Understanding the Mechanism of Direct Activation of AMP-Kinase: Toward a Fine Allosteric Tuning of the Kinase Activity.

Mammalian AMP-activated protein kinase (AMPK) is a Ser/Thr protein kinase with a key role as a sensor in cellular energy homeostasis. It has a major role in numerous metabolic disorders, such as type 2 diabetes, obesity, and cancer, and hence it has gained progressive interest as a potential therapeutic target. AMPK is a heterotrimeric enzyme composed by an α-catalytic subunit and two regulatory subunits, ß and γ. It is regulated by several mechanisms, including indirect activators such as metformin and direct activators such as compound A-769662. The crystal structure of AMPK bound to A-769662 has been recently reported, suggesting a hypothetical allosteric mechanism of AMPK activation assisted by phosphorylated Ser108 at the ß-subunit. Here, we have studied the direct activation mechanism of A-769662 by means of molecular dynamics simulations, suggesting that the activator may act as a glue, coupling the dynamical motion of the ß-subunit and the N-terminal domain of the α-subunit, and assisting the preorganization of the ATP-binding site. This is achieved through the formation of an allosteric network that connects the activator and ATP-binding sites, particularly through key interactions formed between αAsp88 and ßArg83 and between ßpSer108 and αLys29. Overall, these studies shed light into key mechanistic determinants of the allosteric regulation of this cellular energy sensor, and pave the way for the fine-tuning of the rational design of direct activators of this cellular energy sensor.

Multiple Multicomponent Reactions: Unexplored Substrates, Selective Processes, and Versatile Chemotypes in Biomedicine.

Multiple multicomponent reactions rapidly assemble complex structures. Despite being very productive, the lack of selectivity and the reduced number of viable transformations restrict their general application in synthesis. Hereby, we describe a rationale for a selective version of these processes based in the preferential generation of intermediates which are less reactive than the initial substrates. In this way, applying the Groebke-Blackburn-Bienaymé reaction on a range of α-polyamino-polyazines, we prepared a family compact heterocyclic scaffolds with relevant applications in medicinal and biological chemistry (live cell imaging probes, selective binders for DNA quadruplexes, and antiviral agents against human adenoviruses). The approach has general character and yields complex molecular targets in a selective, tunable and direct manner.

Generation and Reactions of an Octacyclic Hindered Pyramidalized Alkene.

Octacyclo[10.6.1.01,10.03,7.04,9.08,19.011,16.013,17]nonadeca-5,8,14-triene (27), a hindered pyramidalized alkene, has been generated from a diiodide precursor. Contrary to the usual behavior of known pyramidalized alkenes, no Diels-Alder adducts were obtained from the present alkene when it was generated by different standard procedures in the presence of different dienes. However, products derived from the reduction, t-BuLi addition, condensation with the solvent, or dimerization were isolated from these reactions, depending on the conditions used to generate it. No [2 + 2] cross product among this pyramidalized alkene and tricyclo[3.3.1.03,7]non-3(7)-ene was formed when a mixture of the corresponding precursor diiodides was reacted with sodium amalgam. The analysis of selected geometrical and orbital parameters determined from quantum mechanical calculations indicates that the degree of pyramidalization of this alkene and its higher steric hindrance compared with other polycyclic pyramidalized alkenes may explain its peculiar reactivity.

Molecular dynamic simulations of protein/RNA complexes: CRISPR/Csy4 endoribonuclease.

BACKGROUND: Many prokaryotic genomes comprise Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) offering defense against foreign nucleic acids. These immune systems are conditioned by the production of small CRISPR-derived RNAs matured from long RNA precursors. This often requires a Csy4 endoribonuclease cleaving the RNA 3'-end. METHODS: We report extended explicit solvent molecular dynamic (MD) simulations of Csy4/RNA complex in precursor and product states, based on X-ray structures of product and inactivated precursor (55 simulations; ~3.7µs in total). RESULTS: The simulations identify double-protonated His29 and deprotonated terminal phosphate as the likely dominant protonation states consistent with the product structure. We revealed potential substates consistent with Ser148 and His29 acting as the general base and acid, respectively. The Ser148 could be straightforwardly deprotonated through solvent and could without further structural rearrangements deprotonate the nucleophile, contrasting similar studies investigating the general base role of nucleobases in ribozymes. We could not locate geometries consistent with His29 acting as general base. However, we caution that the X-ray structures do not always capture the catalytically active geometries and then the reactive structures may be unreachable by the simulation technique. CONCLUSIONS: We identified potential catalytic arrangement of the Csy4/RNA complex but we also report limitations of the simulation technique. Even for the dominant protonation state we could not achieve full agreement between the simulations and the structural data. GENERAL SIGNIFICANCE: Potential catalytic arrangement of the Csy4/RNA complex is found. Further, we provide unique insights into limitations of simulations of protein/RNA complexes, namely, the influence of the starting experimental structures and force field limitations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Insertion of Isocyanides into N-Si Bonds: Multicomponent Reactions with Azines Leading to Potent Antiparasitic Compounds.

Trimethylsilyl chloride is an efficient activating agent for azines in isocyanide-based reactions, which then proceed through a key insertion of the isocyanide into a N-Si bond. The reaction is initiated by N activation of the azine, followed by nucleophilic attack of an isocyanide in a Reissert-type process. Finally, a second equivalent of the same or a different isocyanide inserts into the N-Si bond leading to the final adduct. The use of distinct nucleophiles leads to a variety of α-substituted dihydroazines after a selective cascade process. Based on computational studies, a mechanistic hypothesis for the course of these reactions was proposed. The resulting products exhibit significant activity against Trypanosoma brucei and T. cruzi, featuring favorable drug-like properties and safety profiles.

Short Access to Belt Compounds with Spatially Close C=C Bonds and Their Transannular Reactions.

Two domino Diels-Alder adducts were obtained from 3,7-bis(cyclopenta-2,4-dien-1-ylidene)-cis-bicyclo[3.3.0]octane and dimethyl acetylenedicarboxylate or N-methylmaleimide under microwave irradiation. From the first adduct, a C20H24 diene with C2v symmetry was obtained by Zn/AcOH reduction, hydrolysis, oxidative decarboxylation, and selective hydrogenation. Photochemical [2+2] cycloaddition of this diene gave a thermally unstable cyclobutane derivative, which reverts to the diene. However, both the diene and the cyclobutane derivatives could be identified by X-ray diffraction analysis upon irradiation of the diene crystal. New six-membered rings are formed upon the transannular addition of bromine or iodine to the diene. The N-type selectivity of the addition was examined by theoretical calculations, which revealed the distinct susceptibility of the doubly bonded carbon atoms to the bromine attack.

Stereocontrolled Annulations of Indolo[2,3-a]quinolizidine-Derived Lactams with a Silylated Nazarov Reagent: Access to Allo and Epiallo Yohimbine-Type Derivatives.

The facial selectivity of double Michael addition reactions of the silylated Nazarov reagent 4 to unsaturated indolo[2,3-a]quinolizidine lactams 3 has been studied. Pentacyclic 3-H/15-H trans adducts 5 are generated from Nind -unsubstituted lactams, but the corresponding cis isomers 6 are formed when the indole nitrogen has a tert-butyloxycarbonyl (Boc) substituent. This reversal in the facial selectivity of the annulation has been rationalized by means of theoretical calculations, which indicate that the initial nucleophilic attack under stereoelectronic control is hampered by the presence of the bulky Boc group. The synthetic usefulness of the pentacyclic Nazarov-derived adducts is demonstrated by their conversion into allo and epiallo yohimbine-type targets.

Quantum mechanical-based strategies in drug discovery: Finding the pace to new challenges in drug design.

The expansion of the chemical space to tangible libraries containing billions of synthesizable molecules opens exciting opportunities for drug discovery, but also challenges the power of computer-aided drug design to prioritize the best candidates. This directly hits quantum mechanics (QM) methods, which provide chemically accurate properties, but subject to small-sized systems. Preserving accuracy while optimizing the computational cost is at the heart of many efforts to develop high-quality, efficient QM-based strategies, reflected in refined algorithms and computational approaches. The design of QM-tailored physics-based force fields and the coupling of QM with machine learning, in conjunction with the computing performance of supercomputing resources, will enhance the ability to use these methods in drug discovery. The challenge is formidable, but we will undoubtedly see impressive advances that will define a new era.

Quantification of nitrate-π interactions and selective transport of nitrate using calix[4]pyrroles with two aromatic walls.

Herein we disclose the results of our investigations regarding the interactions between the biologically relevant nitrate oxoanion and several "two-wall" aryl-extended calix[4]pyrroles. There exists a clear relationship between the electronic nature of the aromatic walls of the calix[4]pyrroles and the stability of the nitrate⊂calix[4]pyrrole complex. This suggests that NO3(-)-π interactions have an important electrostatic component. We provide energetic estimates for the interaction of nitrate with several phenyl derivatives. Additionally, we report solid-state evidence for a preferred binding geometry of the nitrate anion included in the calix[4]pyrroles. Finally, the "two-wall" aryl-extended calix[4]pyrroles show excellent activity in ion transport through lipid-based lamellar membranes. Notably the best anion transporters are highly selective for transport of nitrate over other anions.

Anion-π interactions involving [MX(n)](m-) anions: a comprehensive theoretical study.

In this manuscript we perform a systematic study on the geometric and energetic features of anion-π complexes, wherein the anion is a metal complex of variable shapes and charges. Such a study is lacking in the literature. For the calculations we used the ab initio RI-MP2/def2-TZVPP level of theory. A search in the Cambridge Structural Database (CSD) provides the experimental starting point that inspired the subsequent theoretical study. The influence of [MX(n)](m-) on the anion-π interaction was analyzed in terms of energetic, geometric, and charge transfer properties and Bader's theory of "atom-in-molecules" (AIM). The binding energy depends on the coordination index, geometric features and different orientations adopted by the metallic anion. The binding mode resembling a stacking interaction for linear, trigonal planar and square-planar anions is the most favorable. For tetrahedral and octahedral anions the most favorable orientation is the one with three halogen atoms pointing to the ring.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima.

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where 'nerve hemoglobins' (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb 'classic' globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.

Revealing druggable cryptic pockets in the Nsp1 of SARS-CoV-2 and other ß-coronaviruses by simulations and crystallography.

Non-structural protein 1 (Nsp1) is a main pathogenicity factor of α- and ß-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive molecular dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous ß-coronaviruses.

Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK-eNOS-NO pathway.

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1ß1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK-eNOS-NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.

Theoretical study on cooperativity effects between anion-π and halogen-bonding interactions.

This article analyzes the interplay between lone pair-π (lp-π) or anion-π interactions and halogen-bonding interactions. Interesting cooperativity effects are observed when lp/anion-π and halogen-bonding interactions coexist in the same complex, and they are found even in systems in which the distance between the anion and halogen-bond donor molecule is longer than 9 Å. These effects are studied theoretically in terms of energetic and geometric features of the complexes, which are computed by ab initio methods. Bader's theory of "atoms in molecules" is used to characterize the interactions and to analyze their strengthening or weakening depending upon the variation of charge density at critical points. The physical nature of the interactions and cooperativity effects are studied by means of molecular interaction potential with polarization partition scheme. By taking advantage of all aforementioned computational methods, the present study examines how these interactions mutually influence each other. Additionally, experimental evidence for such interactions is obtained from the Cambridge Structural Database (CSD).

On the directionality of anion-π interactions.

The directionality of two important noncovalent interactions involving aromatic rings (namely anion-π and cation-π) is investigated. It has been recently published that the anion-π interactions observed in X-ray structures where the anion is located exactly over the center of the ring are scarce compared to cation-π interactions. To explain this behavior, we have analyzed how the interaction energy (RI-MP2/aug-cc-pVDZ level of theory) is affected by moving the anion from the center of the ring to several directions in anion-π complexes of chloride with either hexafluorobenzene or trifluoro-s-triazine. We have compared the results with the directionality of the cation-π interaction in the sodium-benzene complex. The results are useful to explain the experimental differences between both ion-π interactions. We have also computed the van der Waals radii of several halide anions and we have compared them to the neutral halogen atoms.

Radical cation (C˙(+)-π) and radical anion (A˙(-)-π) interactions with aromatic rings: energetic, orbitalic and spin density considerations.

Cation and anion-π interactions are important binding forces where aromatic rings are involved. In this manuscript we define and compare two related interactions that have not been reported so far, namely radical cation and radical anion-π interactions (C˙(+)-π and A˙(-)-π, respectively). Moreover, we compare the energetic features of these complexes with the related closed-shell interactions. Interestingly the C˙(+)-π interaction is more favourable than the standard cation-π interaction and the contrary is observed for the A˙(-)-π interaction. Changes in the aromatic character of the ring upon complexation of the ion have been studied using the nucleus-independent chemical shift (NICS) criterion. Orbitalic and spin density calculations of the complexes have been computed in order to investigate if spin transfer effects between the radical ions and the aromatic ring exist. We have analyzed the physical nature of the interactions by means of Bader's theory of "atoms-in-molecules" and partitioning the total interaction energy into individual components using the Molecular Interaction Potential with a polarization partition scheme. Finally, we describe an interesting biological example, which involves the tetrahydrobiopterin cofactor, where the presence of a radical ion-π interaction is important.

Unexpected nonadditivity effects in anion-π complexes.

Several complexes of fluorine-substituted ethyne, ethene, butadiene, benzene, and [n]radialenes (n = 3-5) with two anions have been optimized at the RI-MP2/aug-cc-pVTZ level of theory. The additivity of the anion-π interaction was studied depending on the number of double bonds and fluorine atoms. Interesting nonadditivity effects were observed in the aromatic and antiaromatic complexes, which were analyzed by partitioning the total interaction energy into individual components, using Bader's theory of "atoms in molecules" and changes in the aromatic character of the ring upon complexation.

From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus.

With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains.

Structural basis of the selective activation of enzyme isoforms: Allosteric response to activators of ß1- and ß2-containing AMPK complexes.

AMP-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 complexes, which exhibit notable differences in the sensitivity to allosteric activators. To shed light into the molecular determinants of the allosteric regulation of this energy sensor, we have examined the structural and dynamical properties of ß1- and ß2-containing AMPK complexes formed with small molecule activators A-769662 and SC4, and dissected the mechanical response leading to active-like enzyme conformations through the analysis of interaction networks between structural domains. The results reveal the mechanical sensitivity of the α2ß1 complex, in contrast with a larger resilience of the α2ß2 species, especially regarding modulation by A-769662. Furthermore, binding of activators to α2ß1 consistently promotes the pre-organization of the ATP-binding site, favoring the adoption of activated states of the enzyme. These findings are discussed in light of the changes in the residue content of ß-subunit isoforms, particularly regarding the ß1Asn111 â†’ ß2Asp111 substitution as a key factor in modulating the mechanical sensitivity of ß1- and ß2-containing AMPK complexes. Our studies pave the way for the design of activators tailored for improving the therapeutic treatment of tissue-specific metabolic disorders.

Amphiphilic Histidine-Based Oligopeptides Exhibit pH-Reversible Fibril Formation.

We report the design, simulation, synthesis, and reversible self-assembly of nanofibrils using polyhistidine-based oligopeptides. The inclusion of aromatic amino acids in the histidine block produces distinct antiparallel ß-strands that lead to the formation of amyloid-like fibrils. The structures undergo self-assembly in response to a change in pH. This creates the potential to produce well-defined fibrils for biotechnological and biomedical applications that are pH-responsive in a physiologically relevant range.