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Stereospecific recognition of chiral molecules plays a crucial role in biological systems. The μ-opioid receptor (MOR) exhibits binding affinity towards (−)-morphine, a well-established gold standard in pain management, while it shows minimal binding affinity for the (+)-morphine enantiomer, resulting in a lack of analgesic activity. Understanding how MOR stereoselectively recognizes morphine enantiomers has remained a puzzle in neuroscience and pharmacology for over half-a-century due to the lack of direct observation techniques. To unravel this mystery, we constructed the binding and unbinding processes of morphine enantiomers with MOR via molecular dynamics simulations to investigate the thermodynamics and kinetics governing MOR's stereoselective recognition of morphine enantiomers. Our findings reveal that the binding of (−)-morphine stabilizes MOR in its activated state, exhibiting a deep energy well and a prolonged residence time. In contrast, (+)-morphine fails to sustain the activation state of MOR. Furthermore, the results suggest that specific residues, namely D1142.50 and D1473.32, are deprotonated in the active state of MOR bound to (−)-morphine. This work highlights that the selectivity in molecular recognition goes beyond binding affinities, extending into the realm of residence time. [ABSTRACT FROM AUTHOR]

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na+) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na+ translocation through the μ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na+ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na+ translocation in inactive and active MORs, ii) estimates of Na+-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling. [ABSTRACT FROM AUTHOR]

Crystal structures of several bacterial Nav channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Nav channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial NavAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K+ ions can block the entrance to the selectivity filter of NavAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place by modest applied forces. In contrast to K+, three Na+ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na+ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K+ block is equivalent to large applied potentials experimentally measured for two bacterial Nav channels to induce inward currents of K+ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes. [ABSTRACT FROM AUTHOR]

A recentcrystallization of several ion channels hasprovided strongimpetus for efforts aimed at understanding the different strategiesemployed by nature for selective ion transport. In this work, we usedtwo variants of the selectivity filter of NaK channel to explore molecularmechanisms that give rise to K+-selectivity. We computedone-dimensional (1D) and two-dimensional (2D) potentials of mean force(PMFs) for ion permeation across the channel. The results indicatethat the energies for Na+and K+permeationacross the selectivity filter display significant differences in positionsof the binding sites and barriers. One characteristic signature ofa K+-selective channel is the apparent preservation ofthe site analogous to that of S2 in KcsA. The S2-bound ion can bealmost ideally dehydrated and coordinated by 6 to 8 carbonyls. Ina striking contrast, the PMFs controlling transport of ions in a nonselectivevariant show almost identical profiles for either K+orNa+and significant involvement of water molecules in ioncoordination across the entire selectivity filter. An analysis ofdifferences in 1D PMFs for Na+and K+suggeststhat coordination number alone is an insufficient predictor of siteselectivity, while chemical composition (ratio of carbonyls and watermolecules) correlates well with preference for K+. Multi-ioneffects such as dependence of the barriers and wells for permeantion on the type of copermeant ion were found to play a significantrole in the selectivity signature of the channel as well. [ABSTRACT FROM AUTHOR]

Abstract: Most water-soluble and small molecules are taken up by substrate-specific channels belonging to the Gram-negative bacteria. The protein named OpdK, a member of OprD family, plays an important role in transporting the vanillate as the only carbon source of Pseudomonas aeruginosa. P. aeruginosa infections can be serious due to the high intrinsic antibiotic resistance owing to the present of the OprD family. We applied standard molecular dynamics, steered molecular dynamics and umbrella sampling, to investigate the thermodynamics of vanillate passing through the pore of OpdK protein at physiological temperature. The results indicate that hydrogen bonds of vanillate-L3 (L3: Gly92-Gln111) and hydrophobic interactions of vanillate-L7 (Gly252-Asn278) are crucial to the transport of vanillate. Compared to L7, L3 can hardly change the shape of the pore, but its amino acids can effectively affect the transport process. The important role of charged residues in the barrel of the protein for the substrate transport has been proved in the experiment researches and our simulations also determinate that these residues may prevent the vanillate from entering the cell. These results provide detailed information that will facilitate the development of effective drugs. [Copyright &y& Elsevier]

Protein-protein interactions (PPIs) play a critical role in living cells and represent promising targets for the drug discovery and life sciences communities. However, lateral transmembrane PPIs are difficult targets for small-molecule inhibitor development given less structural information is known and fewer ligand discovery methods have been explored compared to soluble proteins. In this study, the interactions of the transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) were disrupted by pentamidine derivatives to curb the committed step of EBV infection. A pentamidine derivative 2 with a 7-atom di-amide linker had the best activity whilst switching the amide regiochemistry in the linker influenced membrane permeability and abolished anti TMD-5 activity. Molecular dynamics simulations were performed to understand the interaction between pentamidine derivatives and TMD-5, and to rationalise the observed structure-activity relationships. This study explicitly demonstrated that the interaction of small molecule with lipid should be considered alongside interaction with the protein target when designing small molecules targeting the PPIs of TMDs. In all, this study provides proof of concept for the rational design of small molecules targeting transmembrane PPIs. Image 1 • Protein-protein interactions are vital in cells and represent promising target. • Protein-protein interfaces are difficult to target due to inherent challenges. • Compound 2 successfully targeted the interfaces and addressed this challenge. • The activity of compound 2 is extremely influenced by the regiochemistry of linker. • Interactions of molecule with lipid are vital when targeting the interfaces. [ABSTRACT FROM AUTHOR]

In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the I f current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance. [ABSTRACT FROM AUTHOR]

Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1, however, was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom molecular dynamics simulations with a swarm of trajectories with the string method for extensive transition path sampling and evolution. A detailed residue–residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular, it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al . (Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A, Kurokawa T, et al . X-ray crystal structure of voltage-gated proton channel. Nature 2014). The mechanisms governing ion transport in the wild-type and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results, we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport. [ABSTRACT FROM AUTHOR]

• There is growing interest in re-purposing drugs already approved for human use. • We demonstrate that lovastatin is a TLR antagonist, selective for TLR4. • Lovastatin blocks a wide array of effects of LPS in BV2 microglia. • Intrathecal lovastatin suppresses neuropathic pain and microglial activation. There is growing interest in drug repositioning to find new therapeutic indications for drugs already approved for use in people. Lovastatin is an FDA approved drug that has been used clinically for over a decade as a lipid-lowering medication. While lovastatin is classically considered to act as a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor, the present series of studies reveal a novel lovastatin effect, that being as a Toll-like receptor 4 (TLR4) antagonist. Lovastatin selectively inhibits lipopolysaccharide (LPS)-induced TLR4-NF-κB activation without affecting signaling by other homologous TLRs. In vitro biophysical binding and cellular thermal shift assay (CETSA) show that lovastatin is recognized by TLR4′s coreceptor myeloid differentiation protein 2 (MD-2). This finding is supported by molecular dynamics simulations that lovastatin targets the LPS binding pocket of MD-2 and lovastatin binding stabilizes the MD-2 conformation. In vitro studies of BV-2 microglial cells revealed that lovastatin inhibits multiple effects of LPS, including activation of NFkB; mRNA expression of tumor necrosis factor-a, interleukin-6 and cyclo-oxygenase 2; production of nitric oxide and reactive oxygen species; as well as phagocytic activity. Furthermore, intrathecal delivery of lovastatin over lumbosacral spinal cord of rats attenuated both neuropathic pain from sciatic nerve injury and expression of the microglial activation marker CD11 in lumbar spinal cord dorsal horn. Given the well-established role of microglia and proinflammatory signaling in neuropathic pain, these data are supportive that lovastatin, as a TLR4 antagonist, may be productively repurposed for treating chronic pain. [ABSTRACT FROM AUTHOR]

[Display omitted] • DHA C-10 derivatives were designed, synthesized and evaluated by TLR4 antagonistic activities. • The electron-rich functional group is important for the DHA derivative's TLR4 antagonistic activity. • 2k with alkynyl as the end functional group exhibit 14 times more potent TLR4 antagonistic activity. • 2k inhibited LPS-induced TLR4 dimerization, endocytosis and NF-κB but not MAPKs activation. • DHA derivative 2k increased and prolonged morphine analgesia. Dihydroartemisinin (DHA), a natural product isolated from the traditional Chinese herb Artemisia annua and one of the clinical frontline drugs against malarial infections, has recently been discovered as a Toll-like Receptor 4 (TLR4) antagonist. However, the TLR4 antagonistic activity of DHA is modest and it exhibits cellular toxicity. In this work, the structure-activity relationship (SAR) of DHA as TLR4 antagonist was explored. Since destroying the sesquiterpene endoperoxide scaffold substantially compromised the TLR4 antagonistic activity and molecular dynamics analysis showed that the C-10 hydroxyl group formed a hydrogen bond with E72 of myeloid differentiation factor 2 (MD2) to prevent it moving deeper into MD2, SAR of DHA was focused on the C-10 hemiacetal position. With extending the length of the linear alkane chain at C10 position, the TLR4 antagonistic activity of DHA analogs increased first and then decreased with the best TLR4 antagonism occurring at the length of the carbon chain of 3–4 carbons. In contrast, the cellular toxicity of DHA analogs was raised with the increasing length of the linear alkane chain. The TLR4 antagonistic activity of DHA derivatives with substituted halogen as the terminal functional group decreased with the decrease of electronegativity of the substituted halogen, which implies the electron-rich functional group at the end of the alkane chain appears preferred. Therefore, DHA derivative 2k with alkynyl as the end functional group, exhibited 14 times more potent TLR4 antagonistic activity than DHA. Moreover, 2k showed less cellular toxicity than DHA. Cellular signaling characterizations indicated that 2k inhibited LPS-induced TLR4 dimerization and endocytosis and suppressed LPS-induced NF-κB but not MAPKs activation, culminating in blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO and IL-1β. Further, 2k was active in vivo ; it significantly increased and prolonged morphine analgesia. Collectively, this study provides a structural guidance to reposition DHA derivatives as TLR4 antagonists. [ABSTRACT FROM AUTHOR]