Substrate‐Dependent Stereospecificity of Tyl‐KR1: An Isolated Polyketide Synthase Ketoreductase Domain from Streptomyces fradiae

The stereospecificity of an enzymatic reaction depends on the way in which a substrate and its enantiomer bind to the active site. These binding modes cannot be easily predicted. We have studied the stereospecificity and stereoselectivity of the ketoreductase domain Tyl‐KR1 of the tylactone polyketide synthase from Streptomyces fradiae by analysing the stereochemical outcome of the reduction of five different keto ester substrates. The absolute configuration of the Tyl‐KR1 reduction products was determined by using vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations. The conversion of only one of the tested substrates, 2‐methyl‐3‐oxovaleric acid N‐acetylcysteamine thioester, afforded the expected anti‐(2R,3R) configuration of the α‐methyl‐β‐hydroxyl ester product, representing the stereochemistry observed for the physiological polyketide product tylactone. For all other substrates, which were modified with respect to the type of ester and/or the chain length (C₄ instead of C₅), the opposite configuration (anti‐(2S,3S)) was obtained with significant enantio‐ and diastereoselectivity. Inversion of both stereocentres suggests completely different binding modes invoked by only minor modifications of the substrate structure.     

Q‐DockLHM: Low‐resolution refinement for ligand comparative modeling

The success of ligand docking calculations typically depends on the quality of the receptor structure. Given improvements in protein structure prediction approaches, approximate protein models now can be routinely obtained for the majority of gene products in a given proteome. Structure‐based virtual screening of large combinatorial libraries of lead candidates against theoretically modeled receptor structures requires fast and reliable docking techniques capable of dealing with structural inaccuracies in protein models. Here, we present Q‐Dock^LHM, a method for low‐resolution refinement of binding poses provided by FINDSITE^LHM, a ligand homology modeling approach. We compare its performance to that of classical ligand docking approaches in ligand docking against a representative set of experimental (both holo and apo) as well as theoretically modeled receptor structures. Docking benchmarks reveal that unlike all‐atom docking, Q‐Dock^LHM exhibits the desired tolerance to the receptor's structure deformation. Our results suggest that the use of an evolution‐based approach to ligand homology modeling followed by fast low‐resolution refinement is capable of achieving satisfactory performance in ligand‐binding pose prediction with promising applicability to proteome‐scale applications. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl‐ACP reductase (FabI) benzimidazole inhibitors

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard‐Jones and Coulomb Softcore potentials parameters in the one‐step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one‐step TI in a protein‐ligand binding system. The best performing model, the one‐step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM‐GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure‐activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well‐known halogen bond favoring enzyme. © 2015 Wiley Periodicals, Inc.     

Limiting the Number of Potential Binding Modes by Introducing Symmetry into Ligands: Structure‐Based Design of Inhibitors for Trypsin‐Like Serine Proteases

In the absence of X‐ray data, the exploration of compound binding modes continues to be a challenging task. For structure‐based design, specific features of active sites in different targets play a major role in rationalizing ligand binding characteristics. For example, dibasic compounds have been reported as potent inhibitors of various trypsin‐like serine proteases, the active sites of which contain several binding pockets that can be targeted by cationic moieties. This results in several possible orientations within the active site, complicating the binding mode prediction of such compounds by docking tools. Therefore, we introduced symmetry in bi‐ and tribasic compounds to reduce conformational space in docking calculations and to simplify binding mode selection by limiting the number of possible pocket occupations. Asymmetric bisbenzamidines were used as starting points for a multistage and structure‐guided optimization. A series of 24 final compounds with either two or three benzamidine substructures was ultimately synthesized and evaluated as inhibitors of five serine proteases, leading to potent symmetric inhibitors for the pharmaceutical drug targets matriptase, matriptase‐2, thrombin and factor Xa. This study underlines the relevance of ligand symmetry for chemical biology.     

Amylose‐grafted Curdlan: A New Class of Semi‐artificial Branched Polysaccharides for Hierarchical Polymeric Superstructures Created by the Action of “Orthogonal” Binding Sites

The semi‐artificial branched‐polysaccharides, amylose‐grafted curdlans, were synthesized utilizing an enzymatic polymerization. Both a curdlan main chain and amylose side chains on the polysaccharides maintain the original helical structure as well as the molecular binding ability. Thanks to the difference in their molecular recognition properties between β‐1,3‐glucan chain and α‐1,4‐glucan chain, the amylose‐grafted curdlans can provide two different orthogonal binding sites within one polymeric system. When a water‐soluble polythiophene was mixed with the amylose‐grafted curdlan, the polythiophene was twisted in two different modes and therein, fluorescence energy of the polythiophene wrapped by the amylose side chains was successfully transferred to the polythiophene wrapped by the curdlan main chain. We thus concluded that in the dendritic superstructure of this polysaccharide, a self‐organized “Janus‐type FRET system” was successfully constructed.     

Fluorescent dyes in the context of DNA‐binding: The case of Thioflavin T

Thioflavin T (ThT) is a dye characterized by a strong fluorescence light‐up on binding to biosubstrates. Although this effect is known to be related to the inhibition of intramolecular torsion on excitation, the binding modes and their role in affecting photoinduced processes are by no means adequately understood. Here, a combined molecular dynamics and quantum chemical modeling is used to study the tuning of the photophysical properties of ThT when moving from solution to DNA binding. The binding mechanism of ThT to B‐DNA was found to be very complex as a result of an uncommon interplay between different binding modes, for example, monomer intercalation and external binding but also groove binding of the dimer. The detailed analysis of the relation between the different binding modes and the structural and electronic properties of ThT can be used to better understand the interaction with other biosubstrates.     

Isatin‐(thio)semicarbazide/oxime‐1H‐1,2,3‐triazole‐coumarin Hybrids: Design,Synthesis, and in vitro Anti‐mycobacterial Evaluation

Isatin and coumarin derivatives with potential anti‐tubercular activity, while (thio)semicarbazide/oxime and 1H‐1,2,3‐triazole moieties exhibited favorable properties such as hydrogen bonding and/or metal chelation capability, so integration of the four pharmacophores into one molecule may provide more effective anti‐tubercular candidates. Based on the consideration earlier, 12 isatin‐(thio)semicarbazide/oxime‐1H‐1,2,3‐triazole‐coumarin hybrids 8a–l were designed, synthesized, and evaluated for their in vitro anti‐mycobacterial activities against M. tuberculosis (MTB) H₃₇Rv and MDR‐TB. The results showed that all the hybrids (MIC: 50–>200 μg/mL) exhibited weak to moderate inhibitory activity against MTB H₃₇Rv and MDR‐TB, which were far less potent than the references isoniazid (MIC: 0.05 μg/mL) and rifampicin (MIC: 0.39 μg/mL) against MTB H₃₇Rv. The most active hybrid 8h (MIC: 50 μg/mL) was comparable with rifampicin (MIC: 32 μg/mL) and more active than isoniazid (MIC: >128 μg/mL) against MDR‐TB, could be act as a lead for further optimization. Moreover, the enriched structure–activity relationship paved the way to the further rational development of this kind of hybrids.     

Determination of ligand efficiency indices in a group of 7H‐purine‐2,6‐dione derivatives with psychotropic activity using micellar electrokinetic chromatography

Discovering hit compounds and optimization processes in medicinal chemistry nowadays could be improved by predictive tools, based on the relationship between structure of molecules and lipophilic properties. Lipophilicity of drug candidate can affect both the pharmacokinetic and pharmacodynamics properties, in particular, the ability of a molecule to cross the cell membrane. Among the new methods for determination of the lipophilicity of compounds, micellar electrokinetic chromatography (MEKC) is considered to be an appropriate one for bioactive molecules, as it closely mimics the physiological conditions. In this paper MEKC was used for the estimation of the lipophilicity of 24 derivatives of 8‐alkoxy‐7H‐purine‐2,6‐dione, designed and synthesized as potential antidepressant/anxiolytic and antipsychotic agents. The results of experimental method were compared with calculated in silico parameters (AlogPs and milogP by Virtual Computational Laboratory website, log P_Pallas by Pallas 3.1, Mlog P by Marvin, log P_ChemS by ChemSketch, log P_ChemDraw by ChemBioUltra) using Principal Component Analysis (PCA) method. Finally, using estimated log P values for selected compounds ligand – lipophilicity efficiency (LLE), per cent efficiency index (PEI), and binding efficiency index (BEI) parameters were calculated. Applied MEKC procedure could be used for selection of potential lead structure in a group of 7H‐purine‐2,6‐dione derivatives.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

Poly­[lead(II)‐μ2‐aqua‐μ4‐tereph­thalato]

The title compound, [Pb(C₈H₄O₄)(H₂O)]_n, forms as an insoluble product in the reaction of sodium terephthalate(2−) with Pb(NO₃)₂ in water. Analysis has shown that the crystal structure is centrosymmetric, with the asymmetric unit containing one formula unit. The lead geometry is hemidirected seven‐coordinate, with both monodentate and bidentate carboxyl­ate coordination modes present. The combination of hydrogen bonds and coordination bonds produces a three‐dimensional structure, including the first example, in a lead complex, of the common metal‐coordinated carboxyl­ate/water (6) graph‐set motif.     

Ligand‐Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free—Towards a Conformational Activity Screening for Drug Discovery

Investigation of protein–ligand interactions is crucial during early drug‐discovery processes. ATR‐FTIR spectroscopy can detect label‐free protein–ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand‐induced secondary structural change. Two specific binding modes of 19 drug‐like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the k_obs values of ligand dissociation were obtained. The results were validated by X‐ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment.     

Convenient One‐Pot Room Temperature Synthesis of 2‐(trifluoromethyl)‐1,2,3,4,7,8‐hexahydroquinolin‐5(6H)‐one derivatives via Catalyst‐Free Multicomponent Reactions

This paper describes an efficient approach for the synthesis of 2‐(trifluoromethyl)‐1,2,3,4,7,8‐hexahydroquinolin‐5(6H)‐one derivatives via a one‐pot four‐component reaction of aromatic aldehyde, 5,5‐dimethylcyclohexane‐1,3‐dione, 4,4,4‐trifluoro‐1‐(thien‐2‐yl)butane‐1,3‐dione, and NH₄OAc with excellent yields at room temperature. In this synthesis, the trifluoromethyl was efficiently introduced in hexahydroquinoline structure, and they may be valuable the drug candidates. This approach also offered several other advantages, such as catalyst‐free, mild conditions, and simple experimental operation.     

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l ‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.     

On the induced‐fit mechanism of substrate‐enzyme binding structures of nylon‐oligomer hydrolase

We present a detailed computational investigation of the induced‐fit motion in a nylon‐oligomer hydrolase (NylB) upon substrate binding. To this aim, we resort on the recently introduced parallel cascade selection molecular dynamics approach, allowing for an accelerated access to the set of conformational changes from an open‐ to a closed‐state structure to form the enzyme‐substrate complex in a specific induce‐fit mechanism. The structural investigation is quantitatively complemented by free energy analyses within the umbrella sampling algorithm accompanied by weighted histogram analysis. We find that the stabilization free energy is about 1.4 kcal/mol, whereas the highest free energy barrier to be overcome is about 2.3 kcal/mol. Conversely, the energetic contribution for the substrate binding is about 20 kcal/mol, as estimated from Generalized Born/Surface Area. This means that the open‐close induced‐fit motion could occur frequently once the substrate binds to the open state of NylB. © 2014 Wiley Periodicals, Inc.     

Single‐Molecule Force Measurement Guides the Design of Multivalent Ligands with Picomolar Affinity

Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purposes. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Herein, we develop a force measurement platform to probe the distribution and separation of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and use this to assess the geometry of appropriate linkers for distinct multivalent binding modes. A tetravalent lead ZD‐4, which was developed from an antitumor drug ZD6474 (Vandetanib) with combined hybrid binding effects, yielded a 2000‐fold improvement in the binding affinity to VEGFR with IC₅₀ value of 25 pm . We confirmed the improved affinity by the associated increase of tumor uptake in the VEGFR‐targeting positron emission tomography (PET) imaging using U87 tumor xenograft mouse model.     

Square Tiling by Square Macrocycles at the Liquid/Solid Interface: Co‐crystallisation with One‐ or Two‐Dimensional Order

We have systematically investigated the self‐assembled monolayers of seven bimolecular mixtures of square‐shaped pyridinophanes and cyclophanes bearing alkoxy or alkoxycarbonyl substituents in the presence of the tropylium ion as a marker of pyridinophanes at liquid/graphite interfaces by means of scanning tunnelling microscopy (STM). The purpose of this work was to elucidate the mixing behaviour of these macrocycles highlighting the formation of one‐ or two‐dimensionally ordered square tilings consisting of alternating alignments of different macrocycles as a result of attractive dipole–dipole or hydrogen‐bonding interactions; four co‐crystals differing in the dimensionality of the ordering of pyridinophane and cyclophane were observed. The different modes of interaction between the functional groups (ether or carbonyl group) in the side‐chains of the pyridinophanes and cyclophanes lead to the formation of co‐crystals with dimensionally different orderings of the two macrocycles. These observations revealed that a slight modification of the molecular structure may dramatically change the mixing behaviour and structures of the co‐crystals.     

Structure‐Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure‐based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein‐bound library member(s) by saturation‐transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.     

DNA‐Binding Properties of Amidine‐Substituted Spiropyran Photoswitches

Two amidine‐substituted spiropyran derivatives have been characterized with respect to the DNA‐binding properties over a broad pH interval. The two derivatives differ in the number of positive charges. By varying the pH, the protonation state of the derivatives is also changed, allowing for additional variations in the charge distribution. We show that the closed spiro isomer does not bind for either of the two derivatives, whereas the open merocyanine forms bind both in the protonated and in the nonprotonated state, but with dramatically different binding constants. Flow‐oriented linear dichroism (LD) measurements also show that there are differences in the binding modes between the various forms. We rationalize these differences in terms of structure and charge distribution.     

Identification and X‐ray Co‐crystal Structure of a Small‐Molecule Activator of LFA‐1‐ICAM‐1 Binding

Stabilization of protein–protein interactions by small molecules is a concept with few examples reported to date. Herein we describe the identification and X‐ray co‐crystal structure determination of IBE‐667, an ICAM‐1 binding enhancer for LFA‐1. IBE‐667 was designed based on the SAR information obtained from an on‐bead screen of tagged one‐bead one‐compound combinatorial libraries by confocal nanoscanning and bead picking (CONA). Cellular assays demonstrate the activity of IBE‐667 in promoting the binding of LFA‐1 on activated immune cells to ICAM‐1.