Dual Binding Mode Research Articles

The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile

The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces crotonyl-CoA.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5’ and 3’ termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Corrigendum: Hydroxy-Directed Ruthenium-Catalyzed Alkene/Alkyne Coupling: Increased Scope, Stereochemical Implications, and Mechanistic Rationale.

The recognition of the dual binding mode of propargyl and allyl alcohols to [Cp*Ru] fragments fostered the development of a highly regioselective intermolecular Alder-ene-type reaction of alkynes with 1,2-disubstituted alkenes. The increased substrate scope opens new perspectives in stereochemical terms. As the loaded catalyst is chiral-at-metal, stereochemical information is efficiently relayed from the propargylic site to the emerging C-C bond. This interpretation is based on the X-ray structure of the first Cp*Ru complex carrying an intact enyne ligand, and provides valuable insights into bonding and activation of the substrates. Computational data draw a clear picture of the principles governing regio- and stereocontrol.

Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes

AbtractCellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Binding mode analyses of NAP derivatives as mu opioid receptor selective ligands through docking studies and molecular dynamics simulation

Mu opioid receptor selective antagonists are highly desirable because of their utility as pharmacological probes for receptor characterization and functional studies. Furthermore, the mu opioid receptors act as an important target in drug abuse and addiction treatment. Previously, we reported NAP as a novel lead compound with high selectivity and affinity towards the mu opioid receptor. Based on NAP, we have synthesized its derivatives and further characterized their binding affinities and selectivity towards the receptor. NMP and NGP were identified as the two most selective MOR ligands among NAP derivatives. In the present study, molecular modeling methods were applied to assess the dual binding modes of NAP derivatives, particularly on NMP and NGP, in three opioid receptors, in order to analyze the effects of structural modifications on the pyridyl ring of NAP on the binding affinity and selectivity. The results indicated that the steric hindrance, electrostatic, and hydrophobic effects caused by the substituents on the pyridyl ring of NAP contributed complimentarily on the binding affinity and selectivity of NAP derivatives to three opioid receptors. Analyses of these contributions provided insights on future design of more potent and selective mu opioid receptor ligands.

Hydroxy-Directed Ruthenium-Catalyzed Alkene/Alkyne Coupling: Increased Scope, Stereochemical Implications, and Mechanistic Rationale.

The recognition of the dual binding mode of propargyl and allyl alcohols to [Cp*Ru] fragments fostered the development of a highly regioselective intermolecular Alder-ene-type reaction of alkynes with 1,2-disubstituted alkenes. The increased substrate scope opens new perspectives in stereochemical terms. As the loaded catalyst is chiral-at-metal, stereochemical information is efficiently relayed from the propargylic site to the emerging C-C bond. This interpretation is based on the X-ray structure of the first Cp*Ru complex carrying an intact enyne ligand, and provides valuable insights into bonding and activation of the substrates. Computational data draw a clear picture of the principles governing regio- and stereocontrol.

Hydroxy‐Directed Ruthenium‐Catalyzed Alkene/Alkyne Coupling: Increased Scope, Stereochemical Implications, and Mechanistic Rationale

AbstractThe recognition of the dual binding mode of propargyl and allyl alcohols to [Cp*Ru] fragments fostered the development of a highly regioselective intermolecular Alder‐ene‐type reaction of alkynes with 1,2‐disubstituted alkenes. The increased substrate scope opens new perspectives in stereochemical terms. As the loaded catalyst is chiral‐at‐metal, stereochemical information is efficiently relayed from the propargylic site to the emerging C−C bond. This interpretation is based on the X‐ray structure of the first Cp*Ru complex carrying an intact enyne ligand, and provides valuable insights into bonding and activation of the substrates. Computational data draw a clear picture of the principles governing regio‐ and stereocontrol.

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome

The assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals.

Aminoalkyl Derivatives of 8-Alkoxypurine-2,6-diones: Multifunctional 5-HT1A /5-HT7 Receptor Ligands and PDE Inhibitors with Antidepressant Activity.

In the search for potential psychotropic agents, a new series of 3,7-dimethyl- and 1,3-dimethyl-8-alkoxypurine-2,6-dione derivatives of arylpiperazines, perhydroisoquinolines, or tetrahydroisoquinolines with flexible alkylene spacers (5-16 and 21-32) were synthesized and evaluated for 5-HT1A /5-HT7 receptor affinities as well as PDE4B1 and PDE10A inhibitory properties. The 1-(4-(4-(2-hydroxyphenyl)piperazin-1-yl)butyl)-3,7-dimethyl-8-propoxypurine-2,6-dione (16) and 7-(2-hydroxyphenyl)piperazinylalkyl-1,3-dimethyl-8-ethoxypurine-2,6-diones (31 and 32) as potent dual 5-HT1A /5-HT7 receptor ligands with antagonistic activity produced an antidepressant-like effect in the forced swim test in mice. This effect was similar to that produced by citalopram. All the tested compounds were stronger phosphodiesterase isoenzyme inhibitors than theophylline and theobromine. The most potent compounds, 15 and 16, were characterized by 51 and 52% inhibition, respectively, of PDE4B1 activity at a concentration of 10-5 M. Concerning the above findings, it may be assumed that the inhibition of PDE4B1 may impact on the signal strength and specificity resulting from antagonism toward the 5-HT1 and 5-HT7 receptors, especially in the case of compounds 15 and 16. This dual receptor and enzyme binding mode was analyzed and explained via molecular modeling studies.

Dual binding mode in cohesin-dockerin complexes as assessed through stretching studies.

A recent experimental study by Jobst et al. of stretching of a wild-type (WT) cohesin-dockerin complex has identified two kinds of the force-displacement patterns, with a single or double-peaked final rupture, which are termed "short" and "long" here. This duality has been interpreted as arising from the existence of two kinds of binding. Here, we analyze the separation of two cohesin-dockerin complexes of C. thermocellum theoretically. We use a coarse-grained structure-based model and the values of the pulling speeds are nearly experimental. In their native states, the two systems differ in the mutual binding orientations of the molecules in the complex. We demonstrate that the WT complex (PDB:1OHZ) unravels along two possible pathways that are qualitatively consistent with the presence of the short and long patterns observed experimentally. On the other hand, the mutated complex (PDB:2CCL) leads only to short trajectories. The short and long stretching pathways also appear in the cohesin-dockerin-Xmodule complex (PDB:4IU3, WT) of R. flavefaciens. Thus the duality in the stretching patterns need not be necessarily due to the duality in binding.

Molecular Basis for the Protein Recognition Specificity of the Dynein Light Chain DYNLT1/Tctex1: CHARACTERIZATION OF THE INTERACTION WITH ACTIVIN RECEPTOR IIB

It has been suggested that DYNLT1, a dynein light chain known to bind to various cellular and viral proteins, can function both as a molecular clamp and as a microtubule-cargo adapter. Recent data have shown that the DYNLT1 homodimer binds to two dynein intermediate chains to subsequently link cargo proteins such as the guanine nucleotide exchange factor Lfc or the small GTPases RagA and Rab3D. Although over 20 DYNLT1-interacting proteins have been reported, the exact sequence requirements that enable their association to the canonical binding groove or to the secondary site within the DYNLT1 surface are unknown. We describe herein the sequence recognition properties of the hydrophobic groove of DYNLT1 known to accommodate dynein intermediate chain. Using a pepscan approach, we have substituted each amino acid within the interacting peptide for all 20 natural amino acids and identified novel binding sequences. Our data led us to propose activin receptor IIB as a novel DYNLT1 ligand and suggest that DYNLT1 functions as a molecular dimerization engine bringing together two receptor monomers in the cytoplasmic side of the membrane. In addition, we provide evidence regarding a dual binding mode adopted by certain interacting partners such as Lfc or the parathyroid hormone receptor. Finally, we have used NMR spectroscopy to obtain the solution structure of human DYNLT1 forming a complex with dynein intermediate chain of ∼74 kDa; it is the first mammalian structure available.

Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4’ phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (−.164 kcal/mol) and B (−.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: −5.872 kcal/mol and B form: −5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.

Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors.

Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3’-deoxy-3,3’-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A–E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from μM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.

Dual binding mode of antithyroid drug methimazole to mammalian heme peroxidases - structural determination of the lactoperoxidase-methimazole complex at 1.97 Å resolution.

Lactoperoxidase (LPO, EC 1.11.1.7) is a member of the mammalian heme peroxidase family which also includes thyroid peroxidase (TPO). These two enzymes have a sequence homology of 76%. The structure of LPO is known but not that of TPO. In order to determine the mode of binding of antithyroid drugs to thyroid peroxidase, we have determined the crystal structure of LPO complexed with an antithyroid drug, methimazole (MMZ) at 1.97 Å resolution. LPO was isolated from caprine colostrum, purified to homogeneity and crystallized with 20% poly(ethylene glycol)‐3350. Crystals of LPO were soaked in a reservoir solution containing MMZ. The structure determination showed the presence of two crystallographically independent molecules in the asymmetric unit. Both molecules contained one molecule of MMZ, but with different orientations. MMZ was held tightly between the heme moiety on one side and the hydrophobic parts of the side chains of Arg255, Glu258, and Leu262 on the opposite side. The back of the cleft contained the side chains of Gln105 and His109 which also interacted with MMZ. In both orientations, MMZ had identical buried areas and formed a similar number of interactions. It appears that the molecules of MMZ can enter the substrate‐binding channel of LPO in two opposite orientations. But once they reach the distal heme pocket, their orientations are frozen due to equally tight packing of MMZ in both orientations. This is a novel example of an inhibitor binding to an enzyme with two orientations at the same site with nearly equal occupancies.

A 4:1 stoichiometric binding and stabilization of mitoxantrone-parallel stranded G-quadruplex complex established by spectroscopy techniques

Small molecule ligands which specifically bind and stabilize G-quadruplex structures in telomeric ends inhibit the activity of telomerase enzyme, an important marker for cancer. Understanding of the binding mode of ligand-G quadruplex complex is important for evaluating relative efficacy of anti-tumor drugs. The present study is focused on interaction of anti-tumor drug mitoxantrone (MTX) with tetra-molecular parallel stranded G-quadruplex sequence d-TTGGGGT using absorbance, fluorescence and circular dichroism spectroscopy techniques. Absorbance of mitoxantrone shows hypochromism up to MTX (D)/DNA quadruplex (N) ratio ~5, followed by hyperchromism up to D/N=0.21 accompanied by a red shift of 15nm. The fluorescence emission of MTX shows decrease up to D/N ~5 and then increases with red shift of 8nm. The two observed fluorescent lifetimes, 0.17ns (91%) and 0.44ns (9%), indicate dual binding mode. Absence of isobestic and isoemissive point indicates presence of multiple complexes. Circular Dichroism spectra showing positive induced band at 645nm and two exciton bands centered at 619 and 664nm suggest binding of mitoxantrone as a dimer. Proton NMR studies show intermolecular MTX-MTX short contacts confirming existence of stacked dimer of MTX. Thermal melting transitions of DNA saturate at D/N=4 with ΔTm=25°C. The results establish highly specific external groove binding of 4 molecules of mitoxantrone as two dimers at two distinct sites of DNA.

Design and synthesis of N-substituted-2-hydroxyiminoacetamides and interactions with cholinesterases

Within this study, we designed and synthesized four new oxime compounds of the N-substituted 2-hydroxyiminoacetamide structure and evaluated their interactions with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our aim was to explore the possibility of extending the dual-binding mode of interaction between the enzyme and the inhibitor to a so-called triple-binding mode of interaction through the introduction of an additional binding moiety. N-substituted 2-hydroxyiminoacetamide 1 was prepared via BOP catalyzed amidation of hydroxyiminoacetic acid with 3-azido-1-phenylpropylamine. An azide group enabled us to prepare more elaborate structures 2–4 by the copper-catalyzed azide-alkyne cycloaddition. The new compounds 1–4 differed in their presumed AChE peripheral site binding moiety, which ranged from an azide group to functionalized heterocycles. Molecular docking studies revealed that all three binding moieties are involved in the non-covalent interactions with ChEs for all of the four compounds, albeit not always in the complete accordance with the proposed hypothesis. All of the four compounds reversibly inhibited the ChEs with their inhibition potency increasing in the same order for both enzymes (1 < 2 < 4 < 3). A higher preference for binding to BChE (KI from 0.30 μmol/L to 130 μmol/L) over AChE (KI from 50 μmol/L to 1200 μmol/L) was observed for all of the compounds. Compounds were screened for reactivation of cyclosarin-, sarin- and VX-inhibited AChE and BChE.

Increased Conformational Flexibility of HLA-B*27 Subtypes Associated With Ankylosing Spondylitis.

Dissimilarities in antigen processing and presentation are known to contribute to the differential association of HLA-B*27 subtypes with the inflammatory rheumatic disease ankylosing spondylitis (AS). In support of this notion, previous x-ray crystallographic data showed that peptides can be displayed by almost identical HLA-B*27 molecules in a subtype-dependent manner, allowing cytotoxic T lymphocytes to distinguish between these subtypes. For example, a human self-peptide derived from vasoactive intestinal peptide receptor type 1 (pVIPR; sequence RRKWRRWHL) is displayed in a single conformation by B*27:09 (which is not associated with AS), while B*27:05 (which is associated with AS) presents the peptide in a dual binding mode. In addition, differences in conformational flexibility between these subtypes might affect their stability or antigen presentation capability. This study was undertaken to investigate B*27:04 and B*27:06, another pair of minimally distinct HLA-B*27 subtypes, to assess whether dual peptide conformations or structural dynamics play a role in the initiation of AS. Using x-ray crystallography, we determined the structures of the pVIPR-B*27:04 and pVIPR-B*27:06 complexes and used isotope-edited infrared (IR) spectroscopy to probe the dynamics of these HLA-B*27 subtypes. As opposed to B*27:05 and B*27:09, B*27:04 (which is associated with AS) displays pVIPR conventionally and B*27:06 (which is not associated with AS) presents the peptide in a dual conformation. Comparison of the 4 HLA-B*27 subtypes using IR spectroscopy revealed that B*27:04 and B*27:05 possess elevated molecular dynamics compared to the nonassociated subtypes B*27:06 and B*27:09. Our results demonstrate that an increase in conformational flexibility characterizes the disease-associated subtypes B*27:04 and B*27:05.

Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions.