Effect of nelfinavir stereoisomers on coronavirus main protease: Molecular docking, molecular dynamics simulation and MM/GBSA study.

In this study, the binding strength of 32 diastereomers of nelfinavir, a proposed drug for the treatment of COVID-19, was considered against main protease. Molecular docking was used to determine the most potent diastereomers. The top three diastereomers along with apo form of protein were then considered via molecular dynamics simulation and MM-GBSA method. During the simulation, the structural consideration of four proteins considered was carried out using RMSD, RMSF, Rg and hydrogen bond analysis tools. Our data demonstrated that the effect of nelfinavir RSRSR stereoisomer on protein stability and compactness is higher than the other. We also found from the hydrogen bond analysis that this important diastereomer form three hydrogen bonds with the residues of Glu166, Gly143 and Hie41. MM/GBSA analysis showed that the binding strength of RSRSR is more than other stereoisomers and that the main contributions to binding energy are vdW and electronic terms. The nelfinavir RSRSR stereoisomer introduced in this study may be effective in the treatment of COVID-19.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

In silico design and molecular basis for the selectivity of Olinone toward the first over the second bromodomain of BRD4.

Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of small-molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.

Understanding allosteric interactions in hMLKL protein that modulate necroptosis and its inhibition.

Mixed Lineage Kinase domain-Like (MLKL), a key player in necroptosis, is a multi-domain protein with an N-terminal 4 helical bundle (4HB) and a pseudokinase domain (PsK) connected by brace helices. Phosphorylation of PsK domain of MLKL is a key step towards oligomerization of 4HB domain that causes cell death. Necrosulfonamide (NSA) binds to the 4HB domain of MLKL to inhibit necroptosis. To understand the molecular details of MLKL function and it's inhibition, we have performed a molecular dynamic study on hMLKL protein in apo, phosphorylated and NSA-bound states for a total 3 µs simulation time. Our simulations show increased inter-domain flexibility, increased rigidification of the activation loop and increased alpha helical content in the brace helix region revealing a form of monomeric hMLKL necessary for oligomerization upon phosphorylation as compared to apo state. NSA binding disrupts this activated form and causes two main effects on hMLKL conformation: (1) locking of the relative orientation of 4HB and PsK domains by the formation of several new interactions and (2) prevention of key 4HB residues to participate in cross-linking for oligomer formation. This new understanding of the effect of hMLKL conformations on phosphorylation and NSA binding suggest new avenues for designing effective allosteric inhibitors of hMLKL.

Structural basis of species-selective antagonist binding to the succinate receptor.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.

Exploring Gatekeeper Mutations in EGFR through Computer Simulations.

The emergence of resistance against drugs that inhibit a particular protein is a major problem in targeted therapy. There is a clear need for rigorous methods to predict the likelihood of specific drug-resistance mutations arising in response to the binding of a drug. In this work we attempt to develop a robust computational protocol for predicting drug resistant mutations at the gatekeeper position (T790) in EGFR. We explore how mutations at this site affects interactions with ATP and three drugs that are currently used in clinics. We found, as expected, that certain mutations are not tolerated structurally, while some other mutations interfere with the natural substrate and hence are unlikely to be selected for. However, we found five possible mutations that are well tolerated structurally and energetically. Two of these mutations were predicted to have increased affinity for the drugs over ATP, as has been reported earlier. By reproducing the trends in the experimental binding affinities of the data, the methods chosen here are able to correctly predict the effects of these mutations on the binding affinities of the drugs. However, the increased affinity does not always translate into increased efficacy, because the efficacy is affected by several other factors such as binding kinetics, competition with ATP, and residence times. The computational methods used in the current study are able to reproduce or predict the effects of mutations on the binding affinities. However, a different set of methods is required to predict the kinetics of drug binding.

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074.

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.

Crystal Structures of Anti-apoptotic BFL-1 and Its Complex with a Covalent Stapled Peptide Inhibitor.

BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.

The Antinociceptive Agent SBFI-26 Binds to Anandamide Transporters FABP5 and FABP7 at Two Different Sites.

Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti-inflammatory effects in mice. The synthesis of SBFI-26 yields several stereoisomers, and it is not known how the inhibitor binds the transporters. Here we report co-crystal structures of SBFI-26 in complex with human FABP5 and FABP7 at 2.2 and 1.9 Å resolution, respectively. We found that only (S)-SBFI-26 was present in the crystal structures. The inhibitor largely mimics the fatty acid binding pattern, but it also has several unique interactions. Notably, the FABP7 complex corroborates key aspects of the ligand binding pose at the canonical site previously predicted by virtual screening. In FABP5, SBFI-26 was unexpectedly found to bind at the substrate entry portal region in addition to binding at the canonical ligand-binding pocket. Our structural and binding energy analyses indicate that both R and S forms appear to bind the transporter equally well. We suggest that the S enantiomer observed in the crystal structures may be a result of the crystallization process selectively incorporating the (S)-SBFI-26-FABP complexes into the growing lattice, or that the S enantiomer may bind to the portal site more rapidly than to the canonical site, leading to an increased local concentration of the S enantiomer for binding to the canonical site. Our work reveals two binding poses of SBFI-26 in its target transporters. This knowledge will guide the development of more potent FABP inhibitors based upon the SBFI-26 scaffold.

Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors.

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

X-ray structures define human P2X(3) receptor gating cycle and antagonist action.

P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the 'cytoplasmic cap', which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

Helix12-Stabilization Antagonist of Vitamin D Receptor.

To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and synthesized vitamin D analogues with bulky side chains based on the "active antagonist" concept in which antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds 3a and 3b showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and static X-ray crystal structure analyses indicated that compound 3a stabilizes H11-H12 but displaces H6-H7 so that 3a is a novel rather than "active" or "passive" type of antagonist. We classified 3a as a third type of antagonist and called it "H11-H12 stabilization antagonist". HDX-MS analysis indicated that antagonist 3b is an "active" antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse depending on each antagonist structure. Additionally, HDX-MS was proven to be very useful for investigations of protein structure alterations resulting from ligand binding.

Interpreting Thermodynamic Profiles of Aminoadamantane Compounds Inhibiting the M2 Proton Channel of Influenza A by Free Energy Calculations.

The development of novel anti-influenza drugs is of great importance because of the capability of influenza viruses to occasionally cross interspecies barriers and to rapidly mutate. One class of anti-influenza agents, aminoadamantanes, including the drugs amantadine and rimantadine now widely abandoned due to virus resistance, bind to and block the pore of the transmembrane domain of the M2 proton channel (M2TM) of influenza A. Here, we present one of the still rare studies that interprets thermodynamic profiles from isothermal titration calorimetry (ITC) experiments in terms of individual energy contributions to binding, calculated by the computationally inexpensive implicit solvent/implicit membrane molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach, for aminoadamantane compounds binding to M2TM of the avian "Weybridge" strain. For all eight pairs of aminoadamantane compounds considered, the trend of the predicted relative binding free energies and their individual components, effective binding energies and changes in the configurational entropy, agrees with experimental measures (ΔΔG, ΔΔH, TΔΔS) in 88, 88, and 50% of the cases. In addition, information yielded by the MM-PBSA approach about determinants of binding goes beyond that available in component quantities (ΔH, ΔS) from ITC measurements. We demonstrate how one can make use of such information to link thermodynamic profiles from ITC with structural causes on the ligand side and, ultimately, to guide decision making in lead optimization in a prospective manner, which results in an aminoadamantane derivative with improved binding affinity against M2TM(Weybridge).

Activation helix orientation of the estrogen receptor is mediated by receptor dimerization: evidence from molecular dynamics simulations.

In recent years, the nuclear receptors (NR) dynamics have been studied extensively by various approaches. However, the transition path of helix 12 (H12) to an agonist or an antagonist conformation and the exchange pathway between these states is not clear yet. A number of accelerated molecular dynamics (aMD) runs were performed on both an ERα monomer and a homodimer with a total length of 2.2 µs. We have been able to sample reasonably well the H12 conformational landscape to reproduce precisely both the agonist and the antagonist conformations, starting from an unfolded position, and to describe the transition path between them, even in the presence of an agonist ligand. These conformations were the most prevalent, suggesting that the extended H12 state is not likely to exist and that the natural ERα H12 position might exist in both the agonist and antagonist states. Remarkably, the H12 transition occurs and is regulated only in a dimer form and the proper agonist or antagonist H12 conformation can be achieved solely in one of the dimer subunits. These results clearly demonstrate that clusters of the two well-known H12 states exist by themselves in the protein free energy landscape, i.e. they are not constituted directly by the ligands, and dimerization favors the switch between them. Conversely, in a monomer, no transitions have been observed. Thus, the dimer formation helps the constitution of populations of discrete H12 conformational states and reshapes the conformational landscape. Further analyses have shown that these observations can be explained by specific interface and long range protein-protein interactions, resulting in conformational fluctuations in helices 5 and 11. Based on these results, a new ERα activation/deactivation mechanism and a sequence of binding events during receptor activity modulation have been suggested according to which ligands control the H12 conformation via alterations of the inter-dimer interactions. These findings agree with the HDX and fluorescence experiments and provide an explanation on a structural basis of these data, demonstrating that the dynamics of H12 are not altered greatly upon ligand binding and large fluctuations at the end of H11 are present.

Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

Structure-based design of pseudopeptidic inhibitors for SIRT1 and SIRT2.

The lack of substrate-bound crystal structures of SIRT1 and SIRT2 complicates the drug design for these targets. In this work, we aim to study whether SIRT3 could serve as a target structure in the design of substrate based pseudopeptidic inhibitors of SIRT1 and SIRT2. We created a binding hypothesis for pseudopeptidic inhibitors, synthesized a series of inhibitors, and studied how well the fulfillment of the binding criteria proposed by the hypothesis correlated with the in vitro inhibitory activities. The chosen approach was further validated by studying docking results between 12 different SIRT3, Sir2Tm, SIRT1 and SIRT2 X-ray structures and homology models in different conformational forms. It was concluded that the created binding hypothesis can be used in the design of the substrate based inhibitors of SIRT1 and SIRT2 although there are some reservations, and it is better to use the substrate-bound structure of SIRT3 instead of the available apo-SIRT2 as the target structure.

Predicted structures and dynamics for agonists and antagonists bound to serotonin 5-HT2B and 5-HT2C receptors.

Subtype 2 serotonin (5-hydroxytryptamine, 5-HT) receptors are major drug targets for schizophrenia, feeding disorders, perception, depression, migraines, hypertension, anxiety, hallucinogens, and gastrointestinal dysfunctions. (1) We report here the predicted structure of 5-HT2B and 5-HT2C receptors bound to highly potent and selective 5-HT2B antagonist PRX-08066 3, (pKi: 30 nM), including the key binding residues [V103 (2.53), L132 (3.29), V190 (4.60), and L347 (6.58)] determining the selectivity of binding to 5-HT2B over 5-HT2A. We also report structures of the endogenous agonist (5-HT) and a HT2B selective antagonist 2 (1-methyl-1-1,6,7,8-tetrahydro-pyrrolo[2,3-g]quinoline-5-carboxylic acid pyridine-3-ylamide). We examine the dynamics for the agonist- and antagonist-bound HT2B receptors in explicit membrane and water finding dramatically different patterns of water migration into the NPxxY motif and the binding site that correlates with the stability of ionic locks in the D(E)RY region.

Preliminary molecular dynamic simulations of the estrogen receptor alpha ligand binding domain from antagonist to apo.

Estrogen receptors (ER) are known as nuclear receptors. They exist in the cytoplasm of human cells and serves as a DNA binding transcription factor that regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. The human estrogen receptor comes in two forms, alpha and beta. This work focuses on the alpha form of the estrogen receptor. The ERalpha is found in breast cancer cells, ovarian stroma cells, endometrium, and the hypothalamus. It has been suggested that exposure to DDE, a metabolite of DDT, and other pesticides causes conformational changes in the estrogen receptor. Before examining these factors, this work examines the protein unfolding from the antagonist form found in the 3ERT PDB crystal structure. The 3ERT PDB crystal structure has the estrogen receptor bound to the cancer drug 4-hydroxytamoxifen. The 4-hydroxytamoxifen ligand was extracted before the simulation, resulting in new conformational freedom due to absence of van der Waals contacts between the ligand and the receptor. The conformational changes that result expose the binding clef of the co peptide beside Helix 12 of the receptor forming an apo conformation. Two key conformations in the loops at either end of the H12 are produced resulting in the antagonist to apo conformation transformation. The results were produced over a 42ns Molecular Dynamics simulation using the AMBER FF99SB force field.

Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose-response in mice infected with Escherichia coli.

OBJECTIVES: Phosphorodiamidate morpholino oligomers (PMOs) are DNA analogues that inhibit translation by an antisense mechanism. Membrane-penetrating peptides attached to PMOs increase PMO efficacy by enhancing penetration through bacterial membranes. The objectives of these experiments are to demonstrate gene-specific efficacy and establish a dose-response relationship of a peptide-PMO conjugate. METHODS: An 11-base PMO (AcpP) targeted at acpP (an essential gene) of Escherichia coli was synthesized and conjugated with the cell-penetrating peptide RFFRFFRFFRXB (X is 6-aminohexanoic acid and B is beta-alanine). Mice were infected by intraperitoneal (i.p.) injection with K-12 E. coli W3110, and treated i.p. at 15 min and 12 h post-infection with various amounts of AcpP peptide-PMO conjugate, AcpP PMO without attached peptide, scrambled base sequence PMOs or ampicillin. A strain (LT1) of E. coli was constructed by replacing acpP with an allele that has four wobble base substitutions in the region targeted by the PMO. RESULTS: Twelve hours after a single treatment, 30 microg of AcpP peptide-PMO or 3 mg of AcpP PMO reduced bacteraemia by 3 orders of magnitude compared with treatment with water. Neither scrambled base sequence PMO controls nor 30 microg of ampicillin reduced bacteraemia. Two treatments with 30 microg of AcpP peptide-PMO reduced cfu significantly more than four treatments with 15 microg at 15 min, 4, 8 and 12 h. Mice treated with doses of AcpP peptide-PMO > 30 microg showed further reductions in plasma cfu. Survival 48 h after treatment with 2 x 30 microg (3 mg/kg) of AcpP peptide-PMO or 2 x 3 mg (300 mg/kg) of AcpP PMO was 100%, compared with 20% for mice treated with water or scrambled base sequence PMO controls. However, survival was reduced to 75% and 0% for mice treated with 2 x 300 microg and 2 x 1 mg of AcpP peptide-PMO, respectively. A conjugate made from the D-isomeric form of each amino acid was less effective than the L-amino acid equivalent, and required 2 x 300 microg treatments for significant reduction in bacteria and survival. Mice infected with LT1 and treated with AcpP peptide-PMO did not survive and had the same amount of bacteria in the blood as mice treated with water, whereas those treated with 2 x 100 microg of AcpPmut4 peptide-PMO (complementary to the mutated allele) survived, and had a 3 orders of magnitude reduction in bacteria in the blood at 24 h post-infection. CONCLUSIONS: Both AcpP peptide-PMO and AcpP PMO significantly reduced bacteraemia and promoted survival of mice infected with E. coli W3110. The conjugate was about 50-100 times more potent than the PMO without attached peptide. The L-isomeric peptide-PMO was 10 times more potent than the D-isomeric equivalent. The conjugate apparently was toxic at doses > or = 2 x 300 microg/mouse (30 mg/kg). PMOs produced a sequence-specific antibiotic effect and the conjugate had a therapeutic index (toxic dose/effective dose) approximately equal to 10 in a mouse model of infection.