Exploring the cocrystallization potential of urea and benzamide

The cocrystallization landscape of benzamide and urea interacting with aliphatic and aromatic carboxylic acids was studied both experimentally and theoretically. Ten new cocrystals of benzamide were synthesized using an oriented samples approach via a fast dropped evaporation technique. Information about types of known bi-component cocrystals augmented with knowledge of simple binary eutectic mixtures was used for the analysis of virtual screening efficiency among 514 potential pairs involving aromatic carboxylic acids interacting with urea or benzamide. Quantification of intermolecular interaction was achieved by estimating the excess thermodynamic functions of binary liquid mixtures under supercooled conditions within a COSMO-RS framework. The smoothed histograms suggest that slightly more potential pairs of benzamide are characterized in the attractive region compared to urea. Finally, it is emphasized that prediction of cocrystals of urea is fairly direct, while it remains ambiguous for benzamide paired with carboxylic acids. The two known simple eutectics of urea are found within the first two quartiles defined by excess thermodynamic functions, and all known cocrystals are outside of this range belonging to the third or fourth quartile. On the contrary, such a simple separation of positive and negative cases of benzamide miscibility in the solid state is not observed. The difference in properties between urea and benzamide R2,2(8) heterosynthons is also documented by alterations of substituent effects. Intermolecular interactions of urea with para substituted benzoic acid analogues are stronger compared to those of benzamide. Also, the amount of charge transfer from amide to aromatic carboxylic acid and vice versa is more pronounced for urea. However, in both cases, the greater the electron withdrawing character of the substituent, the higher the binding energy, and the stronger the supermolecule polarization via the charge transfer mechanism.     

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.     

Thermitase, a thermostable subtilisin: comparison of predicted and experimental structures and the molecular cause of thermostability

The Subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, Subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three-dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserve structural core and less well in seven loop regions involving insertions and deletions relative to Subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular case of thermostability we confront hypotheses erived by calculation of the details of interatomic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main cause of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.     

An enzyme-coupled assay for glyoxylic acid

Herein, we present a new enzyme-linked spectrophotometric assay for glyoxylate that detects glyoxylate via the formation of an intensely colored formazan. This glyoxylate-specific assay is reliant upon the enzymatic conversion of glyoxylate to oxaloacetate coupled to the reduction of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide (NADH). The NADH-dependent reduction of a tetrazolium to the formazan enables the measurement of nanomole quantities of glyoxylate in an assay that is amenable to high-throughput screening methods. Assay validation was accomplished using two methods for glyoxylate generation, the base-catalyzed N-dealkylation of alpha-hydroxyhippurate to benzamide and glyoxylate and the oxidative cleavage of the glycyl Calpha-N bond in N-benzoylglycine (hippurate) by peptidylglycine alpha-amidating monooxygenase to again yield benzamide and glyoxylate. For both reactions, analysis of benzamide produced by reverse-phase high-performance liquid chromatography compared with glyoxylate measured using our glyoxylate assay showed a 1:1 molar ratio of benzamide to glyoxylate. These results indicate that the enzyme-linked spectrophotometric assay can quantitatively measure submicromole quantities of glyoxylate.     

The Calculation of 3D Solubility Parameters Using Molecular Models

Abstract

In this paper we describe the use of molecular mechanics models to examine detailed intermolecular interactions within the liquid state of a common nonionic surfactant system, nonyl phenol ethoxylate (NPE). Using constant energy molecular dynamics simulations we have studied the relative strengths of dispersive interactions versus polar interactions and have estimated three dimensional solubility parameters for NPE systems as a function of temperature and ethylene oxide content. The predictions at 300 K are in good agreement with three dimensional solubility parameters predicted using group contribution tables. Models of the amorphous liquid state were represented by single molecular structures of NPE in a periodic cell. The solubility parameters predicted with these models were in good agreement with those values derived from models having eight NPE molecules packed into a cell with the exception of the electrostatic interactions, which are the most sensitive to system size effects.     

Synthesis,in Silico Study and Biological Evaluation of N-(Benzothiazol/Thiazol-2-yl)benzamide Derivatives as Quorum Sensing Inhibitors against Pseudomonas aeruginosa

The development of bacterial resistance to chemical therapy poses a severe danger to efficacy of treating bacterial infections. One of the key factors for resistance to antimicrobial medications is growth of bacteria in biofilm. Quorum sensing (QS) inhibition was created as an alternative treatment by developing novel anti-biofilm medicines. Cell-cell communication is impeded by QS inhibition, which targets QS signaling pathway. The goal of this work is to develop newer drugs that are effective against Pseudomonas aeruginosa by decreasing QS and acting as anti-biofilm agents. In this investigation, N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h were designed and synthesized in good yields. Further, molecular docking analyses revealed that binding affinity values were founded −11.2 to −7.6 kcal/mol that were moderate to good. The physicochemical properties of these prepared compounds were investigated through in-silico method. Molecular dynamic simulation was also used to know better understanding of stability of the protein and ligand complex. Comparing N-(benzo[d]thiazol-2-yl)benzamide 3a to salicylic acid (4.40±0.10) that was utilised as standard for quorum sensing inhibitor, the anti-QS action was found greater for N-(benzo[d]thiazol-2-yl)benzamide 3a (4.67±0.45) than salicylic acid (4.40±0.10). Overall, research results suggested that N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h may hold to develop new quorum sensing inhibitors.     

Discovering benzamide derivatives as glycogen phosphorylase inhibitors and their binding site at the enzyme

A series of novel benzamide derivatives was designed, synthesized, and their inhibitory activities against glycogen phosphorylase (GP) in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate were evaluated. The structure-activity relationships (SAR) of these compounds are also presented. Within this series of compounds, 4m is the most potent GPa inhibitor (IC(50)=2.68 microM), which is nearly 100 times more potent than the initial compound 1. Analysis of mapping between pharmacophores of different binding sites and each compound demonstrated that these benzamide derivatives bind at the dimer interface of the rabbit muscle enzyme, and possible docking modes of compound 4m were explored by molecular docking simulation.     

Pharmacophore mapping,molecular docking,chemical synthesis of some novel pyrrolyl benzamide derivatives and evaluation of their inhibitory activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis

In an effort to produce new lead antimycobacterial compounds, herein we have reported the synthesis of a sequence of new pyrrolyl benzamide derivatives. The new chemical entities were screened to target enoyl-ACP reductase enzyme, which is one of the key enzymes of M. tuberculosis that are involved in type II fatty acid biosynthetic pathway. Compound 3q exhibited H-bonding interactions with Tyr158, Thr196 and co-factor NAD⁺ that binds the active site of InhA. All the pyrrolyl benzamide compounds were evaluated as inhibitors of M. tuberculosis H₃₇Rv as well as inhibitors of InhA. Among them, few representative compounds were tested for mammalian cell toxicity on the human lung cancer cell-line (A549) and MV cell line that presented no cytotoxicity. Five of these compounds exhibited a good activity against InhA.     

Evaluation of free energy landscape for base-amino acid interactions using ab initio force field and extensive sampling

Structural data of protein-DNA complex show redundancy and flexibility in base-amino acid interactions. To understand the origin of the specificity in protein-DNA recognition, we calculated the interaction free energy, enthalpy, entropy, and minimum energy maps for AT-Asn, GC-Asn, AT-Ser, and GC-Ser by means of a set of ab initio force field with extensive conformational sampling. We found that the most preferable interactions in these pairs are stabilized by hydrogen bonding, and are mainly enthalpy driven. However, minima in the free energy maps are not necessarily the same as those in the minimum energy map or enthalpy maps, due to the entropic effect. The effect of entropy is particularly important in the case of GC-Asn. Experimentally observed structures of base-amino acid interactions are within preferable regions in the calculated free energy maps, where there are many different interaction configurations with similar energy. The full geometry optimization procedure using ab initio molecular orbital method was applied to get the optimal interaction geometries for AT-Asn, GC-Asn, AT-Ser, and GC-Ser. We found that there are various base-amino acid combinations with similar interaction energies. These results suggest that the redundancy and conformational flexibility in the base-amino acid interactions play an important role in the protein-DNA recognition.     

Coarse-Graining Provides Insights on the Essential Nature of Heterogeneity in Actin Filaments

Experiments have shown that actin is structurally polymorphic, but knowledge of the details of molecular level heterogeneity in both the dynamics of a single subunit and the interactions between subunits is still lacking. Here, using atomistic molecular dynamics simulations of the actin filament, we identify domains of atoms that move in a correlated fashion, quantify interactions between these domains using coarse-grained (CG) analysis methods, and perform CG simulations to explore the importance of filament heterogeneity. The persistence length and torsional stiffness calculated from molecular dynamics simulation data agree with experimental values. We additionally observe that distinct actin conformations coexist in actin filaments. The filaments also exhibit random twist angles that are broadly distributed. CG analysis reveals that interactions between equivalent CG pairs vary from one subunit to another. To explore the importance of heterogeneity on filament dynamics, we perform CG simulations using different methods of parameterization to show that only by including heterogeneous interactions can we reproduce the twist angles and related properties. Free energy calculations further suggest that in general the actin filament is best represented as a set of subunits with differing CG sites and interactions, and the incorporating heterogeneity into the CG interactions is more important than including that in the CG sites. Our work therefore presents a systematic method to explore molecular level detail in this large and complex biopolymer.     

Synthesis and structure-activity relationship of disubstituted benzamides as a novel class of antimalarial agents

Malaria is a devastating world health problem. Using a compound library screening approach, we identified a novel series of disubstituted benzamide compounds with significant activity against malaria strains 3D7 and K1. These compounds represent a new antimalarial molecular scaffold exemplified by compound 1, which demonstrated EC(50) values of 60 and 430 nM against strains 3D7 and K1, respectively. Herein we report our findings on the efficient synthesis, structure-activity relationships, and biological activity of this new class of antimalarial agents.     

Specific interactions for ab initio folding of protein terminal regions with secondary structures

Proteins fold into unique three-dimensional structures by specific, orientation-dependent interactions between amino acid residues. Here, we extract orientation-dependent interactions from protein structures by treating each polar atom as a dipole with a direction. The resulting statistical energy function successfully refolds 13 out of 16 fully unfolded secondary-structure terminal regions of 10-23 amino acid residues in 15 small proteins. Dissecting the orientation-dependent energy function reveals that the orientation preference between hydrogen-bonded atoms is not enough to account for the structural specificity of proteins. The result has significant implications on the theoretical and experimental searches for specific interactions involved in protein folding and molecular recognition between proteins and other biologically active molecules.     

Discovery Studio 2.0的模块扩展及应用:残基水平非键相互作用能量的自动批量计算

非键相互作用对于生物体系中的分子识别和结合过程起着关键作用。然而,传统的方法并不能在残基水平自动批量计算非键相互作用。近年来,已经发展了一些方法和工具进行非键相互作用的计算分析。该文研究发展了一种可以自动计算残基间非键相互作用的方法,即用Perl脚本调用Discovery Studio 2.0(DS 2.0,Accelrys Inc.)底层模块中的非键相互作用协议,实现了直接利用命令行批量计算非键相互作用能量,而无需通过DS2.0的图形界面。该方法扩展了DS2.0的计算模块,并于近期运用到了复合结构的研究分析中。     

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide¹ is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.     

Discovery of human autophagy initiation kinase ULK1 inhibitors by multi-directional in silico screening strategies

Autophagy is a self-catabolic mechanism employed by cancer cells to acquire nutrients and energy in times of stress conditions, thereby leading to its progression and survival. Thus, autophagy inhibition has emerged as a new paradigm in the area of cancer treatment. Here, we leverage multi-dimensional screening campaigns aim to identify potent inhibitors against an early and an essential autophagic kinase, ULK1 from DrugBank database. In particular, receptor-based hypothesis, pharmacophore hypothesis, e-pharmacophore hypothesis and shape similarity-based screening algorithm were employed. Of note, the results of the different algorithm were then integrated to eliminate the false positive prediction. Moreover, the inhibitory activities and PK/PD parameters of the leads were tested by Glide and Qikprop algorithm. This resulted in a set of four hits namely; DB12686, DB08341, DB07936, and DB07163. Finally, molecular dynamics simulation was performed using the GROMACS package, to validate the binding kinetics of the hit compound. The compound activity in vitro was assessed by PASS algorithm, highlights the anti-cancer activities of the hits. The structural insights reveal existence of functional moieties such as piperidine carboxamide, benzenesulfonamide, benzamide, and isoindolone in the resultant hits which plays a major role in the anti-cancer activity. Overall, we strongly believe that these ULK1 antagonists could be novel and potent drug candidates for future cancer therapeutics.     

Adamantyl N-benzylbenzamide: new series of depigmentation agents with tyrosinase inhibitory activity

A new series of polyhydroxylated N-benzylbenzamide derivatives containing an adamantyl moiety has been synthesized, and the depigmenting and tyrosinase inhibitory activities of the molecules were evaluated. The lipophilic character of the adamantyl moiety appeared to confer greater depigmentation power on the benzamide derivatives as compared to those lacking adamantyl substitution. Molecular modeling was applied in order to elucidate the interactions between ligands and tyrosinase that led to inhibition.     

Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions

Fluorescence resonance energy transfer (FRET) in association with a time-resolved fluorescence mode of detection was used to design a new homogeneous technology suitable to monitor biomolecular interactions. A lanthanide cryptate characterised by a long lived fluorescence emission was used as donor and a cross-linked allophycocyanine was used as acceptor. This new donor/acceptor pair displayed an exceptionally large Forster radius of 9 nm. This allowed to build up a set of labelling strategies to probe the interactions between biomolecules with an emphasis on fully indirect cassette formats particularly suitable for high throughput screening applications. Herein we describe the basics of the technology, review the latest applications to the study of molecular interactions involved in cells and new oligonucleotides based assays.     

Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11β-HSD1 inhibitors

Discovery and optimization of a piperidyl benzamide series of 11β-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.     

How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme

To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.