Chemically inducible split protein regulators for mammalian cells.

Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited because of unfavorable ligand characteristics or the immunogenicity of xenogeneic protein domains. To address these issues, we present a strategy for engineering inducible split protein regulators (INSPIRE) in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a cognate physiological ligand or clinically approved drug. We show that the INSPIRE platform can be used for dynamic, orthogonal and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of a glucocorticoid-responsive INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small-molecule responsive systems that can be implemented for the construction of new sensors, regulatory networks and therapeutic applications.

Fentanyl and the Fluorinated Fentanyl Derivative NFEPP Elicit Distinct Hydrogen-Bond Dynamics of the Opioid Receptor.

The development of safe therapeutics to manage pain is of central interest for biomedical applications. The fluorinated fentanyl derivative N-(3-fluoro-1-phenethylpiperidin-4-yl)-N-phenylpropionamide (NFEPP) is potentially a safer alternative to fentanyl because unlike fentanyl─which binds to the µ-opioid receptor (MOR) at both physiological and acidic pH─NFEPP might bind to the MOR only at acidic pH typical of inflamed tissue. Knowledge of the protonation-coupled dynamics of the receptor-drug interactions is thus required to understand the molecular mechanism by which receptor activation initiates cell signaling to silence pain. To this end, here we have carried out extensive atomistic simulations of the MOR in different protonation states, in the absence of opioid drugs, and in the presence of fentanyl vs NFEPP. We used graph-based analyses to characterize internal hydrogen-bond networks that could contribute to the activation of the MOR. We find that fentanyl and NFEPP prefer distinct binding poses and that, in their binding poses, fentanyl and NFEPP partake in distinct internal hydrogen-bond networks, leading to the cytoplasmic G-protein-binding region. Moreover, the protonation state of functionally important aspartic and histidine side chains impacts hydrogen-bond networks that extend throughout the receptor, such that the ligand-bound MOR presents at its cytoplasmic G-protein-binding side, a hydrogen-bonding environment where dynamics depend on whether fentanyl or NFEPP is bound, and on the protonation state of specific MOR groups. The exquisite sensitivity of the internal protein-water hydrogen-bond network to the protonation state and to details of the drug binding could enable the MOR to elicit distinct pH- and opioid-dependent responses at its cytoplasmic G-protein-binding site.

ProBiS-Dock: A Hybrid Multitemplate Homology Flexible Docking Algorithm Enabled by Protein Binding Site Comparison.

The protein data bank (PDB) is a rich source of protein ligand structures, but ligands are not explicitly used in current docking algorithms. We have developed ProBiS-Dock, a docking algorithm complementary to the ProBiS-Dock Database (J. Chem. Inf. Model. 2021, 61, 4097-4107) that treats small molecules and proteins as fully flexible entities and allows conformational changes in both after ligand binding. A new scoring function is described that consists of a binding site-specific scoring function (ProBiS-Score) and a general statistical scoring function. ProBiS-Dock enables rapid docking of small molecules to proteins and has been successfully validated in silico against standard benchmarks. It enables rapid search for new active ligands by leveraging existing knowledge in the PDB. The potential of the software for drug development has been confirmed in vitro by the discovery of new inhibitors of human indoleamine 2,3-dioxygenase 1, an enzyme that is an attractive target for cancer therapy and catalyzes the first rate-determining step of l-tryptophan metabolism via the kynurenine pathway. The software is freely available to academic users at http://insilab.org/probisdock.

ProBiS-Dock Database: A Web Server and Interactive Web Repository of Small Ligand-Protein Binding Sites for Drug Design.

We have developed a new system, ProBiS-Dock, which can be used to determine the different types of protein binding sites for small ligands. The binding sites identified this way are then used to construct a new binding site database, the ProBiS-Dock Database, that allows for the ranking of binding sites according to their utility for drug development. The newly constructed database currently has more than 1.4 million binding sites and offers the possibility to investigate potential drug targets originating from different biological species. The interactive ProBiS-Dock Database, a web server and repository that consists of all small-molecule ligand binding sites in all of the protein structures in the Protein Data Bank, is freely available at http://probis-dock-database.insilab.org. The ProBiS-Dock Database will be regularly updated to keep pace with the growth of the Protein Data Bank, and our anticipation is that it will be useful in drug discovery.

CHARMM Force-Field Parameters for Morphine, Heroin, and Oliceridine, and Conformational Dynamics of Opioid Drugs.

Opioid drug binding to specialized G protein-coupled receptors (GPCRs) can lead to analgesia upon activation via downstream Gi protein signaling and to severe side effects via activation of the ß-arrestin signaling pathway. Knowledge of how different opioid drugs interact with receptors is essential, as it can inform and guide the design of safer therapeutics. We performed quantum and classical mechanical computations to explore the potential energy landscape of four opioid drugs: morphine and its derivatives heroin and fentanyl and for the unrelated oliceridine. From potential energy profiles for bond twists and from interactions between opioids and water, we derived a set of force-field parameters that allow a good description of structural properties and intermolecular interactions of the opioids. Potential of mean force profiles computed from molecular dynamics simulations indicate that fentanyl and oliceridine have complex energy landscapes with relatively small energy penalties, suggesting that interactions with the receptor could select different binding poses of the drugs.

Opioid Receptors and Protonation-Coupled Binding of Opioid Drugs.

Opioid receptors are G-protein-coupled receptors (GPCRs) part of cell signaling paths of direct interest to treat pain. Pain may associate with inflamed tissue characterized by acidic pH. The potentially low pH at tissue targeted by opioid drugs in pain management could impact drug binding to the opioid receptor, because opioid drugs typically have a protonated amino group that contributes to receptor binding, and the functioning of GPCRs may involve protonation change. In this review, we discuss the relationship between structure, function, and dynamics of opioid receptors from the perspective of the usefulness of computational studies to evaluate protonation-coupled opioid-receptor interactions.

Protein-water hydrogen-bond networks of G protein-coupled receptors: Graph-based analyses of static structures and molecular dynamics.

Protein and protein-water hydrogen bonds shape the conformational energy landscape of G Protein-Coupled Receptors, GPCRs. As numerous static structures of GPCRs have been solved, the important question arises whether GPCR structures and GPCR conformational dynamics could be described in terms of conserved hydrogen-bond networks, and alterations of these hydrogen-bond networks along the reaction coordinate of the GPCR. To enable efficient analyses of the hydrogen-bond networks of GPCRs we implemented graph-based algorithms, and applied these algorithms to static GPCR structures from structural biology, and from molecular dynamics simulations of two opioid receptors. We find that static GPCR structures tend to have a conserved, core hydrogen-bond network which, when protein and water dynamics are included with simulations, extends to comprise most of the interior of an inactive receptor. In an active receptor, the dynamic protein-water hydrogen-bond network spans the entire receptor, bridging all functional motifs. Such an extensive, dynamic hydrogen-bond network might contribute to the activation mechanism of the GPCR.

Potential Energy Function for Fentanyl-Based Opioid Pain Killers.

Opioids are molecules whose binding to specialized G-Protein Coupled Receptors (GPCRs) triggers a signaling cascade that leads to the downregulation of pain pathways. Binding of an opioid to the membrane-embedded GPCR occurs when the opioid molecule is protonated, which provides a potential strategy to design nontoxic opioids that are protonated and bind to the GPCR only at the low pH of injured or inflamed tissue. Excellent model systems to study protonation-dependent binding of opioids to GPCRs are fentanyl, which is protonated and binds to the GPCR at both physiological and low pH, and the fluorinated fentanyl derivative NFEPP, which is protonated and binds to the GPCR only at low pH. The molecular mechanisms of fentanyl and NFEPP binding to the GPCR are largely unknown. To enable atomistic studies of opioid binding to GPCRs, we have carried out extensive quantum mechanical and classical mechanical computations to derive a potential energy function for fentanyl and NFEPP and present force field parameters for both opioid molecules. We find that fluorination alters the electronic ground state properties of fentanyl. As a consequence, fentanyl and NFEPP have distinct torsional and electrostatic properties likely to impact how they bind to receptors.

Loop Grafting between Similar Local Environments for Fc-Silent Antibodies.

Reduction of the affinity of the fragment crystallizable (Fc) region with immune receptors by substitution of one or a few amino acids, known as Fc-silencing, is an established approach to reduce the immune effector functions of monoclonal antibody therapeutics. This approach to Fc-silencing, however, is problematic as it can lead to instability and immunogenicity of the developed antibodies. We evaluated loop grafting as a novel approach to Fc-silencing in which the Fc loops responsible for immune receptor binding were replaced by loops of up to 20 amino acids from similar local environments in other human and mouse antibodies. Molecular dynamics simulations of the designed variants of an Fc region in a complex with the immune receptor FcγIIIa confirmed that loop grafting potentially leads to a significant reduction in the binding of the antibody variants to the receptor, while retaining their stability. In comparison, standard variants with less than eight substituted amino acids showed possible instability and a lower degree of Fc-silencing due to the occurrence of compensatory interactions. The presented approach to Fc-silencing is general and could be used to modulate undesirable side effects of other antibody therapeutics without affecting their stability or increasing their immunogenicity.

Discovery of Novel Potential Human Targets of Resveratrol by Inverse Molecular Docking.

Resveratrol is a polyphenol known for its antioxidant and anti-inflammatory properties, which support its use as a treatment for variety of diseases. There are already known connections of resveratrol to chemoprevention of cancer because of its ability to prevent tumor initiation and inhibit tumor promotion and progression. Resveratrol is also believed to be important in cardiovascular diseases and neurological disorders, such as Alzheimer's disease. Using an inverse molecular docking approach, we sought to find new potential targets of resveratrol. Docking of resveratrol into each ProBiS predicted binding site of >38 000 protein structures from the Protein Data Bank was examined, and a number of novel potential targets into which resveratrol was docked successfully were found. These explain known actions or predict new effects of resveratrol. The results included three human proteins that are already known to bind resveratrol. A majority of proteins discovered however have no already described connections with resveratrol. We report new potential target human proteins and proteins connected with different organisms into which resveratrol can dock. Our results reveal previously unknown potential target human proteins, whose connection with cardiovascular and neurological disorders could lead to new potential treatments for variety of diseases. We believe that our research could help in future experimental studies on revestratol bioactivity in humans.

Discovery of new MurA inhibitors using induced-fit simulation and docking.

We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1µM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.

Nonpeptidic Selective Inhibitors of the Chymotrypsin-Like (ß5 i) Subunit of the Immunoproteasome.

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (ß5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the ß5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as ß5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

LiSiCA: A Software for Ligand-Based Virtual Screening and Its Application for the Discovery of Butyrylcholinesterase Inhibitors.

We developed LiSiCA (ligand similarity using clique algorithm)--ligand-based virtual screening software that uses a fast maximum clique algorithm to find two- and three-dimensional similarities between pairs of molecules and applied it to the discovery of novel potent butyrylcholinesterase inhibitors. LiSiCA, which runs in parallel on multiple processor cores, was successfully tested on the Database of Useful Decoys-Enhanced, to evaluate its ability to discriminate active molecules from decoys. We then applied LiSiCA for the discovery of novel inhibitors of human butyrylcholinesterase, a promising anti-Alzheimer target, using a known inhibitor as the reference compound. We demonstrated that LiSiCA is capable of finding potent nanomolar inhibitors, whose scaffolds differed from the reference compound, thus proving its ability for scaffold hopping and usefulness in drug discovery.

Molecular dynamics to enhance structure-based virtual screening on cathepsin B.

Molecular dynamics (MD) and molecular docking are commonly used to study molecular interactions in drug discovery. Most docking approaches consider proteins as rigid, which can decrease the accuracy of predicted docked poses. Therefore MD simulations can be used prior to docking to add flexibility to proteins. We evaluated the contribution of using MD together with docking in a docking study on human cathepsin B, a well-studied protein involved in numerous pathological processes. Using CHARMM biomolecular simulation program and AutoDock Vina molecular docking program, we found, that short MD simulations significantly improved molecular docking. Our results, expressed with the area under the receiver operating characteristic curves, show an increase in discriminatory power i.e. the ability to discriminate active from inactive compounds of molecular docking, when docking is performed to selected snapshots from MD simulations.

Mechanistic Insights of Polyphenolic Compounds from Rosemary Bound to Their Protein Targets Obtained by Molecular Dynamics Simulations and Free-Energy Calculations.

Rosemary represents an important medicinal plant that has been attributed with various health-promoting properties, especially antioxidative, anti-inflammatory, and anticarcinogenic activities. Carnosic acid, carnosol, and rosmanol, as well as the phenolic acid ester rosmarinic acid, are the main compounds responsible for these actions. In our earlier research, we carried out an inverse molecular docking at the proteome scale to determine possible protein targets of the mentioned compounds. Here, we subjected the previously identified ligand-protein complexes with HIV-1 protease, K-RAS, and factor X to molecular dynamics simulations coupled with free-energy calculations. We observed that carnosic acid and rosmanol act as viable binders of the HIV-1 protease. In addition, carnosol represents a potential binder of the oncogene protein K-RAS. On the other hand, rosmarinic acid was characterized as a weak binder of factor X. We also emphasized the importance of water-mediated hydrogen-bond networks in stabilizing the binding conformation of the studied polyphenols, as well as in mechanistically explaining their promiscuous nature.

The Effect of the Ala16Val Mutation on the Secondary Structure of the Manganese Superoxide Dismutase Mitochondrial Targeting Sequence.

Manganese Superoxide Dismutase (MnSOD) represents a mitochondrial protein that scavenges reactive oxygen species (ROS) responsible for oxidative stress. A known single nucleotide polymorphism (SNP) rs4880 on the SOD2 gene, causing a mutation from alanine to valine (Ala16Val) in the primary structure of immature MnSOD, has been associated with several types of cancer and other autoimmune diseases. However, no conclusive correlation has been established yet. This study aims to determine the effect of the alanine to valine mutation on the secondary structure of the MnSOD mitochondrial targeting sequence (MTS). A model for each variant of the MTS was prepared and extensively simulated with molecular dynamics simulations using the CHARMM36m force field. The results indicate that the alanine variant of the MTS preserves a uniform α-helical secondary structure favorable for the protein transport into mitochondria, whereas the valine variant quickly breaks down its α-helix. Thus, the alanine MTS represents the more active MnSOD variant, the benefits of which have yet to be determined experimentally.

Mechanistic Insights into Biological Activities of Polyphenolic Compounds from Rosemary Obtained by Inverse Molecular Docking.

Rosemary (Rosmarinus officinalis L.) represents a medicinal plant known for its various health-promoting properties. Its extracts and essential oils exhibit antioxidative, anti-inflammatory, anticarcinogenic, and antimicrobial activities. The main compounds responsible for these effects are the diterpenes carnosic acid, carnosol, and rosmanol, as well as the phenolic acid ester rosmarinic acid. However, surprisingly little is known about the molecular mechanisms responsible for the pharmacological activities of rosemary and its compounds. To discern these mechanisms, we performed a large-scale inverse molecular docking study to identify their potential protein targets. Listed compounds were separately docked into predicted binding sites of all non-redundant holo proteins from the Protein Data Bank and those with the top scores were further examined. We focused on proteins directly related to human health, including human and mammalian proteins as well as proteins from pathogenic bacteria, viruses, and parasites. The observed interactions of rosemary compounds indeed confirm the beforementioned activities, whereas we also identified their potential for anticoagulant and antiparasitic actions. The obtained results were carefully checked against the existing experimental findings from the scientific literature as well as further validated using both redocking procedures and retrospective metrics.

In Silico Laboratory: Tools for Similarity-Based Drug Discovery.

Computational methods that predict and evaluate binding of ligands to receptors implicated in different pathologies have become crucial in modern drug design and discovery. Here, we describe protocols for using the recently developed package of computational tools for similarity-based drug discovery. The ProBiS stand-alone program and web server allow superimposition of protein structures against large protein databases and predict ligands based on detected binding site similarities. GenProBiS allows mapping of human somatic missense mutations related to cancer and non-synonymous single nucleotide polymorphisms and subsequent visual exploration of specific interactions in connection to these mutations. We describe protocols for using LiSiCA, a fast ligand-based virtual screening software that enables easy screening of large databases containing billions of small molecules. Finally, we show the use of BoBER, a web interface that enables user-friendly access to a large database of bioisosteric and scaffold hopping replacements.

Stereoselective Activity of 1-Propargyl-4-styrylpiperidine-like Analogues That Can Discriminate between Monoamine Oxidase Isoforms A and B.

The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.