Lambda-ABF: Simplified, Portable, Accurate, and Cost-Effective Alchemical Free-Energy Computation.

We introduce the lambda-Adaptive Biasing Force (lambda-ABF) method for the computation of alchemical free-energy differences. We propose a software implementation and showcase it on biomolecular systems. The method arises from coupling multiple-walker adaptive biasing force with λ-dynamics. The sampling of the alchemical variable is continuous and converges toward a uniform distribution, making manual optimization of the λ schedule unnecessary. Contrary to most other approaches, alchemical free-energy estimates are obtained immediately without any postprocessing. Free diffusion of λ improves orthogonal relaxation compared to fixed-λ thermodynamic integration or free-energy perturbation. Furthermore, multiple walkers provide generic orthogonal space coverage with minimal user input and negligible computational overhead. We show that our high-performance implementations coupling the Colvars library with NAMD and Tinker-HP can address real-world cases including ligand-receptor binding with both fixed-charge and polarizable models, with a demonstrably richer sampling than fixed-λ methods. The implementation is fully open-source, publicly available, and readily usable by practitioners of current alchemical methods. Thanks to the portable Colvars library, lambda-ABF presents a unified user interface regardless of the back-end (NAMD, Tinker-HP, or any software to be interfaced in the future), sparing users the effort of learning multiple interfaces. Finally, the Colvars Dashboard extension of the visual molecular dynamics (VMD) software provides an interactive monitoring and diagnostic tool for lambda-ABF simulations.

Enforcing Local DNA Kinks by Sequence-Selective Trisintercalating Oligopeptides of a Tricationic Porphyrin: A Polarizable Molecular Dynamics Study.

Bisacridinyl-bisarginyl porphyrin (BABAP) is a trisintercalating derivative of a tricationic porphyrin, formerly designed and synthesized in order to selectively target and photosensitize the ten-base pair palindromic sequence d(CGGGCGCCCG)2 . We resorted to the previously derived (Far et al., 2004) lowest energy-minimized (EM) structure of the BABAP complex with this sequence as a starting point. We performed polarizable molecular dynamics (MD) on this complex. It showed, over a 150 ns duration, the persistent binding of the Arg side-chain on each BABAP arm to the two G bases upstream from the central porphyrin intercalation site. We subsequently performed progressive shortenings of the connector chain linking the Arg-Gly backbone to the acridine, from n=6 methylenes to 4, followed by removal of the Gly backbone and further connector shortenings, from n=4 to n=1. These resulted into progressive deformations ('kinks') of the DNA backbone. In its most accented kinked structure, the DNA backbone was found to have a close overlap with that of DNA bound to Cre recombinase, with, at the level of one acridine intercalation site, negative roll and positive tilt values consistent with those experimentally found for this DNA at its own kinked dinucleotide sequence. Thus, in addition to their photosensitizing properties, some BABAP derivatives could induce sequence-selective, controlled DNA deformations, which are targets for cleavage by endonucleases or for repair enzymes.

Targeting RNA:protein interactions with an integrative approach leads to the identification of potent YBX1 inhibitors.

RNA-protein interactions (RPIs) are promising targets for developing new molecules of therapeutic interest. Nevertheless, challenges arise from the lack of methods and feedback between computational and experimental techniques during the drug discovery process. Here, we tackle these challenges by developing a drug screening approach that integrates chemical, structural and cellular data from both advanced computational techniques and a method to score RPIs in cells for the development of small RPI inhibitors; and we demonstrate its robustness by targeting Y-box binding protein 1 (YB-1), a messenger RNA-binding protein involved in cancer progression and resistance to chemotherapy. This approach led to the identification of 22 hits validated by molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spectroscopy of which 11 were found to significantly interfere with the binding of messenger RNA (mRNA) to YB-1 in cells. One of our leads is an FDA-approved poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor. This work shows the potential of our integrative approach and paves the way for the rational development of RPI inhibitors.

Targeting the Major Groove of the Palindromic d(GGCGCC)2 Sequence by Oligopeptide Derivatives of Anthraquinone Intercalators.

GC-rich sequences are recurring motifs in oncogenes and retroviruses and could be targeted by noncovalent major-groove therapeutic ligands. We considered the palindromic sequence d(G1G2C3G4C5C6)2, and designed several oligopeptide derivatives of the anticancer intercalator mitoxantrone. The stability of their complexes with an 18-mer oligonucleotide encompassing this sequence in its center was validated using polarizable molecular dynamics. We report the most salient structural features of two novel compounds, having a dialkylammonium group as a side chain on both arms. The anthraquinone ring is intercalated in the central d(CpG)2 sequence with its long axis perpendicular to that of the two base pairs. On each strand, this enables each ammonium group to bind in-register to O6/N7 of the two facing G bases upstream. We subsequently designed tris-intercalating derivatives, each dialkylammonium substituted with a connector to an N9-aminoacridine intercalator extending our target range from a six- to a ten-base-pair palindromic sequence, d(C1G2G3G4C5G6C7C8C9G10)2. The structural features of the complex of the most promising derivative are reported. The present design strategy paves the way for designing intercalator-oligopeptide derivatives with even higher selectivity, targeting an increased number of DNA bases, going beyond ten.

Identification of a small molecule splicing inhibitor targeting UHM domains.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

The cooperative binding of TDP-43 to GU-rich RNA repeats antagonizes TDP-43 aggregation.

TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

Quantum-Chemistry Based Design of Halobenzene Derivatives With Augmented Affinities for the HIV-1 Viral G4/C16 Base-Pair.

The HIV-1 integrase (IN) is a major target for the design of novel anti-HIV inhibitors. Among these, three inhibitors which embody a halobenzene ring derivative (HR) in their structures are presently used in clinics. High-resolution X-ray crystallography of the complexes of the IN-viral DNA transient complex bound to each of the three inhibitors showed in all cases the HR ring to interact within a confined zone of the viral DNA, limited to the highly conserved 5'CpA 3'/5'TpG 3' step. The extension of its extracyclic CX bond is electron-depleted, owing to the existence of the "sigma-hole." It interacts favorably with the electron-rich rings of base G4. We have sought to increase the affinity of HR derivatives for the G4/C16 base pair. We thus designed thirteen novel derivatives and computed their Quantum Chemistry (QC) intermolecular interaction energies (ΔE) with this base-pair. Most compounds had ΔE values significantly more favorable than those of the HR of the most potent halobenzene drug presently used in clinics, Dolutegravir. This should enable the improvement in a modular piece-wise fashion, the affinities of halogenated inhibitors for viral DNA (vDNA). In view of large scale polarizable molecular dynamics simulations on the entirety of the IN-vDNA-inhibitor complexes, validations of the SIBFA polarizable method are also reported, in which the evolution of each ΔE(SIBFA) contribution is compared to its QC counterpart along this series of derivatives.

Water Dynamics Around Proteins: T- and R-States of Hemoglobin and Melittin.

The water dynamics, as characterized by the local hydrophobicity (LH), is investigated for tetrameric hemoglobin (Hb) and dimeric melittin. For the T0 to R0 transition in Hb, it is found that LH provides additional molecular-level insight into the Perutz mechanism, i.e., the breaking and formation of salt bridges at the α1/ß2 and α2/ß1 interface is accompanied by changes in LH. For Hb in cubic water boxes with 90 and 120 Å edge length it is observed that following a decrease in LH as a consequence of reduced water density or change of water orientation at the protein/water interface the α/ß interfaces are destabilized; this is a hallmark of the Perutz stereochemical model for the T to R transition in Hb. The present work thus provides a dynamical view of the classical structural model relevant to the molecular foundations of Hb function. For dimeric melittin, earlier results by Cheng and Rossky [ Nature 1998, 392, 696-699] are confirmed and interpreted on the basis of LH from simulations in which the protein structure is frozen. For the flexible melittin dimer, the changes in the local hydration can be as much as 30% greater than for the rigid dimer, reflecting the fact that protein and water dynamics are coupled.

Strong Enrichment of Aromatic and Sulfur-Containing Residues in Ligand-Protein Binding Sites.

While certain residues have clear involvement in determining the 3D structure of a macromolecule because they affect the folding topology or the overall protein stability, the role of different residues in ligand accommodation and binding has attracted less attention. On the basis of the assumption that drug-binding sites on target molecules have specific amino acid compositions, the incidence of each standard amino acid at the binding sites of small molecules and their correlations are calculated for an unprecedented large set of high-quality X-ray structures. Results show, for the first time, strong and highly correlated enrichments of aromatic and sulfur-containing residues, which play an important role in ligand binding and shape the nature of the chemical interactions.

Response to comment on 'Valid molecular dynamics simulations of human hemoglobin require a surprisingly large box size'.

We recently reported that molecular dynamics simulations for hemoglobin require a surprisingly large box size to stabilize the T(0) state relative to R(0), as observed in experiments (El Hage et al., 2018). Gapsys and de Groot have commented on this work but do not provide convincing evidence that the conclusions of El Hage et al., 2018 are incorrect. Here we respond to these concerns, argue that our original conclusions remain valid, and raise our own concerns about some of the results reported in the comment by Gapsys and de Groot that require clarification.

Valid molecular dynamics simulations of human hemoglobin require a surprisingly large box size.

Recent molecular dynamics (MD) simulations of human hemoglobin (Hb) give results in disagreement with experiment. Although it is known that the unliganded (T[Formula: see text]) and liganded (R[Formula: see text]) tetramers are stable in solution, the published MD simulations of T[Formula: see text] undergo a rapid quaternary transition to an R-like structure. We show that T[Formula: see text] is stable only when the periodic solvent box contains ten times more water molecules than the standard size for such simulations. The results suggest that such a large box is required for the hydrophobic effect, which stabilizes the T[Formula: see text] tetramer, to be manifested. Even in the largest box, T[Formula: see text] is not stable unless His146 is protonated, providing an atomistic validation of the Perutz model. The possibility that extra large boxes are required to obtain meaningful results will have to be considered in evaluating existing and future simulations of a wide range of systems.

From in silica to in silico: retention thermodynamics at solid-liquid interfaces.

The dynamics of solvated molecules at the solid/liquid interface is essential for a molecular-level understanding for the solution thermodynamics in reversed phase liquid chromatography (RPLC). The heterogeneous nature of the systems and the competing intermolecular interactions makes solute retention in RPLC a surprisingly challenging problem which benefits greatly from modelling at atomistic resolution. However, the quality of the underlying computational model needs to be sufficiently accurate to provide a realistic description of the energetics and dynamics of systems, especially for solution-phase simulations. Here, the retention thermodynamics and the retention mechanism of a range of benzene-derivatives in C18 stationary-phase chains in contact with water/methanol mixtures is studied using point charge (PC) and multipole (MTP) electrostatic models. The results demonstrate that free energy simulations with a faithful MTP representation of the computational model provide quantitative and molecular level insight into the thermodynamics of adsorption/desorption in chromatographic systems while a conventional PC representation fails in doing so. This provides a rational basis to develop more quantitative and validated models for the optimization of separation systems.

The Role of Water in the Stability of Wild-type and Mutant Insulin Dimers.

Insulin dimerization and aggregation play important roles in the endogenous delivery of the hormone. One of the important residues at the insulin dimer interface is PheB24, which is an invariant aromatic anchor that packs toward its own monomer inside a hydrophobic cavity formed by ValB12, LeuB15, TyrB16, CysB19, and TyrB26. Using molecular dynamics and free-energy simulations within explicit solvent, the structural and dynamical consequences of mutations of Phe at position B24 to glycine (Gly), alanine (Ala), and d-Ala and the des-PheB25 variant are quantified. Consistent with experiments, it is found that the Gly and Ala modifications lead to insulin dimers with reduced stability by 4 and 5 kcal/mol from thermodynamic integration and 4 and 8 kcal/mol from results using molecular mechanics-generalized Born surface area, respectively. Given the experimental difficulties to quantify the thermodynamic stability of modified insulin dimers, such computations provide a valuable complement. Interestingly, the Gly mutant exists as a strongly and a weakly interacting dimer. Analysis of the molecular dynamics simulations shows that this can be explained by water molecules that replace direct monomer-monomer H-bonding contacts at the dimerization interface involving residues B24 to B26. It is concluded that such solvent molecules play an essential role and must be included in future insulin dimerization studies.

Erratum: Publisher's Note: "Implications of short time scale dynamics on long time processes" (Struct. Dyn. 4, 061507 (2017)].

[This corrects the article DOI: 10.1063/1.4996448.].

Molecular Mechanisms Underlying Solute Retention at Heterogeneous Interfaces.

Despite considerable effort, a molecular-level understanding of the mechanisms governing adsorption/desorption in reversed-phase liquid chromatography is still lacking. This impedes rational design of columns and the development of reliable, computationally more efficient approaches to predict the selectivity of a particular column design. Using state-of-the art, validated force fields and free-energy simulations, the adsorption thermodynamics of benzene derivatives is investigated in atomistic detail and provides a quantitative microscopic understanding of retention when compared with experimental data. It is found that pure partitioning or pure adsorption is rather the exception than the rule. Typically, a pronounced ∼1 kcal/mol stabilization on the surface is accompanied by a broad trough indicative of partitioning before the probe molecule incorporates into the mobile phase. The present findings provide a quantitative and rational basis to develop improved effective, coarse-grained computational models and to design columns for specific applications.

A Simple Isomerization of the Purine Scaffold of a Kinase Inhibitor, Roscovitine, Affords a Four- to Seven-Fold Enhancement of Its Affinity for Four CDKs. Could This Be Traced Back to Conjugation-Induced Stiffenings/Loosenings of Rotational Barriers?

Roscovitine is an antitumor purine inhibitor of cyclin-dependent kinase CDK5, for which it displays submicromolar affinity. It reached phase IIb clinical trials in 2007. The search for analogues with improved kinase affinities led recently to an isomer, finisterine, having a nearly 10-fold greater affinity for both CDK5 and CDK9. It solely differs by the displacement of one nitrogen atom in the purine ring, from position 6 to position 9. This has no incidence on the intermolecular interaction of either drug with the neighboring sites that anchor the ring in the recognition site. Quantum chemistry calculations combined with conformational and topological analyses of the impact of the purine ring isomerization of roscovitine and finisterine on its conformational stability show that the modified electronic conjugation, on the other hand, results in a stiffening of the rotational barrier around the extracyclic C-NH bond of finisterine, vicinal to N9, and to which an aryl ring is connected, along with a loosening of the barrier around an extracyclic C6-C bond connecting to a shorter, hydrophobic arm. The first effect is proposed to lead to a lesser hydration entropy of solvation in the case of finisterine, thus to a facilitated desolvation term in the overall energy balances.

Implications of short time scale dynamics on long time processes.

This review provides a comprehensive overview of the structural dynamics in topical gas- and condensed-phase systems on multiple length and time scales. Starting from vibrationally induced dissociation of small molecules in the gas phase, the question of vibrational and internal energy redistribution through conformational dynamics is further developed by considering coupled electron/proton transfer in a model peptide over many orders of magnitude. The influence of the surrounding solvent is probed for electron transfer to the solvent in hydrated I-. Next, the dynamics of a modified PDZ domain over many time scales is analyzed following activation of a photoswitch. The hydration dynamics around halogenated amino acid side chains and their structural dynamics in proteins are relevant for iodinated TyrB26 insulin. Binding of nitric oxide to myoglobin is a process for which experimental and computational analyses have converged to a common view which connects rebinding time scales and the underlying dynamics. Finally, rhodopsin is a paradigmatic system for multiple length- and time-scale processes for which experimental and computational methods provide valuable insights into the functional dynamics. The systems discussed here highlight that for a comprehensive understanding of how structure, flexibility, energetics, and dynamics contribute to functional dynamics, experimental studies in multiple wavelength regions and computational studies including quantum, classical, and more coarse grained levels are required.