scholarly journals Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor

2005 ◽  
Vol 388 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Luís GALES ◽  
Sandra MACEDO-RIBEIRO ◽  
Gemma ARSEQUELL ◽  
Gregorio VALENCIA ◽  
Maria João SARAIVA ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Structural Features ◽  
Familial Amyloidotic Polyneuropathy ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
The Stability ◽  
New Interactions

Ex vivo and in vitro studies have revealed the remarkable amyloid inhibitory potency and specificity of iododiflunisal in relation to transthyretin [Almeida, Macedo, Cardoso, Alves, Valencia, Arsequell, Planas and Saraiva (2004) Biochem. J. 381, 351–356], a protein implicated in familial amyloidotic polyneuropathy. In the present paper, the crystal structure of transthyretin complexed with this diflunisal derivative is reported, which enables a detailed analysis of the protein–ligand interactions. Iododiflunisal binds very deep in the hormone-binding channel. The iodine substituent is tightly anchored into a pocket of the binding site and the fluorine atoms provide extra hydrophobic contacts with the protein. The carboxylate substituent is involved in an electrostatic interaction with the Nζ of a lysine residue. Moreover, ligand-induced conformational alterations in the side chain of some residues result in the formation of new intersubunit hydrogen bonds. All these new interactions, induced by iododiflunisal, increase the stability of the tetramer impairing the formation of amyloid fibrils. The crystal structure of this complex opens perspectives for the design of more specific and effective drugs for familial amyloidotic polyneuropathy patients.

scholarly journals Phytoconstituents from Markhamia Tomentosa Bind To HPV Oncoprotein with Apoptogenic Potential: A Molecular Modeling Approach

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mutiat B. Ibrahim ◽  
Adeola T. Kola-Mustapha ◽  
Niyi S. Adelakun ◽  
Neil A. Koorbanally
Keyword(s):  
Drug Targets ◽  
Active Sites ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Hpv 16 ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Potent Growth ◽  
The Stability

Abstract Markhamia tomentosa crude extract and fractions exhibited potent growth inhibitory effects capable to induce apoptosis in cervical (HeLa) cancer cell line via in vitro model. Presently, interaction of M. tomentosa phytoconstituents with molecular drug targets to exert its anticancer property is evaluated via in silico study. Identified phytoconstituents from M. tomentosa were retrieved from PubChem database and docked in active sites of HPV 16 E6, caspase -3 and caspase -8 targets using AutoDockVina from PyRx software. Screening for druglikeness; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions was carried out with the use of SwissADME and pkCSM web servers. Standard melphalan and co-crystallized ligands of caspases -3 and -8 enzymes were used to validate protein-ligand interactions. Molecular dynamic simulation was used to validate the stability of the hit molecules complexed with caspases -3 and -8. All identified phytoconstituents from M. tomentosa showed binding affinity for HPV with docking scores range of - 5.4 to -2.6 kcal/mol. Ajugol, carnosol, luteolin and phytol showed good docking energy range of -6.8 to -3.6 kcal/mol; and -4.8 to -1.9 kcal/mol for the active sites of caspases -3 and -8 targets respectively. Based on docking scores; drug-likeliness; and ADMET predictions; luteolin and carnosol were selected as hit compounds. These molecules were found to be stable within the binding site of caspase -3 target throughout the 40ns simulation time. These findings identified hit ligands from M. tomentosa phytoconstituents that inhibit HPV 16 E6 oncogene expression with stimulation of caspases -3 and -8 targets.

scholarly journals Novel Protease Inhibitors (PIs) Containing Macrocyclic Components and 3(R),3a(S),6a(R)-bis-Tetrahydrofuranylurethane That Are Potent against Multi-PI-Resistant HIV-1 Variants In Vitro

2010 ◽  
Vol 54 (8) ◽  
pp. 3460-3470 ◽  
Author(s):  
Yasushi Tojo ◽  
Yasuhiro Koh ◽  
Masayuki Amano ◽  
Manabu Aoki ◽  
Debananda Das ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Molecular Design ◽  
Antiviral Agent ◽  
Structural Features ◽  
Biological Properties ◽  
Van Der Waals Interactions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Hiv 1

ABSTRACT Natural products with macrocyclic structural features often display intriguing biological properties. Molecular design incorporating macrocycles may lead to molecules with unique protein-ligand interactions. We generated novel human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a macrocycle and bis-tetrahydrofuranylurethane. Four such compounds exerted potent activity against HIV-1LAI and had 50% effective concentrations (EC50s) of as low as 0.002 μM with minimal cytotoxicity. GRL-216 and GRL-286 blocked the replication of HIV-1NL4-3 variants selected by up to 5 μM saquinavir, ritonavir, nelfinavir, lopinavir, or atazanavir; they had EC50s of 0.020 to 0.046 μM and potent activities against six multi-PI-resistant clinical HIV-1 (HIVmPIr ) variants with EC50s of 0.027 to 0.089 μM. GRL-216 and -286 also blocked HIV-1 protease dimerization as efficiently as darunavir. When HIV-1NL4-3 was selected by GRL-216, it replicated progressively more poorly and failed to replicate in the presence of >0.26 μM GRL-216, suggesting that the emergence of GRL-216-resistant HIV-1 variants is substantially delayed. At passage 50 with GRL-216 (the HIV isolate selected with GRL-216 at up to 0.16 μM [HIV216-0.16 μM]), HIV-1NL4-3 containing the L10I, L24I, M46L, V82I, and I84V mutations remained relatively sensitive to PIs, including darunavir, with the EC50s being 3- to 8-fold-greater than the EC50 of each drug for HIV-1NL4-3. Interestingly, HIV216-0.16 μM had 10-fold increased sensitivity to tipranavir. Analysis of the protein-ligand X-ray structures of GRL-216 revealed that the macrocycle occupied a greater volume of the binding cavity of protease and formed greater van der Waals interactions with V82 and I84 than darunavir. The present data warrant the further development of GRL-216 as a potential antiviral agent for treating individuals harboring wild-type and/or HIVmPIr .

scholarly journals Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative

2004 ◽  
Vol 381 (2) ◽  
pp. 351-356 ◽  
Author(s):  
Maria Rosário ALMEIDA ◽  
Bárbara MACEDO ◽  
Isabel CARDOSO ◽  
Isabel ALVES ◽  
Gregorio VALENCIA ◽  
...  
Keyword(s):  
Plasma Proteins ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Fibril Formation ◽  
Familial Amyloidotic Polyneuropathy ◽  
Flufenamic Acid ◽  
Amyloid Fibril Formation ◽  
Transmission Electron ◽  
Partially Unfolded

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

scholarly journals Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiuye Li ◽  
W. Michael Babinchak ◽  
Witold K. Surewicz
Keyword(s):  
Amyloid Fibrils ◽  
Low Complexity ◽  
Structural Features ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Backbone Conformation ◽  
Hydrophobic Amino Acids ◽  
Insight Into ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

Methods to study the structure of misfolded protein states in systemic amyloidosis

2021 ◽  
Vol 49 (2) ◽  
pp. 977-985
Author(s):  
Marcus Fändrich ◽  
Matthias Schmidt
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Systemic Amyloidosis ◽  
Structural Basis ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics

2011 ◽  
Vol 435 (3) ◽  
pp. 771-781 ◽  
Author(s):  
Tatu J. K. Haataja ◽  
M. Kristian Koski ◽  
J. Kalervo Hiltunen ◽  
Tuomo Glumoff
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Structural Features ◽  
Full Length ◽  
Multifunctional Enzyme ◽  
Domain Composition ◽  
Substrate Channelling

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure–function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

scholarly journals Olive Oil Based Organogels for Effective Topical Delivery of Fluconazole: In-vitro Antifungal Study

2020 ◽  
pp. 29-36
Author(s):  
Mohammad Muqtader Ahmed ◽  
Farhat Fatima ◽  
Abdul Bari Mohammed
Keyword(s):  
Olive Oil ◽  
Ex Vivo ◽  
Similarity Index ◽  
Ex Vivo Permeation ◽  
Efficient Delivery ◽  
In Vitro Diffusion ◽  
Permeation Studies ◽  
The Stability ◽  
Hot Melt

The objective of the study was to formulate olive oil based organogels for the topical application of fluconazole (FLZ), to ensure the efficient delivery of the drug deeper in to the skin layers. Methods: Nine formulations developed by hot-melt method using olive oil, sorbitan monostearate (SMS) and FLZ. Prepared formulations characterized for macro evaluations, pH, spreadibility, viscosity, gel-sol transition, in-vitro diffusion study. Further optimized formulation evaluated for ex-vivo percutaneous permeation, in-vitro antifungal studies and stability studies by similarity index. Results: The results of evaluated parameters ensure the stability and effectiveness of the prepared olive oil based organogels. In-vitro diffusion studied reflects decrease in drug release with increase in surfactant concentration due to increase in viscosity. Moreover, ex-vivo permeation studies revealed that the permeation of FLZ was enhanced for optimized formulations (F6) as compared to the marketed gel formulation. Further, the optimized formulation exhibits the broad zone of inhibition against fungal strains in comparison to control and marketed product during in-vitro antifungal study. Conclusion: The olive oil based organogels formulation shown the enhanced permeation of FLZ from organogel network structure with good antifungal activity as compared to the marketed formulation. Henceforth, the FLZ organogel formulations could be used topically for the effective treatment of fungal infection.

scholarly journals Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Thomas Heerde ◽  
Matthies Rennegarbe ◽  
Alexander Biedermann ◽  
Dilan Savran ◽  
Peter B. Pfeiffer ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Proteinase K ◽  
Seed Structure ◽  
In Vitro Proliferation ◽  
Amyloid A ◽  
Fibril Structure ◽  
Proteolytic Stability ◽  
Fibril Morphology

AbstractSeveral studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

scholarly journals DEVELOPMENT AND EVALUATION OF MUCOADHESIVE NANOGEL OF NEVIRAPINE FOR VAGINAL APPLICATION

2019 ◽  
pp. 144-149
Author(s):  
Sheikh Sofiur Rahman ◽  
ABDUL BAQUEE AHMED
Keyword(s):  
Hiv Infection ◽  
Ex Vivo ◽  
Vaginal Fluid ◽  
Content Uniformity ◽  
Vaginal Mucosa ◽  
Salting Out ◽  
Zero Order Kinetics ◽  
The Stability ◽  
Ph Range

Objectives: The main objective of this study was to develop and evaluate Nevirapine nanoparticle loaded mucoadhesive gel (NVP-Np mucoadhesive gel) for vaginal application for the treatment of HIV infection.  Methods: NVP loaded nanoparticles were prepared by salting out method followed by incorporation in different gel bases to produce NVP-Np mucoadhesive gel The prepared gels were evaluated for their physicochemical parameters, rheological characteristics, mucoadhesion, in-vitro drug release and ex-vivo permeation of drug across porcine vaginal mucosa.  Results: The result of FT-IR and DSC study confirmed the absence of incompatibility of NVP with excipients used in the formulations. The particle size of the prepared NVP-Np was found to be 243.8 ± 3.15 nm, a polydispersity index (PI) of 0.787± 0.002 and zeta potential value -17.12 mV, which revealed the stability of nanoparticles. All the formulations showed good homogeneity, spreadability, physical appearance and content uniformity. The pH of the mucoadhesive gel formulations was in the range of 3.70 ± 0.03 to 4.56 ± 0.02, which lies in the normal pH range of the vaginal fluid.  The cumulative amounts permeated at 6 h were 832.23 ± 63.45 μg/cm2 , 592.13 ± 82.55 μg/cm2 and 941.32 ± 81.10 μg/cm2 from F1(1% Chitosan), F2(1% Carbopol 974P) and F3 (1% HPMC K100M )  respectively. A linear relationship [r2 > 0.9 (0.97 n 0.99)] was observed between the percentage cumulative amount permeated and time, indicating zero order kinetics. Conclusion: In conclusion, NVP-Np mucoadhesive gel was prepared successfully using salting out followed by a homogenization technique for vaginal application of NVP for the prophylaxis of HIV infection.

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