p38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

Regioswitchable Bingel Bis-Functionalization of Fullerene C70 via Supramolecular Masks.

Isomer-pure functionalized fullerenes are required to boost the development of fullerene chemistry in any field, but their multiple functionalization renders a mixture of regioisomers that are very difficult to purify by chromatography. For the specific case of C70, its nonspherical geometry makes its regioselective functionalization more challenging than that of spherical C60. In this work, the supramolecular mask approach is applied for the first time to C70, which is encapsulated in two different nanocapsules to achieve the Bingel bis-cyclopropanation at α-bonds of opposite poles. Based on the tetragonal prismatic geometry imposed by the smaller supramolecular mask tested, the obtained major bis-adduct is completely reversed (major 5 o'clock) compared to bare C70 functionalization (major 2 o'clock). Moreover, by further restricting the accessibility of C70 using a three-shell Matryoshka mask and dibenzyl-bromomalonate, a single regiospecific 2 o'clock bis-isomer is obtained, owing to the perfect complementarity of the mask and the addend steric properties. The outcome of the reactions is fully explained at the molecular level by means of a thorough molecular dynamics (MD) study of the accessibility of the α-bonds to produce the different bis-adducts.

Time Evolution of the Millisecond Allosteric Activation of Imidazole Glycerol Phosphate Synthase.

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we combine extensive molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the substrate-free form to the active ternary complex. IGPS is a heterodimeric bienzyme complex whose HisH subunit is responsible for hydrolyzing glutamine and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with oxyanion hole formation, in line with the experimentally observed 4500-fold activity increase in glutamine hydrolysis. The allosteric activation is controlled by correlated time-evolving dynamic networks connecting the effector and substrate binding sites. This computational strategy tailored to describe millisecond events can be used to rationalize the effect of mutations on the allosteric regulation and guide IGPS engineering efforts.

Three-centre electron sharing indices (3c-ESIs) as a tool to differentiate among (an)agostic interactions and hydrogen bonds in transition metal complexes.

The agostic bond plays an important role in chemistry, not only in transition metal chemistry but also in main group chemistry. In some complexes with M⋯H-X (X = C, N) interactions, differentiation among agostic, anagostic, and hydrogen bonds is challenging. Here we propose the use of three-centre electron sharing indices to classify M⋯H-X (X = C, N) interactions.

Machine Learning Enables Selection of Epistatic Enzyme Mutants for Stability Against Unfolding and Detrimental Aggregation.

Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov'SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside-the-box, predictions not found in other state-of-the-art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness.

Comprehensive Characterization of the Self-Folding Cavitand Dynamics.

The conformational equilibria and guest exchange process of a resorcin[4]arene derived self-folding cavitand receptor have been characterized in detail by molecular dynamics simulations (MD) and 1 H EXSY NMR experiments. A multi-timescale strategy for exploring the fluxional behaviour of this system has been constructed, exploiting conventional MD and accelerated MD (aMD) techniques. The use of aMD allows the reconstruction of the folding/unfolding process of the receptor by sampling high-energy barrier processes unattainable by conventional MD simulations. We obtained MD trajectories sampling events occurring at different timescales from ns to s: 1) rearrangement of the directional hydrogen bond seam stabilizing the receptor, 2) folding/unfolding of the structure transiting partially open intermediates, and 3) guest departure from different folding stages. Most remarkably, reweighing of the biased aMD simulations provided kinetic barriers that are in very good agreement with those determined experimentally by 1 H NMR. These results constitute the first comprehensive characterization of the complex dynamic features of cavitand receptors. Our approach emerges as a valuable rational design tool for synthetic host-guest systems.

Complete Dynamic Reconstruction of C60, C70, and (C59N)2 Encapsulation into an Adaptable Supramolecular Nanocapsule.

The dynamic adaptability of tetragonal prismatic nanocapsule 18+ in the selective separation of fullerenes and endohedral metallofullerenes (EMFs) remains unexplored. Therefore, the essential molecular details of the fullerene recognition and binding process into the coordination capsule and the origins of fullerene selectivity remain elusive. In this work, the key steps of fullerene recognition and binding processes have been deciphered by designing a protocol which combines 1H-1H exchange spectroscopy (2D-EXSY) NMR experiments, long time-scale Molecular Dynamics (MD) and accelerated Molecular Dynamics (aMD) simulations, which are combined to completely reconstruct the spontaneous binding and unbinding pathways from nanosecond to second time-range. On one hand, binding (k'on) and unbinding (koff) rate constants were extracted from 1H-1H exchange spectroscopy (EXSY) NMR experiments for both C60 and C70. On the other hand, MD and aMD allowed monitoring the molecular basis of the encapsulation and guest competition processes at a very early stage under nonequilibrium conditions. The receptor capsule displays dynamical adaptability features similar to those observed in the process of biomolecular recognition in proteins. In addition, the encapsulation of bis-aza[60]fullerene (C59N)2 within a supramolecular coordination capsule has been studied for the first time, showcasing the pros and cons of the dumbbell-shaped guest in the dynamics of the encapsulation process and in the stability of the final bound adduct. The powerful combination of NMR, MD, and aMD methodologies allows to obtain a precise picture of the subtle events directing the encapsulation and is thus a predictive tool for understanding host-guest encapsulation and interactions in numerous supramolecular systems.

Hidden Conformations in Aspergillus niger Monoamine Oxidase are Key for Catalytic Efficiency.

Enzymes exist as an ensemble of conformational states, whose populations can be shifted by substrate binding, allosteric interactions, but also by introducing mutations to their sequence. Tuning the populations of the enzyme conformational states through mutation enables evolution towards novel activity. Herein, Markov state models are used to unveil hidden conformational states of monoamine oxidase from Aspergillus niger (MAO-N). These hidden conformations, not previously observed by any other technique, play a crucial role in substrate binding and enzyme activity. This reveals how distal mutations regulate MAO-N activity by stabilizing these hidden, catalytically important conformational states, but also by modulating the communication pathway between both MAO-N subunits.

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates.

Metal Cluster Electrides: A New Type of Molecular Electride with Delocalised Polyattractor Character.

Electrides are ionic substances containing isolated electrons. These confined electrons are topologically characterised by a quasi-atom, that is, a non-nuclear attractor (NNA) of the electron density. The electronic structure of the octahedral 4 A1g Li6+ and 5 A1g Be6 species shows that these species have a large number of NNAs. These NNAs have highly delocalised electron densities and, as a result, the chemical bonding pattern of these systems is reminiscent of that in solid metals, in which metal cations are surrounded by a "sea" of delocalised valence electrons. We propose the term metal cluster electrides to refer to this new class of compounds. In this study, we establish a computational protocol to identify, characterize, and design metal cluster electrides and we elucidate the intricate bonding patterns of this particular type of species.

Electron correlation effects in third-order densities.

The electronic energy of a system of fermions can be obtained from the second-order reduced density matrix through the contracted Schrödinger equation or its anti-Hermitian counterpart. Both energy expressions depend on the third-order reduced density matrix (3-RDM) which is usually approximated from lower-order densities. The accuracy of these methods depends critically on the set of N-representability conditions enforced in the calculation and the quality of the approximate 3-RDM. There are no benchmark studies including most 3-RDM approximations and, thus far, no assessment of the deterioration of the approximations with correlation effects has been performed. In this paper we introduce a series of tests to assess the performance of 3-RDM approximations in a model system with varying electron correlation effects, the three-electron harmonium atom. The results of this work put forward several limitations of the currently most used 3-RDM approximations for systems with important electron correlation effects.

Analysis of a Compound Class with Triplet States Stabilized by Potentially Baird Aromatic [10]Annulenyl Dicationic Rings.

The low-lying triplet state of a recently published compound (TMTQ) was analyzed quantum chemically in light of suggestions that it is influenced by Baird aromaticity. Two mesomeric structures describe this state: 1) a zwitterionic Baird aromatic structure with a triplet diradical 8π-electron methano[10]annulene (M10A) dicationic ring and 2) a Hückel aromatic with a neutral closed-shell 10π-electron ring. According to charge and spin density distributions, the Hückel aromatic structure dominates the triplet state (the Baird aromatic contributes at most 12 %), and separation of the aromatic fluctuation index (FLU) into α and ß electron contributions emphasizes this finding. The small singlet-triplet energy gap is due to Hückel aromaticity of the M10A ring, clarified by comparison to the smaller analogues of TMTQ. Yet, TMTQ and its analogues are Hückel-Baird hybrids allowing for tuning between closed-shell 4n+2 Hückel aromaticity and open-shell 4n Baird aromaticity.

Octahedral aromaticity in (2S+1)A1g X6(q) clusters (X = Li-C and Be-Si, S = 0-3, and q = -2 to +4).

It is generally observed that quintessential aromatic compounds have delocalised electronic configurations that are of closed-shells or open-shells half-filled with the same spin electrons. Guided by this property, we search for aromatic octahedral clusters of the type X6(q) (X = Li-C and Be-Si, q = -2 to +4) in (2S+1)A1g electronic states with spin multiplicities ranging from the singlet to the septet. With some exceptions, we find that closed-shells or open-shells half-filled with same spin electron systems have large multicentre indices and negative NICS values that are characteristic patterns of aromatic compounds. Our results confirm the existence of octahedral aromaticity but do not allow us to define a general rule for octahedral aromaticity because the ordering of molecular orbitals does not remain the same for different octahedral clusters.

Quantifying aromaticity with electron delocalisation measures.

Aromaticity cannot be measured directly by any physical or chemical experiment because it is not a well-defined magnitude. Its quantification is done indirectly from the measure of different properties that are usually found in aromatic compounds such as bond length equalisation, energetic stabilisation, and particular magnetic behaviour associated with induced ring currents. These properties have been used to set up the myriad of structural-, energetic-, and magnetic-based indices of aromaticity known to date. The cyclic delocalisation of mobile electrons in two or three dimensions is probably one of the key aspects that characterise aromatic compounds. However, it has not been until the last decade that electron delocalisation measures have been widely employed to quantify aromaticity. Some of these new indicators of aromaticity such as the PDI, FLU, ING, and INB were defined in our group. In this paper, we review the different existing descriptors of aromaticity that are based on electron delocalisation properties, we compare their performance with indices based on other properties, and we summarise a number of applications of electronic-based indices for the analysis of aromaticity in interesting chemical problems.

Accelerated molecular dynamics simulations of protein folding.

Folding of four fast-folding proteins, including chignolin, Trp-cage, villin headpiece and WW domain, was simulated via accelerated molecular dynamics (aMD). In comparison with hundred-of-microsecond timescale conventional molecular dynamics (cMD) simulations performed on the Anton supercomputer, aMD captured complete folding of the four proteins in significantly shorter simulation time. The folded protein conformations were found within 0.2-2.1 Å of the native NMR or X-ray crystal structures. Free energy profiles calculated through improved reweighting of the aMD simulations using cumulant expansion to the second-order are in good agreement with those obtained from cMD simulations. This allows us to identify distinct conformational states (e.g., unfolded and intermediate) other than the native structure and the protein folding energy barriers. Detailed analysis of protein secondary structures and local key residue interactions provided important insights into the protein folding pathways. Furthermore, the selections of force fields and aMD simulation parameters are discussed in detail. Our work shows usefulness and accuracy of aMD in studying protein folding, providing basic references in using aMD in future protein-folding studies.

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel.

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

Changes in Protonation States of In-Pathway Residues can Alter Ligand Binding Pathways Obtained From Spontaneous Binding Molecular Dynamics Simulations.

Protein-ligand binding processes often involve changes in protonation states that can be key to recognize and orient the ligand in the binding site. The pathways through which (bio)molecules interplay to attain productively bound complexes are intricate and involve a series of interconnected intermediate and transition states. Molecular dynamics (MD) simulations and enhanced sampling techniques are commonly used to characterize the spontaneous binding of a ligand to its receptor. However, the effect of protonation state changes of in-pathway residues in spontaneous binding MD simulations remained mostly unexplored. Here, we used molecular dynamics simulations to reconstruct the trypsin-benzamidine binding pathway considering different protonation states of His57. This residue is part of the trypsin catalytic triad and is located more than 10 Å away from Asp189, which is responsible for benzamidine binding in the trypsin S1 pocket. Our MD simulations showed that the binding pathways that benzamidine follow to target the S1 binding site are critically dependent on the His57 protonation state. Binding of benzamidine frequently occurs when His57 is protonated in the delta nitrogen while the binding process is significantly less frequent when His57 is positively charged. Constant-pH MD simulations retrieved the equilibrium populations of His57 protonation states at trypsin active pH offering a clearer picture of benzamidine recognition and binding. These results indicate that properly accounting for protonation states of distal residues can be important in spontaneous binding MD simulations.

Discovery and In Vivo Proof of Concept of a Highly Potent Dual Inhibitor of Soluble Epoxide Hydrolase and Acetylcholinesterase for the Treatment of Alzheimer's Disease.

With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer's disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)-TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, human microsomal stability, lack of neurotoxicity, and it rescued memory, synaptic plasticity, and neuroinflammation in an AD mouse model, after low dose chronic oral administration.

Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors.

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

A dissected ring current model for assessing magnetic aromaticity: a general approach for both organic and inorganic rings.

A model based on classical electrodynamics is used to measure the strength of ring currents of different molecular orbitals, i.e., σ- and π-orbitals, and characteristics of ring current loops, i.e., ring current radii and height of current loops above/below the ring planes, among a number of organic as well as inorganic molecules. For the π-current, the present model represents an improvement of previous approaches to determine ring current intensity. It is proven that the present model is more precise than previous models as they could not explain presence of the minimum in the plot of NICS(πzz) versus distance close to the ring plane. Variations in the charge of molecules and the types of constituent atoms of each species affect the ring current radii of both σ- and π-current loops as well as the height of π-current loops above/below the ring plane. It is suggested that variation in the distribution of the one-electron density in different systems is the main source of differences of the ring current characteristics.