- Toremifene interacts with and destabilizes the Ebola virus glycoprotein. [Journal Article]
- NatNature 2016 Jul 7; 535(7610):169-72
- Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines fo...
Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.
- Restoration of dietary-fat induced blood-brain barrier dysfunction by anti-inflammatory lipid-modulating agents. [Journal Article]
- LHLipids Health Dis 2012; 11:117
- CONCLUSIONS: Our findings suggest that BBB dysfunction induced by chronic ingestion of SFA is reversible with timely introduction and sustained treatment with agents that suppress inflammation.
- Preparation of human drug metabolites using fungal peroxygenases. [Journal Article]
- BPBiochem Pharmacol 2011 Oct 1; 82(7):789-96
- The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromat...
The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 220.127.116.11) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 3.1±0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.
- Cochlear Implant : Anaesthesia Challenges. [Journal Article]
- MJMed J Armed Forces India 2004; 60(4):351-6
- Cochlear implants are now an acceptable therapeutic option for those patients with irreversible hearing loss and deaf-mutism. The surgery is time consuming and complicated. Hence, the technique of an...
Cochlear implants are now an acceptable therapeutic option for those patients with irreversible hearing loss and deaf-mutism. The surgery is time consuming and complicated. Hence, the technique of anaesthesia plays a crucial role in success of cochlear implant surgery. Cochlear implant patients have various types of syndromes which are important from anaesthetic as well as surgical point of view. Pre-implant preparation requires objective assessment of hearing, plain X-rays of skull and a CT scan of the temporal bone. Anaesthesia is required for objective assessment of hearing in children under five years of age, to obtain X-rays of skull, magnetic resonance imaging (MRI), CT scan, brain evoked response audiometry (BERA) testing and finally for cochlear implant surgery. Anaesthetic considerations include preoperative familiarisation with the patient and his family. Syndromal illnesses have specific anaesthetic significance such as presence of difficult airway or prolonged QT interval. Parental presence is highly desirable during induction of anaesthesia. Electro-surgical instruments especially monopolar ones, should not be used once the cochlear implant is in place.
- Endothelium-dependent vasorelaxation in the aorta of transgenic mice expressing human apolipoprotein(a) or lipoprotein(a). [Journal Article]
- EEndothelium 2000; 7(4):253-64
- Elevated plasma level of lipoprotein(a) (Lp(a)) is a well established risk factor for premature atherosclerosis and coronary artery disease. Recent studies showed impaired endothelium-dependent vasod...
Elevated plasma level of lipoprotein(a) (Lp(a)) is a well established risk factor for premature atherosclerosis and coronary artery disease. Recent studies showed impaired endothelium-dependent vasodilatation in humans with elevated plasma Lp(a). However, these human studies could not determine whether (1) elevated Lp(a) levels alone are the cause of endothelial dysfunction (these patients had multiple risk factors), and (2) native or oxidatively modified Lp(a) contributes to endothelial dysfunction (no measurements of native/oxidized Lp(a) ratio was reported in humans). In order to test whether apo(a) (an essential component of Lp(a) which is required for binding to endothelial cells) and native Lp(a) cause endothelial dysfunction, in the present study we tested endothelium-dependent vasorelaxation in aortic rings isolated from control and transgenic male mice either expressing the human apo(a) gene (TgA) or both the human apo(a) and human apo B100 genes (TgL). The TgA mice had plasma apo(a) levels of 8.8 +/- 1.2 mg/dl (n=6) and the double transgenic TgL mice had plasma Lp(a) levels of 15.3 +/- 1.4 mg/dl (n=8). Isolated aortic rings with and without endothelium were mounted in organ chambers and contracted with U46619 (10(-8) M) in the presence of ibuprofen (10(-5) M). Acetylcholine caused concentration-dependent (10(-9)-10(-5) M) relaxation, which could be prevented by endothelium removal and by NG-L-nitro-arginine (10(-4) M). Basal and acetylcholine-stimulated endothelium-dependent relaxation and endothelium-independent relaxation to nitroglycerin (10(-6) M) were not significantly different in aortic rings isolated from control and TgA or TgL mice. Twenty-four hour incubation of aortic rings isolated from control mice with recombinant human apo(a) or native Lp(a) (up to 300 microg/ml) caused no impairment of endothelium-dependent relaxations. In contrast, incubation with oxidized Lp(a) (50 microg/ml) or oxidized LDL (250 microg/ml) caused significant suppression of acetylcholine-induced endothelium-dependent vasorelaxation. These results show for the first time that elevated plasma levels of apo(a) and Lp(a) do not cause endothelial dysfunction in transgenic mice.
- Inhibitor-induced changes in the intrinsic fluorescence of human cyclooxygenase-2. [Journal Article]
- BBiochemistry 1996 Aug 20; 35(33):10974-84
- The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-...
The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.