Influence of EGCG on α‐synuclein (αS) aggregation and identification of their possible binding mode: A computational study using molecular dynamics simulation

The accumulation of intrinsically disordered α‐synuclein (αS) protein that can form β‐sheet‐rich fibrils is linked to Parkinson's disease. (−)‐Epigallocatechin‐3‐gallate (EGCG) is the most abundant active component in green tea and can inhibit the fibrillation of αS. The elucidation of this molecular mechanism will be helpful to understand the inhibition mechanism of EGCG to the fibrillation of αS and also to find more potential small molecules that can inhibit the aggregation of αS. In this work, to study the influence of EGCG on the structure of β‐sheet‐rich fibrils of αS and identification of their possible binding mode, molecular dynamics simulations of pentamer and decamer aggregates of αS in complex with EGCG were performed. The obtained results indicate that EGCG can remodel the αS fibrils and break the initial ordered pattern by reducing the β‐sheet content. EGCG can also break the Greek conformation of αS by the disappeared H‐bond in the secondary structure of turn. The results from our study can not only reveal the specific interaction between EGCG and β‐sheet‐rich fibrils of αS, but also provide the useful guidance for the discovery of other potential inhibitors.     

A comparative study for detecting CYP3A induction by CYP3A probe drugs and endogenous markers in cynomolgus monkeys

CYP3A probe drugs such as midazolam and endogenous markers, and plasma 4β‐hydroxycholesterol (4β‐OHC) and urinary 6β‐hydroxycortisol‐to‐cortisol ratios (6β‐OHC/C) have been used as markers of CYP3A induction in cynomolgus monkeys, as with humans. However, there is limited information on their sensitivity and ability to detect CYP3A induction, as most studies were evaluated only at a high dose of the inducer, rifampicin (RIF; 20 mg/kg). In the present study, the CYP3A induction by RIF over a range doses of 0.2, 2 and 20 mg/kg (n = 4) was examined using CYP3A probe drugs (midazolam, triazolam and alprazolam) and the plasma and urinary endogenous CYP3A markers (4β‐OHC and 6β‐OHC/C). The sensitivity and relationship for detecting CYP3A induction was compared among the markers. Four days repeated oral administration of rifampicin to cynomolgus monkeys reduced the area under the plasma concentration–time curve of all CYP3A probe drugs in a rifampicin dose‐dependent manner. Although the endogenous CYP3A markers (4β‐OHC and 6β‐OHC/C) were also changed for the middle (2 mg/kg) and high (20 mg/kg) doses of rifampicin, the fold‐changes were relatively small, and CYP3A induction could not be detected at the lowest dose of rifampicin (0.2 mg/kg). In conclusion, CYP3A probe drugs are more sensitive for detecting CYP3A induction than endogenous CYP3A markers in cynomolgus monkeys, even for a short experimental period.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.     

Pharmacokinetics of nebivolol in the rat: low oral absorption,loss in the gut and systemic stereoselectivity

Nebivolol is a third‐generation β1‐adrenoceptor blocker with β3 agonistic properties (AR). It has a low oral bioavailability that is speculated to be due to its hepatic first‐pass metabolism. Inflammation is known to suppress the clearance of drugs with efficient hepatic metabolism. However, inflammation does not influence nebivolol clearance. Therefore, we looked into the mechanism involved in the drug's low bioavailability and stereoselectivity. Single 1 mg/kg i.v. or intraperitoneal (i.p.) or 2 mg/kg oral doses were administered to male Sprague‐Dawley rats and the plasma nebivolol concentration was measured using chiral and achiral assays. The passage of nebivolol enantiomers through the gut was also measured using everted rat sacs. The serum protein binding of the enantiomers was studied in vitro using the ultrafiltration method. Plasma nebivolol concentrations were significantly lower after p.o., but not after i.p., compared with i.v. doses suggesting the gut as the site of pre‐systemic loss. Approximately 50% of the enantiomers were lost during 90 min incubation in the presence of gut. Only 0.1% of the added drug crossed the gut wall with no evidence of stereoselectivity. Thus stereoselectivity in the pharmacokinetics of nebivolol (+ > ‐) is likely at the level of plasma protein binding. The low nebivolol bioavailability is due to its loss in the gut as well as its limited permeability through the intestinal wall. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse

Hemapolin (2α,3α‐epithio‐17α‐methyl‐5α‐androstan‐17β‐ol) is a designer steroid that is an ingredient in several “dietary” and “nutritional” supplements available online. As an unusual chemical modification to the steroid A‐ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC‐MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α‐methyl‐5α‐androst‐2‐en‐17β‐ol), reduced and dihydroxylated madol (17α‐methyl‐5α‐androstane‐2β,3α,17β‐triol), and the isomeric enone metabolites 17β‐hydroxy‐17α‐methyl‐5α‐androst‐3‐en‐2‐one and 17β‐hydroxy‐17α‐methyl‐5α‐androst‐2‐en‐4‐one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell‐based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC‐MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.     

Protective effects of lithium treatment for spatial memory deficits induced by tau hyperphosphorylation in splenectomized rats

1. Postoperative cognitive dysfunction has become more prevalent in recent years. We used a splenectomized rat model with postoperative spatial learning and memory deficits to investigate the role of tau hyperphosphorylation and glycogen synthase kinase‐3β (GSK‐3β) within the hippocampus. 2. Cognitive function was assessed in a Y‐maze 1 day before and 1, 3 and 7 days after surgery. We measured site‐specific phosphorylation of hippocampal tau (Thr‐205 and Ser‐396), GSK‐3β activity and expression of interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) mRNA and protein as markers of inflammation. We also tested the effects of treatment with lithium chloride (LiCl), a GSK‐3β inhibitor. 3. Splenectomy was associated with learning and memory impairment 3 days later, as well as a rapid and massive hyperphosphorylation of hippocampal tau at Thr‐205 and Ser‐396, activated GSK‐3β, and increased IL‐1β and TNF‐α expression. LiCl completely restored tau hyperphosphorylation to control levels. 4. These data from the splenectomized rat model suggest that inflammatory factors affect tau pathology through the GSK‐3β signalling pathway and that LiCl is a promising treatment for postoperative cognitive deficits.     

Combined 1H‐NMR and Molecular Dynamics Studies on Conformational Behavior of a Model Heptapeptide,GRGDSPC

Among various strategies, the de novo design and in silico approaches are being used to develop the short peptides, models of modified peptides, and mimetics as clinically useful drugs with improved stability and bioavailability. The resulting models will help to isolate the factors behind the folded structure formation and contribute useful information about de novo peptide design. The combined ¹H‐NMR spectroscopic and molecular dynamics methods were used to investigate the conformational behavior of an Arg‐Gly‐Asp (RGD)‐containing peptide, GRGDSPC, the cell‐binding heptapeptide of extracellular matrix protein, fibronectin. The formation of two fused weak β‐turns of type II (HB, 4→1) and type II’ (HB, 7→4) from simulation studies has been consistent with NMR data. The sustainable ‘S’‐shaped molecular structure (which remained unchanged during the entire simulation) and the conformational transitions due to interconversions between multiple turns initiated at Asp⁴, Ser⁵, and Cys⁷ imply that the peptide is flexible in nature. Thus, the model of ‘S’‐shaped structure with flexible multiple turns for GRGDSPC peptide may provide the structural rationale for antagonistic properties of this heptapeptide toward the treatment of integrin‐mediated cellular abnormal behaviors such as thrombosis and metastasis.     

A peptide derived from the C‐terminal part of a plant cysteine protease folds into a stack of two β‐hairpins,a scaffold present in the emerging family of granulin‐like growth factors

Abstract: A 35 amino acid residue peptide corresponding to the N‐terminal subdomain of the granulin‐like repeat from rice oryzain β was synthesized and regioselectively oxidized to produce a species with a [1–3, 2–4] disulfide‐pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two β‐hairpins found in the emerging family of granulin‐like growth factors. Because of the longer second β‐hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well‐structured carp granulin‐1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin‐like growth factors from the animal kingdom and the granulin‐like repeats at the C‐termini of plant cysteine proteases. Therefore, the stack of two β‐hairpins may be a conserved three‐dimensional organization of the granulin‐like repeats from evolutionary distant sources with a significant role in specific protein–protein interactions.     

Structure‐based approaches for the design of benzimidazole‐2‐carbamate derivatives as tubulin polymerization inhibitors

Microtubules are highly dynamic assemblies of α/β‐tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β‐tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β‐tubulin isotype in normal blood cells have increased the interest for designing specific tubulin‐binding anticancer therapies. To this end, we employed our previously proposed model of the β‐tubulin–nocodazole complex, supported by the recently determined X‐ray structure, to identify the fundamental structural differences between β‐tubulin isotypes. Moreover, we employed docking and molecular dynamics (MD) simulations to determine the binding mode of a series of benzimidazole‐2‐carbamete (BzC) derivatives in the βI‐, βIII‐, and βVI‐tubulin isotypes. Our results demonstrate that Ala198 in the βVI isotype reduces the affinity of BzCs, explaining the low bone marrow toxicity for nocodazole. Additionally, no significant differences in the binding modes between βI‐ and βIII‐BzC complexes were observed; however, Ser239 in the βIII isotype might be associated with the low affinity of BzCs to this isotype. Finally, our study provides insight into the β‐tubulin–BzC interaction features essential for the development of more selective and less toxic anticancer therapeutics.     

Approaches for the discovery of metallo‐β‐lactamase inhibitors: A review

After more than 80 years of development, β‐lactam drugs have become the most widely used high‐efficiency, low‐toxic broad‐spectrum antibacterial drugs. However, with the widespread use and even abuse of those drugs, the resistance of major pathogens to β‐lactam drugs has increased over years, which has become a thorny problem to the public health. A common mechanism of the resistance to β‐lactams is the producing of β‐lactamases, which can hydrolyze the β‐lactam ring and inactivate these drugs. Metallo‐β‐lactamases (MBLs) are one kind of β‐lactamases that require metal ions for their catalytic activities. Although it is a well‐known strategy to recover the efficacy of β‐lactams by the combination of β‐lactamase inhibitors, there are still no MBL inhibitors that can be used in clinical practice. Therefore, it is urgent to develop MBL inhibitors. This review outlines the currently discovered MBL inhibitors with an emphasis on various strategies and approaches taken to discover MBL inhibitors, which may lead to diverse classes of inhibitors. Recent progress, particularly new screening methods, and the rational design of potent MBL inhibitors are discussed.     

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Abstract: Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, C^α‐methylated α‐amino acid l ‐(αMe)Nva with a short, linear side‐chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side‐chain hydrogenation of the corresponding C^α‐methyl, C^α‐allylglycine (Mag) peptides. A detailed solution and crystal‐state conformational analysis based on FT‐IR absorption, ¹H NMR and X‐ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β‐turn and 3₁₀‐helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l ‐(αMe)Nva induces the preferential formation of right‐handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α‐amino acids with a C^α‐methyl substituent and a C^α‐linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Unraveling the Role of Arg4 and Arg6 in the Auto‐Inhibition Mechanism of GSK3β From Molecular Dynamics Simulation

Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. GSK3β can be phosphorylated by the protein kinase B (PKB). The mutations of Arg4 and Arg6 to alanine at N‐terminal GSK3β have been reported to impair its ability to autophosphorylate at Ser9. Despite the extensive experimental observations, the detailed mechanism for the auto‐inhibition of GSK3β has not been rationalized at the molecular level. In this study, we have demonstrated the structural consequences of GSK3β R4A and R6A mutations and the atomic changes that influenced the loss of PKB‐binding affinity. Molecular dynamics simulation results suggested significant loss in atomic contacts in the R4A and R6A mutant systems compared to the wild‐type system. Furthermore, we observed many notable changes (such as conformation, residues motions, hydrogen bonds, and binding free energy) in the mutated GSK3β–PKB complexes. Loss of binding affinity in the mutated systems rendered the decrease in GSK3β phosphorylation, which, in turn, impaired the auto‐inhibition of GSK3β. The significant outcomes obtained from this study can explain the auto‐inhibition of GSK3β and maybe facilitate type 2 diabetes mellitus researches and in developing the potent drug therapies.     

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin. A selective inhibitor in rats of the uterine response to oxytocin

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin was synthesized by a 6 + 3 fragment condensation from precursors which had been formed by solution methods. This analog inhibited uterine responses to oxytocin (pA₂ 7.37, 7.9, 6.17; uterus in vitro without Mg++, in vitro with Mg++, and in vivo, respectively) and showed little or no activity in other bioassays.     

A new [2H]‐labelled α‐trichloroimidate glucuronic ester for the synthesis of deuterated drug conjugates

A new reaction pathway for the synthesis of a [²H]‐labelled trichloroacetimidate precursor for the preparation of glucuronides is described. Therewith, stable isotope‐labelled drug glucuronides become accessible on a preparative scale, which can further be used as internal standards for quantitative analysis.     

Radiosynthesis of the α4β2 nicotinic acetylcholine receptor ligand: 5‐((1‐[11C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)vinyl)pyridine

5‐((1‐[¹¹C]‐methyl‐2‐(S)‐pyrrolidinyl)methoxy)‐2‐chloro‐3‐((E)‐2‐(2‐fluoropyridin‐4‐yl)‐vinyl)pyridine ([¹¹C]‐FPVC) was synthesized from [¹¹C]‐methyl iodide and the corresponding normethyl precursor. The average time of synthesis, purification, and formulation was 42 min with an average non‐decay‐corrected radiochemical yield of 19%. The average specific radioactivity was 359 GBq/µmol (9691 mCi/µmole) at end of synthesis (EOS). Copyright © 2006 John Wiley & Sons, Ltd.     

Discovery of Narlaprevir (SCH 900518): A Potent,Second Generation HCV NS3 Serine Protease Inhibitor

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Molecular dynamics simulation of the antimicrobial salivary peptide histatin‐5 in water and in trifluoroethanol: a microscopic description of the water destructuring effect

Abstract: The results of 520 ps molecular dynamics simulation of histatin‐5, a small peptide present in human saliva and possessing antimicrobial activity, dissolved in water and in 2,2,2‐trifluoroethanol, are reported. The simulations indicate that histatin‐5 is destabilized in water and begins to unfold after 250 ps, while in organic solvent it maintains a regular secondary structure throughout the trajectory. Analysis of the peptide?solvent hydrogen bonds indicates that 2,2,2‐trifluoroethanol is a poorer proton acceptor than water. The fluorine atom of the alcohol is almost never engaged in a hydrogen bond and the organic solvent interacts mainly with the peptide through its hydroxyl group. For some residues analysis of the solvent residence time indicated longer values for 2,2,2‐trifluoroethanol than for water. The most striking difference is related to the number of times the solvent enters and leaves the first coordination shell of the peptide. This value was more than one order of magnitude higher for water than for the alcohol, suggesting that this may be the main cause of α‐helix destabilization perpetrated by water.