Virtual Screening Strategy to Identify Retinoic Acid-Related Orphan Receptor γt Modulators.

Molecular docking is a key method used in virtual screening (VS) campaigns to identify small-molecule ligands for drug discovery targets. While docking provides a tangible way to understand and predict the protein-ligand complex formation, the docking algorithms are often unable to separate active ligands from inactive molecules in practical VS usage. Here, a novel docking and shape-focused pharmacophore VS protocol is demonstrated for facilitating effective hit discovery using retinoic acid receptor-related orphan receptor gamma t (RORγt) as a case study. RORγt is a prospective target for treating inflammatory diseases such as psoriasis and multiple sclerosis. First, a commercial molecular database was flexibly docked. Second, the alternative docking poses were rescored against the shape/electrostatic potential of negative image-based (NIB) models that mirror the target's binding cavity. The compositions of the NIB models were optimized via iterative trimming and benchmarking using a greedy search-driven algorithm or brute force NIB optimization. Third, a pharmacophore point-based filtering was performed to focus the hit identification on the known RORγt activity hotspots. Fourth, free energy binding affinity evaluation was performed on the remaining molecules. Finally, twenty-eight compounds were selected for in vitro testing and eight compounds were determined to be low µM range RORγt inhibitors, thereby showing that the introduced VS protocol generated an effective hit rate of ~29%.

Optimization of Cavity-Based Negative Images to Boost Docking Enrichment in Virtual Screening.

Molecular docking is a key in silico method used routinely in modern drug discovery projects. Although docking provides high-quality ligand binding predictions, it regularly fails to separate the active compounds from the inactive ones. In negative image-based rescoring (R-NiB), the shape/electrostatic potential (ESP) of docking poses is compared to the negative image of the protein's ligand binding cavity. While R-NiB often improves the docking yield considerably, the cavity-based models do not reach their full potential without expert editing. Accordingly, a greedy search-driven methodology, brute force negative image-based optimization (BR-NiB), is presented for optimizing the models via iterative editing and benchmarking. Thorough and unbiased training, testing and stringent validation with a multitude of drug targets, and alternative docking software show that BR-NiB ensures excellent docking efficacy. BR-NiB can be considered as a new type of shape-focused pharmacophore modeling, where the optimized models contain only the most vital cavity information needed for effectively filtering docked actives from the inactive or decoy compounds. Finally, the BR-NiB code for performing the automated optimization is provided free-of-charge under MIT license via GitHub (https://github.com/jvlehtonen/brutenib) for boosting the success rates of docking-based virtual screening campaigns.

Ligand-Enhanced Negative Images Optimized for Docking Rescoring.

Despite the pivotal role of molecular docking in modern drug discovery, the default docking scoring functions often fail to recognize active ligands in virtual screening campaigns. Negative image-based rescoring improves docking enrichment by comparing the shape/electrostatic potential (ESP) of the flexible docking poses against the target protein's inverted cavity volume. By optimizing these negative image-based (NIB) models using a greedy search, the docking rescoring yield can be improved massively and consistently. Here, a fundamental modification is implemented to this shape-focused pharmacophore modelling approach-actual ligand 3D coordinates are incorporated into the NIB models for the optimization. This hybrid approach, labelled as ligand-enhanced brute-force negative image-based optimization (LBR-NiB), takes the best from both worlds, i.e., the all-roundedness of the NIB models and the difficult to emulate atomic arrangements of actual protein-bound small-molecule ligands. Thorough benchmarking, focused on proinflammatory targets, shows that the LBR-NiB routinely improves the docking enrichment over prior iterations of the R-NiB methodology. This boost can be massive, if the added ligand information provides truly essential binding information that was lacking or completely missing from the cavity-based NIB model. On a practical level, the results indicate that the LBR-NiB typically works well when the added ligand 3D data originates from a high-quality source, such as X-ray crystallography, and, yet, the NIB model compositions can also sometimes be improved by fusing into them, for example, with flexibly docked solvent molecules. In short, the study demonstrates that the protein-bound ligands can be used to improve the shape/ESP features of the negative images for effective docking rescoring use in virtual screening.

Getting Docking into Shape Using Negative Image-Based Rescoring.

The failure of default scoring functions to ensure virtual screening enrichment is a persistent problem for the molecular docking algorithms used in structure-based drug discovery. To remedy this problem, elaborate rescoring and postprocessing schemes have been developed with a varying degree of success, specificity, and cost. The negative image-based rescoring (R-NiB) has been shown to improve the flexible docking performance markedly with a variety of drug targets. The yield improvement is achieved by comparing the alternative docking poses against the negative image of the target protein's ligand-binding cavity. In other words, the shape and electrostatics of the binding pocket is directly used in the similarity comparison to rank the explicit docking poses. Here, the PANTHER/ShaEP-based R-NiB methodology is tested with six popular docking softwares, including GLIDE, PLANTS, GOLD, DOCK, AUTODOCK, and AUTODOCK VINA, using five validated benchmark sets. Overall, the results indicate that R-NiB outperforms the default docking scoring consistently and inexpensively, demonstrating that the methodology is ready for wide-scale virtual screening usage.

A Practical Perspective: The Effect of Ligand Conformers on the Negative Image-Based Screening.

Negative image-based (NIB) screening is a rigid molecular docking methodology that can also be employed in docking rescoring. During the NIB screening, a negative image is generated based on the target protein's ligand-binding cavity by inverting its shape and electrostatics. The resulting NIB model is a drug-like entity or pseudo-ligand that is compared directly against ligand 3D conformers, as is done with a template compound in the ligand-based screening. This cavity-based rigid docking has been demonstrated to work with genuine drug targets in both benchmark testing and drug candidate/lead discovery. Firstly, the study explores in-depth the applicability of different ligand 3D conformer generation software for acquiring the best NIB screening results using cyclooxygenase-2 (COX-2) as the example system. Secondly, the entire NIB workflow from the protein structure preparation, model build-up, and ligand conformer generation to the similarity comparison is performed for COX-2. Accordingly, hands-on instructions are provided on how to employ the NIB methodology from start to finish, both with the rigid docking and docking rescoring using noncommercial software. The practical aspects of the NIB methodology, especially the effect of ligand conformers, are discussed thoroughly, thus, making the methodology accessible for new users.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-ß-hydroxysteroid dehydrogenase 2 - a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

Key role of water in proton transfer at the Qo-site of the cytochrome bc1 complex predicted by atomistic molecular dynamics simulations.

Cytochrome (cyt) bc1 complex, which is an integral part of the respiratory chain and related energy-conserving systems, has two quinone-binding cavities (Qo- and Qi-sites), where the substrate participates in electron and proton transfer. Due to its complexity, many of the mechanistic details of the cyt bc1 function have remained unclear especially regarding the substrate binding at the Qo-site. In this work we address this issue by performing extensive atomistic molecular dynamics simulations with the cyt bc1 complex of Rhodobacter capsulatus embedded in a lipid bilayer. Based on the simulations we are able to show the atom-level binding modes of two substrate forms: quinol (QH2) and quinone (Q). The QH2 binding at the Qo-site involves a coordinated water arrangement that produces an exceptionally close and stable interaction between the cyt b and iron sulfur protein subunits. In this arrangement water molecules are positioned suitably in relation to the hydroxyls of the QH2 ring to act as the primary acceptors of protons detaching from the oxidized substrate. In contrast, water does not have a similar role in the Q binding at the Qo-site. Moreover, the coordinated water molecule is also a prime candidate to act as a structural element, gating for short-circuit suppression at the Qo-site.

Atomistic simulations indicate cardiolipin to have an integral role in the structure of the cytochrome bc1 complex.

The reaction mechanism of the cytochrome (cyt) bc1 complex relies on proton and electron transfer to/from the substrate quinone/quinol, which in turn generate a proton gradient across the mitochondrial membrane. Cardiolipin (CL) have been suggested to play an important role in cyt bc1 function by both ensuring the structural integrity of the protein complex and also by taking part in the proton uptake. Yet, the atom-scale understanding of these highly charged four-tail lipids in the cyt bc1 function has remained quite unclear. We consider this issue through atomistic molecular dynamics simulations that are applied to the entire cyt bc1 dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer. We find CLs to spontaneously diffuse to the dimer interface to the immediate vicinity of the higher potential heme b groups of the complex's catalytic Qi-sites. This observation is in full agreement with crystallographic studies of the complex, and supports the view that CLs are key players in the proton uptake. The simulation results also allow us to present a refined picture for the dimer arrangement in the cyt bc1 complex, the novelty of our work being the description of the role of the surrounding lipid environment: in addition to the specific CL-protein interactions, we observe the protein domains on the positive side of the membrane to settle against the lipids. Altogether, the simulations discussed in this article provide novel views into the dynamics of cyt bc1 with lipids, complementing previous experimental findings.

Examining the Effect of Charged Lipids on Mitochondrial Outer Membrane Dynamics Using Atomistic Simulations.

The outer mitochondrial membrane (OMM) is involved in multiple cellular functions such as apoptosis, inflammation and signaling via its membrane-associated and -embedded proteins. Despite the central role of the OMM in these vital phenomena, the structure and dynamics of the membrane have regularly been investigated in silico using simple two-component models. Accordingly, the aim was to generate the realistic multi-component model of the OMM and inspect its properties using atomistic molecular dynamics (MD) simulations. All major lipid components, phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), were included in the probed OMM models. Because increased levels of anionic PS lipids have potential effects on schizophrenia and, more specifically, on monoamine oxidase B enzyme activity, the effect of varying the PS concentration was explored. The MD simulations indicate that the complex membrane lipid composition (MLC) behavior is notably different from the two-component PC-PE model. The MLC changes caused relatively minor effects on the membrane structural properties such as membrane thickness or area per lipid; however, notable effects could be seen with the dynamical parameters at the water-membrane interface. Increase of PS levels appears to slow down lateral diffusion of all lipids and, in general, the presence of anionic lipids reduced hydration and slowed down the PE headgroup rotation. In addition, sodium ions could neutralize the membrane surface, when PI was the main anionic component; however, a similar effect was not seen for high PS levels. Based on these results, it is advisable for future studies on the OMM and its protein or ligand partners, especially when wanting to replicate the correct properties on the water-membrane interface, to use models that are sufficiently complex, containing anionic lipid types, PI in particular.

Full and partial agonism of ionotropic glutamate receptors indicated by molecular dynamics simulations.

Ionotropic glutamate receptors (iGluRs) are synaptic proteins that facilitate signal transmission in the central nervous system. Extracellular iGluR cleft closure is linked to receptor activation; however, the mechanism underlying partial agonism is not entirely understood. Full agonists close the bilobed ligand-binding domain (LBD), while antagonists prevent closure; the transmembrane ion channel either opens or stays closed, respectively. Although some bulky partial agonists produce intermediate iGluR-LBD closure, the available crystal structures also imply that the cleft can be shut with certain partial agonists. Recently, we have shown that the iGluR-LBD closure stage can be recreated by inserting a ligand into the closed cleft and simulating the ligand-receptor complex with molecular dynamics. Our simulations indicate that partial agonist binding does not necessarily prevent full receptor cleft closure; instead, it destabilizes cleft closure. Interdomain hydrogen bonds were studied thoroughly, and one hydrogen bond, in particular, was consistently disrupted by bound partial agonists. Accordingly, the simulation protocol presented here can be used to categorize compounds in silico as partial or full agonists for iGluRs.

Comparison of virtual high-throughput screening methods for the identification of phosphodiesterase-5 inhibitors.

Reliable and effective virtual high-throughput screening (vHTS) methods are desperately needed to minimize the expenses involved in drug discovery projects. Here, we present an improvement to the negative image-based (NIB) screening: the shape, the electrostatics, and the solvation state of the target protein's ligand-binding site are included into the vHTS. Additionally, the initial vHTS results are postprocessed with molecular mechanics/generalized Born surface area (MMGBSA) calculations to estimate the favorability of ligand-protein interactions. The results show that docking produces very good early enrichment for phosphodiesterase-5 (PDE-5); however, in general, the NIB and the ligand-based screening performed better with or without the added electrostatics. Furthermore, the postprocessing of the NIB screening results using MMGBSA calculations improved the early enrichment for the PDE-5 considerably, thus, making hit discovery affordable.

Negative Image-Based Rescoring: Using Cavity Information to Improve Docking Screening.

Molecular docking produces often lackluster results in real-life virtual screening assays that aim to discover novel drug candidates or hit compounds. The problem lies in the inability of the default docking scoring to properly estimate the Gibbs free energy of binding, which impairs the recognition of the best binding poses and the separation of active ligands from inactive compounds. Negative image-based rescoring (R-NiB) provides both effective and efficient way for re-ranking the outputted flexible docking poses to improve the virtual screening yield. Importantly, R-NiB has been shown to work with multiple genuine drug targets and six popular docking algorithms using demanding benchmark test sets. The effectiveness of the R-NiB methodology relies on the shape/electrostatics similarity between the target protein's ligand-binding cavity and the docked ligand poses. In this chapter, the R-NiB method is described with practical usability in mind.

Negative Image-Based Screening: Rigid Docking Using Cavity Information.

Rational drug discovery relies heavily on molecular docking-based virtual screening, which samples flexibly the ligand binding poses against the target protein's structure. The upside of flexible docking is that the geometries of the generated docking poses are adjusted to match the residue alignment inside the target protein's ligand-binding pocket. The downside is that the flexible docking requires plenty of computing resources and, regardless, acquiring a decent level of enrichment typically demands further rescoring or post-processing. Negative image-based screening is a rigid docking technique that is ultrafast and computationally light but also effective as proven by vast benchmarking and screening experiments. In the NIB screening, the target protein cavity's shape/electrostatics is aligned and compared against ab initio-generated ligand 3D conformers. In this chapter, the NIB methodology is explained at the practical level and both its weaknesses and strengths are discussed candidly.

A Perspective: Active Role of Lipids in Neurotransmitter Dynamics.

Synaptic neurotransmission is generally considered as a function of membrane-embedded receptors and ion channels in response to the neurotransmitter (NT) release and binding. This perspective aims to widen the protein-centric view by including another vital component-the synaptic membrane-in the discussion. A vast set of atomistic molecular dynamics simulations and biophysical experiments indicate that NTs are divided into membrane-binding and membrane-nonbinding categories. The binary choice takes place at the water-membrane interface and follows closely the positioning of the receptors' binding sites in relation to the membrane. Accordingly, when a lipophilic NT is on route to a membrane-buried binding site, it adheres on the membrane and, then, travels along its plane towards the receptor. In contrast, lipophobic NTs, which are destined to bind into receptors with extracellular binding sites, prefer the water phase. This membrane-based sorting splits the neurotransmission into membrane-independent and membrane-dependent mechanisms and should make the NT binding into the receptors more efficient than random diffusion would allow. The potential implications and notable exceptions to the mechanisms are discussed here. Importantly, maintaining specific membrane lipid compositions (MLCs) at the synapses, especially regarding anionic lipids, affect the level of NT-membrane association. These effects provide a plausible link between the MLC imbalances and neurological diseases such as depression or Parkinson's disease. Moreover, the membrane plays a vital role in other phases of the NT life cycle, including storage and release from the synaptic vesicles, transport from the synaptic cleft, as well as their synthesis and degradation.

Membrane-Dependent Binding and Entry Mechanism of Dopamine into Its Receptor.

Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too.

Fragment- and negative image-based screening of phosphodiesterase 10A inhibitors.

A novel virtual screening methodology called fragment- and negative image-based (F-NiB) screening is introduced and tested experimentally using phosphodiesterase 10A (PDE10A) as a case study. Potent PDE10A-specific small-molecule inhibitors are actively sought after for their antipsychotic and neuroprotective effects. The F-NiB combines features from both fragment-based drug discovery and negative image-based (NIB) screening methodologies to facilitate rational drug discovery. The selected structural parts of protein-bound ligand(s) are seamlessly combined with the negative image of the target's ligand-binding cavity. This cavity- and fragment-based hybrid model, namely its shape and electrostatics, is used directly in the rigid docking of ab initio generated ligand 3D conformers. In total, 14 compounds were acquired using the F-NiB methodology, 3D quantitative structure-activity relationship modeling, and pharmacophore modeling. Three of the small molecules inhibited PDE10A at ~27 to ~67 µM range in a radiometric assay. In a larger context, the study shows that the F-NiB provides a flexible way to incorporate small-molecule fragments into the drug discovery.

Negatively Charged Gangliosides Promote Membrane Association of Amphipathic Neurotransmitters.

Lipophilic neurotransmitters (NTs) such as dopamine are chemical messengers enabling neurotransmission by adhering onto the extracellular surface of the post-synaptic membrane in a synapse, followed by binding to their receptors. Previous studies have shown that the strength of the NT-membrane association is dependent on the lipid composition of the membrane. Negatively charged lipids such as phosphatidylserine, phosphatidylglycerol, and phosphatidic acid have been indicated to promote NT-membrane binding, however these anionic lipids reside almost exclusively in the intracellular leaflet of the post-synaptic membrane instead of the extracellular leaflet facing the synaptic cleft. Meanwhile, the extracellular leaflet is relatively rich in biologically relevant anionic gangliosides such as monosialotetrahexosylganglioside (GM1), yet the role of gangliosides in NT-membrane association is not clear. Here, we explored the role of GM1 in modulating the binding of dopamine and histamine (as amphipathic/cationic NTs) as well as acetylcholine (as a hydrophilic/cationic NT) with the post-synaptic membrane surface. Atomistic molecular dynamics simulations and free energy calculations indicated that GM1 fosters membrane association of histamine and dopamine. For acetylcholine, this effect was not observed. The in silico results suggest that gangliosides form a charge-based vestibule in front of the post-synaptic membrane, attracting amphipathic NTs to the vicinity of the membrane. The results also stress the importance to understand the significance of the structural details of NTs, as exemplified by the GM1-acetylcholine interaction. In a larger context, the NT-membrane adherence, coupled to lateral diffusion in the membrane plane, is proposed to improve neurotransmission efficiency by advancing NT entry into the membrane-embedded ligand-binding sites.

Discovery of Retinoic Acid-Related Orphan Receptor γt Inverse Agonists via Docking and Negative Image-Based Screening.

Retinoic acid-related orphan receptor γt (RORγt) has a vital role in the differentiation of T-helper 17 (TH17) cells. Potent and specific RORγt inverse agonists are sought for treating TH17-related diseases such as psoriasis, rheumatoid arthritis, and type 1 diabetes. Here, the aim was to discover novel RORγt ligands using both standard molecular docking and negative image-based screening. Interestingly, both of these in silico techniques put forward mostly the same compounds for experimental testing. In total, 11 of the 34 molecules purchased for testing were verified as RORγt inverse agonists, thus making the effective hit rate 32%. The pIC50 values for the compounds varied from 4.9 (11 µM) to 6.2 (590 nM). Importantly, the fact that the verified hits represent four different cores highlights the structural diversity of the RORγt inverse agonism and the ability of the applied screening methodologies to facilitate much-desired scaffold hopping for drug design.

Improving Docking Performance Using Negative Image-Based Rescoring.

Despite the large computational costs of molecular docking, the default scoring functions are often unable to recognize the active hits from the inactive molecules in large-scale virtual screening experiments. Thus, even though a correct binding pose might be sampled during the docking, the active compound or its biologically relevant pose is not necessarily given high enough score to arouse the attention. Various rescoring and post-processing approaches have emerged for improving the docking performance. Here, it is shown that the very early enrichment (number of actives scored higher than 1% of the highest ranked decoys) can be improved on average 2.5-fold or even 8.7-fold by comparing the docking-based ligand conformers directly against the target protein's cavity shape and electrostatics. The similarity comparison of the conformers is performed without geometry optimization against the negative image of the target protein's ligand-binding cavity using the negative image-based (NIB) screening protocol. The viability of the NIB rescoring or the R-NiB, pioneered in this study, was tested with 11 target proteins using benchmark libraries. By focusing on the shape/electrostatics complementarity of the ligand-receptor association, the R-NiB is able to improve the early enrichment of docking essentially without adding to the computing cost. By implementing consensus scoring, in which the R-NiB and the original docking scoring are weighted for optimal outcome, the early enrichment is improved to a level that facilitates effective drug discovery. Moreover, the use of equal weight from the original docking scoring and the R-NiB scoring improves the yield in most cases.

Structure-Activity Relationship Analysis of 3-Phenylcoumarin-Based Monoamine Oxidase B Inhibitors.

Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson's disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM-1 µM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.