Efficient Refinement of Complex Structures of Flexible Histone Peptides Using Post-Docking Molecular Dynamics Protocols.

Histones are keys to many epigenetic events and their complexes have therapeutic and diagnostic importance. The determination of the structures of histone complexes is fundamental in the design of new drugs. Computational molecular docking is widely used for the prediction of target-ligand complexes. Large, linear peptides like the tail regions of histones are challenging ligands for docking due to their large conformational flexibility, extensive hydration, and weak interactions with the shallow binding pockets of their reader proteins. Thus, fast docking methods often fail to produce complex structures of such peptide ligands at a level appropriate for drug design. To address this challenge, and improve the structural quality of the docked complexes, post-docking refinement has been applied using various molecular dynamics (MD) approaches. However, a final consensus has not been reached on the desired MD refinement protocol. In this present study, MD refinement strategies were systematically explored on a set of problematic complexes of histone peptide ligands with relatively large errors in their docked geometries. Six protocols were compared that differ in their MD simulation parameters. In all cases, pre-MD hydration of the complex interface regions was applied to avoid the unwanted presence of empty cavities. The best-performing protocol achieved a median of 32% improvement over the docked structures in terms of the change in root mean squared deviations from the experimental references. The influence of structural factors and explicit hydration on the performance of post-docking MD refinements are also discussed to help with their implementation in future methods and applications.

Synthesis and estrogenic activity of BODIPY-labeled estradiol conjugates.

Novel BODIPY-estradiol conjugates have been synthesized by selecting position C-3-O for labeling. The conjugation strategy was based on Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or etherification. Estradiol derivatives used as azide partners bearing an ω-azidoalkyl function through C4-C8-long linkers have been prepared. CuAAC reactions of estradiol azides with BODIPY alkyne furnished fluorescent 3-O-labeled conjugates bearing the triazole ring as a coupling moiety. Williamson etherifications of 3-O-(ω-bromoalkyl)-17ß-estradiol derivatives with BODIPY-OH resulted in labeled conjugates connected with an ether moiety. Interactions of the conjugates with estrogen receptor (ER) were investigated using molecular docking calculations in comparison with estradiol. The conjugates occupied both the classical and alternative binding sites on human ERα, with slightly lower binding affinity to references estradiol and diethystilbestrol. All compounds have displayed reasonable estrogenic activity. They increased the proliferation of ER-positive breast cancer cell line MCF7 contrary to ER-negative SKBR-3 cell line. The most potent compound 13a induced the transcriptional activity of ER in dose-dependent manner in dual luciferase recombinant reporter model and increased progesterone receptor's expression, proving the retained estrogenic activity. The fluorescence of candidate compound 13a co-localised with the ERα. The newly synthesized labeled compounds might serve as good starting point for further development of fluorescent probes for modern biological applications. In addition to studying steroid uptake and transport in cells, e.g. in the processes of biodegradation of estrogen-hormones micropollutants, they could also be utilized in examination of estrogen-binding proteins.

Target-ligand binding affinity from single point enthalpy calculation and elemental composition.

Reliable target-ligand binding thermodynamics data are essential for successful drug design and molecular engineering projects. Besides experimental methods, a number of theoretical approaches have been introduced for the generation of binding thermodynamics data. However, available approaches often neglect electronic effects or explicit water molecules influencing target-ligand interactions. To handle electronic effects within a reasonable time frame, we introduce a fast calculator QMH-L using a single target-ligand complex structure pre-optimized at the molecular mechanics level. QMH-L is composed of the semi-empirical quantum mechanics calculation of binding enthalpy with predicted explicit water molecules at the complex interface, and a simple descriptor based on the elemental composition of the ligand. QMH-L estimates the target-ligand binding free energy with a root mean square error (RMSE) of 0.94 kcal mol-1. The calculations also provide binding enthalpy values and they were compared with experimental binding thermodynamics data collected from the most reliable isothermal titration calorimetry studies of systems including various protein targets and challenging, large peptide ligands with a molecular weight of up to 2-3 thousand. The single point enthalpy calculations of QMH-L require modest computational resources and are based on short runs with open source and/or free software like Gromacs, Mopac, MobyWat, and Fragmenter. QMH-L can be applied for fast, automated scoring of drug candidates during a virtual screen, enthalpic engineering of new ligands or thermodynamic explanation of complex interactions.

Inhibition of xanthine oxidase-catalyzed xanthine and 6-mercaptopurine oxidation by luteolin, naringenin, myricetin, ampelopsin and their conjugated metabolites.

Luteolin, naringenin, myricetin, and ampelopsin are abundant flavonoids in nature, and several dietary supplements also contain them at very high doses. After the peroral intake, flavonoids go through extensive presystemic biotransformation; therefore, typically their sulfate/glucuronic acid conjugates reach high concentrations in the circulation. Xanthine oxidase (XO) enzyme is involved in uric acid production, and it also takes part in the elimination of certain drugs (e.g., 6-mercaptopurine). The inhibitory effects of flavonoid aglycones on XO have been widely studied; however, only limited data are available regarding their sulfate and glucuronic acid conjugates. In this study, we examined the impacts of luteolin, naringenin, myricetin, ampelopsin, and their sulfate/glucuronide derivatives on XO-catalyzed xanthine and 6-mercaptopurine oxidations employing in vitro enzyme incubation assays and molecular modeling studies. Our major results/conclusions are the following: (1) Sulfate metabolites were stronger while glucuronic acid derivatives were weaker inhibitors of XO compared to the parent flavonoids. (2) Naringenin, ampelopsin, and their metabolites were weak inhibitors of the enzyme. (3) Luteolin, myricetin, and their sulfates were highly potent inhibitors of XO, and the glucuronides of luteolin showed moderate inhibitory impacts. (4) Conjugated metabolites of luteolin and myricetin can be involved in the inhibitory effects of these flavonoids on XO enzyme.

Construction of Histone-Protein Complex Structures by Peptide Growing.

The structures of histone complexes are master keys to epigenetics. Linear histone peptide tails often bind to shallow pockets of reader proteins via weak interactions, rendering their structure determination challenging. In the present study, a new protocol, PepGrow, is introduced. PepGrow uses docked histone fragments as seeds and grows the full peptide tails in the reader-binding pocket, producing atomic-resolution structures of histone-reader complexes. PepGrow is able to handle the flexibility of histone peptides, and it is demonstrated to be more efficient than linking pre-docked peptide fragments. The new protocol combines the advantages of popular program packages and allows fast generation of solution structures. AutoDock, a force-field-based program, is used to supply the docked peptide fragments used as structural seeds, and the building algorithm of Modeller is adopted and tested as a peptide growing engine. The performance of PepGrow is compared to ten other docking methods, and it is concluded that in situ growing of a ligand from a seed is a viable strategy for the production of complex structures of histone peptides at atomic resolution.

The Advances and Limitations of the Determination and Applications of Water Structure in Molecular Engineering.

Water is a key actor of various processes of nature and, therefore, molecular engineering has to take the structural and energetic consequences of hydration into account. While the present review focuses on the target-ligand interactions in drug design, with a focus on biomolecules, these methods and applications can be easily adapted to other fields of the molecular engineering of molecular complexes, including solid hydrates. The review starts with the problems and solutions of the determination of water structures. The experimental approaches and theoretical calculations are summarized, including conceptual classifications. The implementations and applications of water models are featured for the calculation of the binding thermodynamics and computational ligand docking. It is concluded that theoretical approaches not only reproduce or complete experimental water structures, but also provide key information on the contribution of individual water molecules and are indispensable tools in molecular engineering.

Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor.

Introduction: Previous studies have established that endogenous inorganic polysulfides have significant biological actions activating the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor. Organic polysulfides exert similar effects, but they are much more stable molecules, therefore these compounds are more suitable as drugs. In this study, we aimed to better understand the mechanism of action of organic polysulfides by identification of their binding site on the TRPA1 receptor. Methods: Polysulfides can readily interact with the thiol side chain of the cysteine residues of the protein. To investigate their role in the TRPA1 activation, we replaced several cysteine residues by alanine via site-directed mutagenesis. We searched for TRPA1 mutant variants with decreased or lost activating effect of the polysulfides, but with other functions remaining intact (such as the effects of non-electrophilic agonists and antagonists). The binding properties of the mutant receptors were analyzed by in silico molecular docking. Functional changes were tested by in vitro methods: calcium sensitive fluorescent flow cytometry, whole-cell patch-clamp and radioactive calcium-45 liquid scintillation counting. Results: The cysteines forming the conventional binding site of electrophilic agonists, namely C621, C641 and C665 also bind the organic polysulfides, with the key role of C621. However, only their combined mutation abolished completely the organic polysulfide-induced activation of the receptor. Discussion: Since previous papers provided evidence that organic polysulfides exert analgesic and anti-inflammatory actions in different in vivo animal models, we anticipate that the development of TRPA1-targeted, organic polysulfide-based drugs will be promoted by this identification of the binding site.

Interaction of Fumonisin B1, N-Palmitoyl-Fumonisin B1, 5-O-Palmitoyl-Fumonisin B1, and Fumonisin B4 Mycotoxins with Human Serum Albumin and Their Toxic Impacts on Zebrafish Embryos.

Fumonisins are frequent food contaminants. The high exposure to fumonisins can cause harmful effects in humans and animals. Fumonisin B1 (FB1) is the most typical member of this group; however, the occurrence of several other derivatives has been reported. Acylated metabolites of FB1 have also been described as possible food contaminants, and the very limited data available suggest their significantly higher toxicity compared to FB1. Furthermore, the physicochemical and toxicokinetic properties (e.g., albumin binding) of acyl-FB1 derivatives may show large differences compared to the parent mycotoxin. Therefore, we tested the interactions of FB1, N-palmitoyl-FB1 (N-pal-FB1), 5-O-palmitoyl-FB1 (5-O-pal-FB1), and fumonisin B4 (FB4) with human serum albumin as well as the toxic effects of these mycotoxins on zebrafish embryos were examined. Based on our results, the most important observations and conclusions are the following: (1) FB1 and FB4 bind to albumin with low affinity, while palmitoyl-FB1 derivatives form highly stable complexes with the protein. (2) N-pal-FB1 and 5-O-pal-FB1 likely occupy more high-affinity binding sites on albumin. (3) Among the mycotoxins tested, N-pal-FB1 showed the most toxic effects on zebrafish, followed by 5-O-pal-FB1, FB4, and FB1. (4) Our study provides the first in vivo toxicity data regarding N-pal-FB1, 5-O-pal-FB1, and FB4.

Probing the Interactions of 31 Mycotoxins with Xanthine Oxidase: Alternariol, Alternariol-3-Sulfate, and α-Zearalenol Are Allosteric Inhibitors of the Enzyme.

Mycotoxins are frequent toxic contaminants in foods and beverages, causing a significant health threat. Interactions of mycotoxins with biotransformation enzymes (e.g., cytochrome P450 enzymes, sulfotransferases, and uridine 5'-diphospho-glucuronosyltransferases) may be important due to their possible detoxification or toxic activation during enzymatic processes. Furthermore, mycotoxin-induced enzyme inhibition may affect the biotransformation of other molecules. A recent study described the strong inhibitory effects of alternariol and alternariol-9-methylether on the xanthine oxidase (XO) enzyme. Therefore, we aimed to test the impacts of 31 mycotoxins (including the masked/modified derivatives of alternariol and alternariol-9-methylether) on XO-catalyzed uric acid formation. Besides the in vitro enzyme incubation assays, mycotoxin depletion experiments and modeling studies were performed. Among the mycotoxins tested, alternariol, alternariol-3-sulfate, and α-zearalenol showed moderate inhibitory actions on the enzyme, representing more than tenfold weaker impacts compared with the positive control inhibitor allopurinol. In mycotoxin depletion assays, XO did not affect the concentrations of alternariol, alternariol-3-sulfate, and α-zearalenol in the incubates; thus, these compounds are inhibitors but not substrates of the enzyme. Experimental data and modeling studies suggest the reversible, allosteric inhibition of XO by these three mycotoxins. Our results help the better understanding of the toxicokinetic interactions of mycotoxins.

The heptapeptide somatostatin analogue TT-232 exerts analgesic and anti-inflammatory actions via SST4 receptor activation: In silico, in vitro and in vivo evidence in mice.

Since the conventional and adjuvant analgesics have limited effectiveness frequently accompanied by serious side effects, development of novel, potent pain killers for chronic neuropathic and inflammatory pain conditions is a big challenge. Somatostatin (SS) regulates endocrine, vascular, immune and neuronal functions, cell proliferation through 5 Gi protein-coupled receptors (SST1-SST5). SS released from the capsaicin-sensitive peptidergic sensory nerves mediates anti-inflammatory and antinociceptive effects without endocrine actions via SST4. The therapeutic use of the native SS is limited by its diverse biological actions and short plasma elimination half-life. Therefore, SST4 selective SS analogues could be promising analgesic and anti-inflammatory drug candidates with new mode of action. TT-232 is a cyclic heptapeptide showing great affinity to SST4 and SST1. Here, we report the in silico SST4 receptor binding mechanism, in vitro binding (competition assay) and cAMP- decreasing effect of TT-232 in SST4-expressing CHO cells, as well as its analgesic and anti-inflammatory actions in chronic neuropathic pain and arthritis models using wildtype and SST4-deficient mice. TT-232 binds to SST4 with similar interaction energy (-11.03 kcal/mol) to the superagonist J-2156, displaces somatostatin from SST4 binding (10 nM to 30 µM) and inhibits forskolin-stimulated cAMP accumulation (EC50: 371.6 ± 58.03 nmol; Emax: 78.63 ± 2.636 %). Its i.p. injection (100, 200 µg/kg) results in significant, 35.7 % and 50.4 %, analgesic effects upon single administration in chronic neuropathic pain and repeated injection in arthritis models in wildtype, but not in SST4-deficient mice. These results provide evidence that the analgesic effect of TT-232 is mediated by SST4 activation, which might open novel drug developmental potentials. Chemical compounds Chemical compounds studied in this article TT-232 (PubChem CID: 74053735).

Testing Serum Albumins and Cyclodextrins as Potential Binders of the Mycotoxin Metabolites Alternariol-3-Sulfate, Alternariol-9-Monomethylether and Alternariol-9-Monomethylether-3-Sulfate.

Alternaria mycotoxins, including alternariol (AOH), alternariol-9-monomethylether (AME), and their masked/modified derivatives (e.g., sulfates or glycosides), are common food contaminants. Their acute toxicity is relatively low, while chronic exposure can lead to the development of adverse health effects. Masked/modified metabolites can probably release the more toxic parent mycotoxin due to their enzymatic hydrolysis in the intestines. Previously, we demonstrated the complex formation of AOH with serum albumins and cyclodextrins; these interactions were successfully applied for the extraction of AOH from aqueous matrices (including beverages). Therefore, in this study, the interactions of AME, alternariol-3-sulfate (AS), and alternariol-9-monomethylether-3-sulfate (AMS) were investigated with albumins (human, bovine, porcine, and rat) and with cyclodextrins (sulfobutylether-ß-cyclodextrin, sugammadex, and cyclodextrin bead polymers). Our major results/conclusions are the following: (1) The stability of mycotoxin-albumin complexes showed only minor species dependent variations. (2) AS and AMS formed highly stable complexes with albumins in a wide pH range, while AME-albumin interactions preferred alkaline conditions. (3) AME formed more stable complexes with the cyclodextrins examined than AS and AMS. (4) Beta-cyclodextrin bead polymer proved to be highly suitable for the extraction of AME, AS, and AMS from aqueous solution. (5) Albumins and cyclodextrins are promising binders of the mycotoxins tested.

The Structural Effects of Phosphorylation of Protein Arginine Methyltransferase 5 on Its Binding to Histone H4.

The protein arginine methyltransferase 5 (PRMT5) enzyme is responsible for arginine methylation on various proteins, including histone H4. PRMT5 is a promising drug target, playing a role in the pathomechanism of several diseases, especially in the progression of certain types of cancer. It was recently proved that the phosphorylation of PRMT5 on T80 residue increases its methyltransferase activity; furthermore, elevated levels of the enzyme were measured in the case of human hepatocellular carcinoma and other types of tumours. In this study, we constructed the complexes of the unmodified human PRMT5-methylosome protein 50 (MEP50) structure and its T80-phosphorylated variant in complex with the full-length histone H4 peptide. The full-length histone H4 was built in situ into the human PRMT5-MEP50 enzyme using experimental H4 fragments. Extensive molecular dynamic simulations and structure and energy analyses were performed for the complexed and apo protein partners, as well. Our results provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type; (2) the PRMT5 methylates only the free form of histone H4 not bound in the nucleosome. The atomic level complex structure H4-PRMT5-MEP50 will help the design of new inhibitors and in uncovering further structure-function relationships of PRMT enzymes.

Interaction of the Emerging Mycotoxins Beauvericin, Cyclopiazonic Acid, and Sterigmatocystin with Human Serum Albumin.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA-HSA (Ka ≈ 103 L/mol) and moderately strong CPA-HSA and STC-HSA complexes (Ka ≈ 104 L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow's site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA-HSA interaction may have toxicological importance.

Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design.

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin-SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Incorporation of Oxidized Phenylalanine Derivatives into Insulin Signaling Relevant Proteins May Link Oxidative Stress to Signaling Conditions Underlying Chronic Insulin Resistance.

A link between oxidative stress and insulin resistance has been suggested. Hydroxyl free radicals are known to be able to convert phenylalanine (Phe) into the non-physiological tyrosine isoforms ortho- and meta-tyrosine (o-Tyr, m-Tyr). The aim of our study was to examine the role of o-Tyr and m-Tyr in the development of insulin resistance. We found that insulin-induced uptake of glucose was blunted in cultures of 3T3-L1 grown on media containing o- or m-Tyr. We show that these modified amino acids are incorporated into cellular proteins. We focused on insulin receptor substrate 1 (IRS-1), which plays a role in insulin signaling. The activating phosphorylation of IRS-1 was increased by insulin, the effect of which was abolished in cells grown in m-Tyr or o-Tyr media. We found that phosphorylation of m- or o-Tyr containing IRS-1 segments by insulin receptor (IR) kinase was greatly reduced, PTP-1B phosphatase was incapable of dephosphorylating phosphorylated m- or o-Tyr IRS-1 peptides, and the SH2 domains of phosphoinositide 3-kinase (PI3K) bound the o-Tyr IRS-1 peptides with greatly reduced affinity. According to our data, m- or o-Tyr incorporation into IRS-1 modifies its protein-protein interactions with regulating enzymes and effectors, thus IRS-1 eventually loses its capacity to play its role in insulin signaling, leading to insulin resistance.

Prerequisite Binding Modes Determine the Dynamics of Action of Covalent Agonists of Ion Channel TRPA1.

Transient receptor potential ankyrin 1 (TRPA1) is a transmembrane protein channeling the influx of calcium ions. As a polymodal nocisensor, TRPA1 can be activated by thermal, mechanical stimuli and a wide range of chemically damaging molecules including small volatile environmental toxicants and endogenous algogenic lipids. After activation by such compounds, the ion channel opens up, its central pore widens allowing calcium influx into the cytosol inducing signal transduction pathways. Afterwards, the calcium influx desensitizes irritant evoked responses and results in an inactive state of the ion channel. Recent experimental determination of structures of apo and holo forms of TRPA1 opened the way towards the design of new agonists, which can activate the ion channel. The present study is aimed at the elucidation of binding dynamics of agonists using experimental structures of TRPA1-agonist complexes at the atomic level applying molecular docking and dynamics methods accounting for covalent and non-covalent interactions. Following a test of docking methods focused on the final, holo structures, prerequisite binding modes were detected involving the apo forms. It was shown how reversible interactions with prerequisite binding sites contribute to structural changes of TRPA1 leading to covalent bonding of agonists. The proposed dynamics of action allowed a mechanism-based forecast of new, druggable binding sites of potent agonists.

Determination of Ligand Binding Modes in Hydrated Viral Ion Channels to Foster Drug Design and Repositioning.

Target-based design and repositioning are mainstream strategies of drug discovery. Numerous drug design and repositioning projects have been launched to fight the ongoing COVID-19 pandemic. The resulting drug candidates have often failed due to the misprediction of their target-bound structures. The determination of water positions of such structures is particularly challenging due to the large number of possible drugs and the diversity of their hydration patterns. To answer this challenge and help correct predictions, we introduce a new protocol HydroDock, which can build hydrated drug-target complexes from scratch. HydroDock requires only the dry target and drug structures and produces their complexes with appropriately positioned water molecules. As a test application of the protocol, we built the structures of amantadine derivatives in complex with the influenza M2 transmembrane ion channel. The repositioning of amantadine derivatives from this influenza target to the SARS-CoV-2 envelope protein was also investigated. Excellent agreement was observed between experiments and the structures determined by HydroDock. The atomic resolution complex structures showed that water plays a similar role in the binding of amphipathic amantadine derivatives to transmembrane ion channels of both influenza A and SARS-CoV-2. While the hydrophobic regions of the channels capture the bulky hydrocarbon group of the ligand, the surrounding waters direct its orientation parallel with the axes of the channels via bridging interactions with the ionic ligand head. As HydroDock supplied otherwise undetermined structural details, it can be recommended to improve the reliability of future design and repositioning of antiviral drug candidates and many other ligands with an influence of water structure on their mechanism of action.

Small molecule somatostatin receptor subtype 4 (sst4) agonists are novel anti-inflammatory and analgesic drug candidates.

We provided strong proof of concept evidence that somatostatin mediates potent analgesic and anti-inflammatory actions via its receptor subtype 4 (sst4) located both at the periphery and the central nervous system. Therefore, sst4 agonists are promising novel drug candidates for neuropathic pain and neurogenic inflammation, but rational drug design was not possible due to the lack of knowledge about its 3-dimensional structure. We modeled the sst4 receptor structure, described its agonist binding properties, and characterized the binding of our novel small molecule sst4 agonists (4-phenetylamino-7H-pyrrolo[2,3-d]pyrimidine derivatives) using an in silico platform. In addition to the in silico binding data, somatostatin displacement by Compound 1 was demonstrated in the competitive binding assay on sst4-expressing cells. In vivo effects were investigated in rat models of neurogenic inflammation and chronic traumatic neuropathic pain. We defined high- and low-affinity binding pockets of sst4 for our ligands, binding of the highest affinity compounds were similar to that of the reference ligand J-2156. We showed potent G-protein activation with the highest potency of 10 nM EC50 value and highest efficacy of 342%. Oral administration of 100 µg/kg of 5 compounds significantly inhibited acute neurogenic plasma protein extravasation in the paw skin by 40-60%, one candidate abolished and 3 others diminished sciatic nerve-ligation induced neuropathic hyperalgesia by 28-62%. The in silico predictions on sst4-ligands were tested in biological systems. Low oral dose of our novel agonists inhibit neurogenic inflammation and neuropathic pain, which opens promising drug developmental perspectives for these unmet medical need conditions.