| 1. |
- Begum, Afshan, et al.
(författare)
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Transthyretin Binding Mode Dichotomy of Fluorescent trans-Stilbene Ligands
- 2023
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 14:5, s. 820-828
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Tidskriftsartikel (refereegranskat)abstract
- The orientations of ligands bound to the transthyretin (TTR) thyroxine (T4) binding site are difficult to predict. Conflicting binding modes of resveratrol have been reported. We previously reported two resveratrol based trans-stilbene fluorescent ligands, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (SB-11) and (E)-4-(2-(naphthalen-2-yl)vinyl)-benzene-1,2-diol (SB-14), that bind native and misfolded protofibrillar TTR. The binding orientations of these two analogous ligands to native tetrameric TTR were predicted to be opposite. Herein we report the crystal structures of these TTR:ligand complexes. Opposite binding modes were verified but were different than predicted. The reverse binding mode (SB14) placing the naphthalene moiety toward the opening of the binding pocket renders the fluorescent ligand pH sensitive due to changes in Lys15 amine protonation. Conversely, the forward binding mode (SB-11) placing the naphthalene inward mediates a stabilizing conformational change, allowing intersubunit H-bonding between Ser117 of different monomers across the dimer interface. Our structures of TTR complexes answer important questions in ligand design and interpretation of trans-stilbene binding modes to the TTR T4 binding site.
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| 2. |
- Good, James A. D., 1985-, et al.
(författare)
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Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
- 2016
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Ingår i: Cell chemical biology. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 23:3, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.
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| 3. |
- Hall, Michael, et al.
(författare)
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Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:51, s. 14733-14738
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Tidskriftsartikel (refereegranskat)abstract
- Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
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| 4. |
- Iakovleva, Irina, et al.
(författare)
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Enthalpic Forces Correlate with Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma
- 2015
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 58:16, s. 6507-6515
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Tidskriftsartikel (refereegranskat)abstract
- The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design.
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| 5. |
- Iakovleva, Irina, 1977-, et al.
(författare)
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Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer
- 2016
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR’s native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed.
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| 6. |
- Iakovleva, Irina, et al.
(författare)
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The flavonoid luteolin, but not luteolin-7-o-glucoside, prevents a transthyretin mediated toxic response
- 2015
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 10:5
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Tidskriftsartikel (refereegranskat)abstract
- Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.
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| 7. |
- Kulén, Martina, et al.
(författare)
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Structure-based design of inhibitors targeting PrfA, the master virulence regulator of Listeria monocytogenes
- 2018
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 61:9, s. 4165-4175
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein "tunnel" as the main inhibitor binding site (A1), where the compounds participate in an extensive hydrophobic network that restricts the protein's ability to form functional DNA-binding helix−turn−helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-A1 selective PrfA inhibitors with potent antivirulence properties.
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| 8. |
- Minh, Nghia Nguyen Thi, et al.
(författare)
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Binding of a Pyrene-Based Fluorescent Amyloid Ligand to Transthyretin : A Combined Crystallographic and Molecular Dynamics Study
- 2023
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 127:30, s. 6628-6635
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Tidskriftsartikel (refereegranskat)abstract
- Misfolding and aggregation of transthyretin (TTR) causeseveralamyloid diseases. Besides being an amyloidogenic protein, TTR hasan affinity for bicyclic small-molecule ligands in its thyroxine (T4)binding site. One class of TTR ligands are trans-stilbenes. The trans-stilbenescaffold is also widely applied for amyloid fibril-specific ligandsused as fluorescence probes and as positron emission tomography tracersfor amyloid detection and diagnosis of amyloidosis. We have shownthat native tetrameric TTR binds to amyloid ligands based on the trans-stilbenescaffold providing a platform for the determination of high-resolutionstructures of these important molecules bound to protein. In thisstudy, we provide spectroscopic evidence of binding and X-ray crystallographicstructure data on tetrameric TTR complex with the fluorescent salicylicacid-based pyrene amyloid ligand (Py1SA), an analogue of the Congored analogue X-34. The ambiguous electron density from the X-ray diffraction,however, did not permit Py1SA placement with enough confidence likelydue to partial ligand occupancy. Instead, the preferred orientationof the Py1SA ligand in the binding pocket was determined by moleculardynamics and umbrella sampling approaches. We find a distinct preferencefor the binding modes with the salicylic acid group pointing intothe pocket and the pyrene moiety outward to the opening of the T4binding site. Our work provides insight into TTR binding mode preferencefor trans-stilbene salicylic acid derivatives as well as a frameworkfor determining structures of TTR-ligand complexes.
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| 9. |
- Mishra, Rajesh, et al.
(författare)
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Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation
- 2019
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : ELSEVIER. - 1570-9639 .- 1878-1454. ; 1867:10, s. 909-921
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Tidskriftsartikel (refereegranskat)abstract
- Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125-209) comprising a small two-stranded beta-sheet and three alpha-helices. The N-terminal domain (residues 23-124) is intrinsically disordered. Expression of truncated PrP (residues 90-231) is sufficient to cause prion disease and residues 90/100-231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self -chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards beta-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular beta-sheet model structure of the PrP90-231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.
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| 10. |
- Nilsson, Lina, et al.
(författare)
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Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity
- 2016
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment.
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| 11. |
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| 12. |
- Zhang, Jin, et al.
(författare)
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Structure-based Virtual Screening Protocol for in silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin
- 2016
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Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 50:21, s. 11984-11993
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Tidskriftsartikel (refereegranskat)abstract
- Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 mu M. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.
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