.     

PDB ligand conformational energies calculated quantum-mechanically

We present here a greatly updated version of an earlier study on the conformational energies of protein-ligand complexes in the Protein Data Bank (PDB) [Nicklaus et al. Bioorg. Med. Chem. 1995, 3, 411-428], with the goal of improving on all possible aspects such as number and selection of ligand instances, energy calculations performed, and additional analyses conducted. Starting from about 357,000 ligand instances deposited in the 2008 version of the Ligand Expo database of the experimental 3D coordinates of all small-molecule instances in the PDB, we created a "high-quality" subset of ligand instances by various filtering steps including application of crystallographic quality criteria and structural unambiguousness. Submission of 640 Gaussian 03 jobs yielded a set of about 415 successfully concluded runs. We used a stepwise optimization of internal degrees of freedom at the DFT level of theory with the B3LYP/6-31G(d) basis set and a single-point energy calculation at B3LYP/6-311++G(3df,2p) after each round of (partial) optimization to separate energy changes due to bond length stretches vs bond angle changes vs torsion changes. Even for the most "conservative" choice of all the possible conformational energies-the energy difference between the conformation in which all internal degrees of freedom except torsions have been optimized and the fully optimized conformer-significant energy values were found. The range of 0 to ~25 kcal/mol was populated quite evenly and independently of the crystallographic resolution. A smaller number of "outliers" of yet higher energies were seen only at resolutions above 1.3 ?. The energies showed some correlation with molecular size and flexibility but not with crystallographic quality metrics such as the Cruickshank diffraction-component precision index (DPI) and R(free)-R, or with the ligand instance-specific metrics such as occupancy-weighted B-factor (OWAB), real-space R factor (RSR), and real-space correlation coefficient (RSCC). We repeated these calculations with the solvent model IEFPCM, which yielded energy differences that were generally somewhat lower than the corresponding vacuum results but did not produce a qualitatively different picture. Torsional sampling around the crystal conformation at the molecular mechanics level using the MMFF94s force field typically led to an increase in energy.