Illuminating HIV gp120-Ligand Recognition through Computationally-Driven Optimization of Antibody-Recruiting Molecules.

Exploration of binding sites of ligands (drug candidates) on macromolecular targets is a central question of molecular design. Although there are experimental and theoretical methods available for the determination of atomic resolution structure of drug-target complexes, they are often limited to identify only the primary binding mode (site and conformation). Systematic exploration of multiple (allosteric or prerequisite) binding modes is a challenge for present methods. The Wrapper module of our new method, Wrap 'n' Shake, answers this challenge by a fast, computational blind docking approach. Beyond the primary (orthosteric) binding mode, Wrapper systematically produces all possible binding modes of a drug scanning the entire surface of the target. In several fast blind docking cycles, the entire surface of the target molecule is systematically wrapped in a monolayer of N ligand copies. The resulted target-ligandN complex structure can be used as it is, or important ligand binding modes can be further distinguished in molecular dynamics shakers. Wrapper has been tested on important protein targets of drug design projects on cellular signaling and cancer. Here, we provide a practical description of the application of Wrapper.

The present article describes a conceptual view on the design of reusable bioderived high-value-added materials. The translation of a highly complex irregular structure of natural biopolymer into a well-defined hierarchically organized molecular chain led to the discovery of unique adhesive properties enhanced by a novel multiple binding effect. For practical applications, biomass-derived furanic polyesters were found as reusable thermoplastic adhesives. Examined poly(ethylene-2,5-furandicarboxylate) (PEF) and poly(hexamethylene-2,5-furandicarboxylate) (PHF) showed strong adhesion to aluminum in single-lap shear tests (1.47 ± 0.1 and 1.18 ± 0.1 kN/cm2, respectively). After the separation, the joints could be easily restored by reheating of the metal parts. Three consecutive cycles of regluing were successfully performed without a significant drop in the adhesive strength. Strong adhesion of the biomass-derived polymers to glass surfaces was also observed (0.93 ± 0.11 kN/cm2 for PEF and 0.84 ± 0.06 kN/cm2 for PHF). An in-depth study of the surfaces after the shear tests, carried out by means of scanning electron microscopy, revealed predominantly cohesive failure in the case of aluminum samples and adhesive failure in the case of glass samples. Computational modeling revealed a multiple oxygen binding mode for the interaction of furanic polyester molecules with the glass surface and metal atoms. Only sustainable materials were used as a carbon source for the production of target polymers, which showed excellent compatibility with the practically most demanding constructing materials (a universal reusable hot-melt adhesive for copper, brass, Be-copper, Mn-bronze, zinc, aluminum, titanium, and glass).

Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1DBD forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7-2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS·tRNACUAPyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.

Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes

Feiner Unterschied: Einzelmolekülkraftspektroskopie wurde zur Untersuchung von DNA-Intercalierungen in An- und Abwesenheit ungebundener Farbstoffmoleküle genutzt (siehe Bild). Die kraftinduzierte Intercalierung, traditionell der Farbstoffbindung an DNA aus Lösung zugeschrieben, war auch noch 2 h nach dem Entfernen freier Farbstoffmoleküle zu beobachten. Bindung/Abspaltung und Intercalierung/Desintercalierung sind offenbar eigenständige Prozesse auf verschiedenen Zeitskalen. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation...

In silico modeling of toluene binding site in the pore of voltage-gate sodium channel Thomas RF Scior1, Evelyn Martínez-Morales2, Silvia L Cruz3, Eduardo M Salinas-Stefanon21Departamento de Farmacia; 2Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla, Pue, México; 3Departamento de Farmacobiología, CINVESTAV-IPN, Calz. Col. Granjas Coapa, MéxicoAbstract: Toluene is a commonly used organic solvent in commercial products and is sometimes abused as an inhalative hallucinogen, causing arrythmogenic toxicity. At a molecular level we investigated whether a hypothetical interaction model could be devised for the reported myo- and cardiotoxic effects of toluene. Three lines of computed evidence support our hypothesis on the interaction mechanism: (i) Toluene binds at the local anesthetic binding site (LABS), on the wild type (WT) but not on its F1579A mutation, confirming our experimental findings that it inhibits only the WT of skeletal muscle or cardiac isoforms (Nav 1.4 or 1.5). (ii) Typically for small alkylaryl moiety, multiple binding modes were detected during docking. Toluene is trapped in the tryptophane-rich area at the extracellular vestibule by hydrophobic interaction, mainly π–π stacking, or bound to the LABS with equal binding strength and number of solved poses, mostly by edge-to-face contacts. (iii) The computed loss of toluene binding at the LABS on the mutant model parallels clearly the observed loss of toluene effects on Nav 1.4. Moreover, we inspected the complete primary sequences with the omitted loops in the 3D models to identify the possible interacting amino acids among the 16% nonidentical ones, and thus confirmed the observed toxicity effects.Keywords: Toluene, Na+ channel, Nav1.4 isoform, Nav1.5 isoform, in-silico simulation, cardiotoxicity

The current mini-review explains how fragmental methods (FMs) can be used in the analysis and prediction of physicochemical properties and biological activities. The considered properties include log P, solubility, pK(a), intestinal permeability, P-gp substrate specificity and toxicity. The focus will be a description of a "mechanistic" approach, which implies a gradual reduction of alternative explanations for any property or activity. This means a flexible construction of fragmental parameters using large amounts of experimental data. Since biological activities involve multiple (unknown) target macromolecules with multiple binding modes, a stepwise classification (C-SAR) analysis is most useful. It involves the following procedures: (i). construction of physicochemical profiles using parameters that can be reliably predicted, (ii). identification of reactive functional groups and the largest active skeletons, (iii). generalization of these groups and skeletons in terms of "site-specific physicochemical profiling". This entails a dynamic construction of 2D pharmacophores that can be converted into 3D models.

The binding of a platinum intercalator complex [Pt(1C3)(dien)](2+) (1C3 = 1-[(3-aminopropyl)amino]-anthracene-9,10-dione, dien = 3-azapentane-1,5-diamine) to DNA and to the self-complementary oligonucleotide 5'-d(TGGCCA)-3' has been investigated by UV-visible spectrophotometry and 2D NMR spectroscopy, respectively. The uncomplexed anthraquinone, 1C3, has an apparent DNA binding constant of 1.4 x 10(4), similar to that of ethidium bromide. Addition of the coordinatively saturated {Pt(dien)} moiety increases the binding constant to 3.7 x 10(5) M(-1), showing the effect of the increased positive charge introduced by this moiety. Multiple binding modes are evident from the lack of isosbestic points in the titration spectra and the non-linear nature of the half-reciprocal plot used to calculate the binding constant. [Pt(1C3)(dien)](2+) forms a 2 : 1 adduct with 5'-d(TGGCCA)-3' and is shown by 2D NMR to intercalate primarily in the TG:CA base pairs at the ends of the oligonucleotide with the side chain and {Pt(dien)} situated in the minor groove.

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes.

Microsecond Dynamics of Protein–DNA Interactions: Direct Observation of the Wrapping/Unwrapping Kinetics of Single-stranded DNA around the E. coli SSB Tetramer

Recentadvances in improved force fields and sampling methods have made itpossible for the accurate calculation of protein–ligand bindingfree energies. Alchemical free energy perturbation (FEP) using anexplicit solvent model is one of the most rigorous methods to calculaterelative binding free energies. However, for cases where there arehigh energy barriers separating the relevant conformations that areimportant for ligand binding, the calculated free energy may dependon the initial conformation used in the simulation due to the lackof complete sampling of all the important regions in phase space.This is particularly true for ligands with multiple possible bindingmodes separated by high energy barriers, making it difficult to sampleall relevant binding modes even with modern enhanced sampling methods.In this paper, we apply a previously developed method that providesa corrected binding free energy for ligands with multiple bindingmodes by combining the free energy results from multiple alchemicalFEP calculations starting from all enumerated poses, and the resultsare compared with Glide docking and MM-GBSA calculations. From thesecalculations, the dominant ligand binding mode can also be predicted.We apply this method to a series of ligands that bind to c-Jun N-terminalkinase-1 (JNK1) and obtain improved free energy results. The dominantligand binding modes predicted by this method agree with the availablecrystallography, while both Glide docking and MM-GBSA calculationsincorrectly predict the binding modes for some ligands. The methodalso helps separate the force field error from the ligand samplingerror, such that deviations in the predicted binding free energy fromthe experimental values likely indicate possible inaccuracies in theforce field. An error in the force field for a subset of the ligandsstudied was identified using this method, and improved free energyresults were obtained by correcting the partial charges assigned tothe ligands. This improved the root-mean-square error (RMSE) for thepredicted binding free energy from 1.9 kcal/mol with the originalpartial charges to 1.3 kcal/mol with the corrected partial charges.

Certain phenylethylamines, such as 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane (DOB; 1a), are high-affinity 5-HT(2) agonists. Previous structure-affinity studies have concluded that both the 2,5-dimethoxy substitution pattern and the nature of substituents at the 4-position are important determinants of high affinity. We recently demonstrated that replacement of the bromo group of DOB with a 3-(phenyl)propyl substituent results in retention of affinity and that, counter to established structure-affinity relationships, the 2,5-dimethoxy substitution pattern is no longer a requirement for the binding. The present investigation extends these findings by examining a series of analogues, 3, lacking a 5-methoxy group. It was additionally found that shifting the phenylalkyl substituent from the 4- to the 5-position (e.g., 4i) also results in retention of affinity. For example, 1-(2-methoxy-5-(3-phenylpropyl)-2-aminopropane (6; the alpha-methyl derivative of 4i) binds at 5-HT(2A) receptors with high affinity (K(i) = 13 nM) and possesses 5-HT(2A) antagonist character. Thus, not only is the 2,5-dimethoxy substitution pattern not a requirement for the binding of certain phenylethylamines at 5-HT(2A) receptors, the presence of a 4-position substituent (previously thought to serve as a modulator of affinity of DOB-like agents) is also not required. Striking differences in the 5-HT(2A) binding requirements of the present compounds as compared to DOB-like agents suggest multiple substituent-dependent modes of binding.

The DNA-encoded library (DEL) discovery platform has emerged as a powerful technology for hit identification in recent years. It has become one of the major parallel workstreams for small molecule drug discovery along with other strategies such as HTS and data mining. For many researchers working in the DEL field, it has become increasingly evident that many hits and leads discovered via DEL screening bind to target proteins with unique and unprecedented binding modes. This Perspective is our attempt to analyze reports of DEL screening with the purpose of providing a rigorous and useful account of the binding modes observed for DEL-derived ligands with a focus on binding mode novelty.