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.

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.

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.

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.

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.

Extending Halogen-based Medicinal Chemistry to Proteins: IODO-INSULIN AS A CASE STUDY.

Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (TyrB26) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-TyrB26]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (TyrB16, PheB24, PheB25, 3-I-TyrB26, and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-TyrB26]insulin analog (determined as an R6 zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-TyrB26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-TyrB26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.

A Toolkit to Fit Nonbonded Parameters from and for Condensed Phase Simulations.

The quality of atomistic simulations depends decisively on the accuracy of the underlying energy function (force field). Of particular importance for condensed-phase properties are nonbonded interactions, including the electrostatic and Lennard-Jones terms. Permanent atomic multipoles (MTPs) are an extension to common point-charge (PC) representations in atomistic simulations. MTPs are commonly determined from and fitted to an ab initio Electrostatic Potential (ESP), and Lennard-Jones (LJ) parameters are obtained from comparison of experimental and computed observables using molecular dynamics (MD) simulations. For this a set of thermodynamic observables such as density, heat of vaporization, and hydration free energy is chosen, to which the parametrization is fitted. The current work introduces a comprehensive computing environment (Fitting Wizard (FW)) for optimizing nonbonded interactions for atomistic force fields of different qualities. The FW supports fitting of standard PC-based force fields and more physically motivated multipolar (MTP) force fields. A broader study including 20 molecules ranging from N-methyl-acetamide and benzene to halogenated benzenes, phenols, anilines, and pyridines yields a root mean squared deviation for hydration free energies of 0.36 kcal/mol over a range of 8 kcal/mol. It is furthermore shown that PC-based force fields are not necessarily inferior compared to MTP parametrizations depending on the molecule considered.

Could the "Janus-like" properties of the halobenzene CX bond (X=Cl, Br) be leveraged to enhance molecular recognition?

The CX bond in halobenzenes (XCl, Br) exhibits a dual character, being electron-deficient along the CX direction, and electron-rich on its flanks. We sought to amplify both features by resorting to electron-withdrawing and electron-donating substituents, respectively. This was done by quantum chemistry (QC) computations in the recognition sites of three protein targets: farnesyl transferase, coagulation factor Xa, and the HIV-1 integrase. In this context, some substituents, notably fluorine, CF3 , and NHCH3 , afforded significant overall gains in the binding energies as compared to the parent halobenzene, in the 2-5 kcal/mol range. In fact, we found that some di- and up to tetra-substitutions enabled even larger gains than those they contribute separately owing to many-body effects. Moreover, desolvation was also found to be a key contributor to the energy balances. As a consequence, some particular substituents, contributing to reduce the halobenzene dipole moment, accordingly reduced solvation: this factor acted in synergy with their enhancement of the intermolecular interaction energies along and around the CX bond. We could thus leverage the "Janus-like" properties of such a bond and the fact that it can be tuned and possibly amplified by well-chosen substituents. We propose a simple yet rigorous computational strategy resorting to QC to prescreen novel substituted halobenzenes. The QC results on the recognition sites then set benchmarks to validate polarizable molecular mechanics/dynamics approaches used to handle the entirety of the inhibitor-protein complex.

Polarizable molecular mechanics studies of Cu(I)/Zn(II) superoxide dismutase: bimetallic binding site and structured waters.

The existence of a network of structured waters in the vicinity of the bimetallic site of Cu/Zn-superoxide dismutase (SOD) has been inferred from high-resolution X-ray crystallography. Long-duration molecular dynamics (MD) simulations could enable to quantify the lifetimes and possible interchanges of these waters between themselves as well as with a ligand diffusing toward the bimetallic site. The presence of several charged or polar ligands makes it necessary to resort to second-generation polarizable potentials. As a first step toward such simulations, we benchmark in this article the accuracy of one such potential, sum of interactions between fragments Ab initio computed (SIBFA), by comparisons with quantum mechanics (QM) computations. We first consider the bimetallic binding site of a Cu/Zn-SOD, in which three histidines and a water molecule are bound to Cu(I) and three histidines and one aspartate are bound to Zn(II). The comparisons are made for different His6 complexes with either one or both cations, and either with or without Asp and water. The total net charges vary from zero to three. We subsequently perform preliminary short-duration MD simulations of 296 waters solvating Cu/Zn-SOD. Six representative geometries are selected and energy-minimized. Single-point SIBFA and QM computations are then performed in parallel on model binding sites extracted from these six structures, each of which totals 301 atoms including the closest 28 waters from the Cu metal site. The ranking of their relative stabilities as given by SIBFA is identical to the QM one, and the relative energy differences by both approaches are fully consistent. In addition, the lowest-energy structure, from SIBFA and QM, has a close overlap with the crystallographic one. The SIBFA calculations enable to quantify the impact of polarization and charge transfer in the ranking of the six structures. Five structural waters, which connect Arg141 and Glu131, are endowed with very high dipole moments (2.7-3.0 Debye), equal and larger than the one computed by SIBFA in ice-like arrangements (2.7 D).

Substituent-modulated affinities of halobenzene derivatives to the HIV-1 integrase recognition site. Analyses of the interaction energies by parallel quantum chemical and polarizable molecular mechanics.

The C-X bond of halobenzenes (X = Cl, Br) has a dual character, its electron density being depleted in its prolongation and built-up on its sides. We have recently considered three protein or nucleic acid recognition sites of halobenzenes and quantified the energy gains that either electron-attracting substituents or electron-donating ones contribute due to such a character (El Hage et al., paper in revision). Nonadditivity was found to impact the total interaction energies. We focus here on one recognition site, that of the HIV-1 integrase, in which the halobenzene ring of the drug elvitegravir is sandwiched between a guanine and a cytosine base. We perform energy-decomposition analyses of the ab initio quantum-chemistry (QC) binding energies of the parent halobenzene ring and its derivatives with this G-C base pair. In these complexes, the nonadditivity of ΔE could be traced back mostly to the polarization contribution Epol. In view of large-scale applications to the entirety of the complex formed between the integrase, the viral DNA, and the whole drug, the analyses were performed in parallel with a polarizable molecular mechanics method, SIBFA. This method could faithfully reproduce most features of the QC energies. This is due to its use of QC-derived distributed multipoles and polarizabilities, which enable us to account for both nonisotropy and nonadditivity.

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.

Could an anisotropic molecular mechanics/dynamics potential account for sigma hole effects in the complexes of halogenated compounds?

Halogenated compounds are gaining an increasing importance in medicinal chemistry and materials science. Ab initio quantum chemistry (QC) has unraveled the existence of a "sigma hole" along the C-X (X = F, Cl, Br, I) bond, namely, a depletion of electronic density prolonging the bond, concomitant with a build-up on its sides, both of which are enhanced along the F < Cl < Br < I series. We have evaluated whether these features were intrinsically built-in in an anisotropic, polarizable molecular mechanics (APMM) procedure such as SIBFA (sum of interactions between fragments ab initio computed). For that purpose, we have computed the interaction energies of fluoro-, chloro-, and bromobenzene with two probes: a divalent cation, Mg(II), and water approaching X through either one H or its O atom. This was done by parallel QC energy-decomposition analyses (EDA) and SIBFA computations. With both probes, the leading QC contribution responsible for the existence of the sigma hole is the Coulomb contribution E(c). For all three halogenated compounds, and with both probes, the in- and out-of-plane angular features of E(c) were closely mirrored by the SIBFA electrostatic multipolar contribution (E(MTP)). Resorting to such a contribution thus dispenses with empirically-fitted "extra", off-centered partial atomic charges as in classical molecular mechanics/dynamics.

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.

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.

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.

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.

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.

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.