Block of the hERG channel by bupivacaine: Electrophysiological and modeling insights towards stereochemical optimization

The hERG voltage-gated potassium channel mediates the cardiac I(Kr) current, which is crucial for the duration of the cardiac action potential. Undesired block of the channel by certain drugs may prolong the QT interval and increase the risk of malignant ventricular arrhythmias. Although the molecular determinants of hERG block have been intensively studied, not much is known about its stereoselectivity. Levo-(S)-bupivacaine was the first drug reported to have a higher affinity to block hERG than its enantiomer. This study strives to understand the principles underlying the stereoselectivity of bupivacaine block with the help of mutagenesis analyses and molecular modeling simulations. Electrophysiological measurements of mutated hERG channels allowed for the identification of residues involved in bupivacaine binding and stereoselectivity. Docking and molecular mechanics simulations for both enantiomers of bupivacaine and terfenadine (a non-stereoselective blocker) were performed inside an open-state model of the hERG channel. The predicted binding modes enabled a clear depiction of ligand-protein interactions. Estimated binding affinities for both enantiomers were consistent with electrophysiological measurements. A similar computational procedure was applied to bupivacaine enantiomers towards two mutated hERG channels (Tyr652Ala and Phe656Ala). This study confirmed, at the molecular level, that bupivacaine stereoselectively binds the hERG channel. These results help to lay the foundation for structural guidelines to optimize the cardiotoxic profile of drug candidates in silico.

Supramolecular Organization of Heptapyrenotide Oligomers—An in Depth Investigation by Molecular Dynamics Simulations

We present a molecular modeling study based on ab initio and classical molecular dynamics calculations, for the investigation of the tridimensional structure and supramolecular assembly formation of heptapyrenotide oligomers in water solution. Our calculations show that free oligomers self-assemble in helical structures characterized by an inner core formed by π- stacked pyrene units, and external grooves formed by the linker moieties. The coiling of the linkers has high ordering, dominated by hydrogen-bond interactions among the phosphate and amide groups. Our models support a mechanism of longitudinal supramolecular oligomerization based on interstrand pyrene intercalation. Only a minimal number of pyrene units intercalate at one end, favoring formation of very extended longitudinal chains, as also detected by AFM experiment. Our results provide a structural explanation of the mechanism of chirality amplification in 1:1 mixtures of standard heptapyrenotides and modified oligomers with covalently linked deoxycytidine, based on selective molecular recognition and binding of the nucleotide to the groove of the left-wound helix.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Bipyridyl- and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking

The synthesis and incorporation into oligonucleotides of C-nucleosides containing the two aromatic, non-hydrogen-bonding nucleobase substitutes biphenyl (I) and bipyridyl (Y) are described. Their homo- and hetero-recognition properties in different sequential arrangements were then investigated via UV-melting curve analysis, gel mobility assays, CD- and NMR spectroscopy. An NMR analysis of a dodecamer duplex containing one biphenyl pair in the center, as well as CD data on duplexes with multiple insertions provide further evidence for the zipper-like interstrand stacking motif that we proposed earlier based on molecular modeling. UV-thermal melting experiments with duplexes containing one to up to seven I- or Y base pairs revealed a constant increase in T(m) in the case of I and a constant decrease for Y. Mixed I/Y base pairs lead to stabilities in between the homoseries. Insertion of alternating I/abasic site- or Y/abasic site pairs strongly decreases the thermal stability of duplexes. Asymmetric distribution of I- or Y residues on either strand of the duplex were also investigated in this context. Duplexes with three natural base pairs at both ends and 50 % of I pairs in the center are still readily formed, while duplexes with blunt ended I pairs tend to aggregate unspecifically. Duplexes with one natural overhang at the end of a I-I base pair tract can both aggregate or form ordered duplexes, depending on the nature of the natural bases in the overhang

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real-time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440-13_c.1440-1del in intron 14 of ITGA2B causing exon skipping in 7 unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits thereby interfering with integrin maturation and/or function. Our study extends knowledge of Glanzmann thrombasthenia and the pathophysiology of an integrin. This article is protected by copyright. All rights reserved.

Oligodeoxynucleotide Duplexes Containing (5′S)-5′-C-Alkyl-Modified 2′-Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor-Groove Enhance Duplex Stability?

10.1002/hlca.200390311.abs A series of oligonucleotides containing (5′S)-5′-C-butyl- and (5′S)-5′-C-isopentyl-substituted 2′-deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl-zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA-duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I–III, Fig. 2) could experimentally be realized and their duplex-forming properties analyzed by UV-melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in Tm. We found that, within the proposed duplexes with more loosely packed alkyl groups (type-III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B-DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type-II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type-III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl-zipper formation presumably by loss of structured H2O in the minor groove.

Watson-Crick base-pairing properties of tricyclo-DNA

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the corresponding phosphoramidite building blocks is described, as well as a structural description of a representative of an alpha- and a beta-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and tcd(paactgtcacg) showed increased thermal stabilities of up to DeltaT(m)/mod. = +1.2 degrees C with complementary DNA and +2.4 degrees C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of +3.1 degrees C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in an A-conformation, a fact which is in agreement with results from molecular modeling

Bicyclo[3.2.1]amide-DNA: a chiral, non-chiroselective base-pairing system

The design, synthesis and base-pairing properties of bicyclo[3.2.1]amide-(bca)DNA, a novel phosphodiester based DNA analogue, is reported. This analogue consists of a conformationally constrained backbone entity which emulates a B-DNA geometry, to which the nucleobases were attached via an extended, acyclic amide linker. Homobasic adenine-containing bca-decamers form duplexes with complementary oligonucleotides containing the bca-, the DNA the RNA and, surprisingly, also the L-RNA backbone. UV- and CD-spectroscopic investigations revealed the duplexes with D- or L-complement to be of similar stability and enantiomorphic in structure. Bca-oligonucleotides containing all four bases form strictly antiparallel, left-handed complementary duplexes with itself and complementary DNA but not with RNA. Base-mismatch discrimination is comparable to that of DNA while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior relative to that of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association

In vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli ST131: Characterization of an Acquired Extended-Spectrum AmpC (ESAC) Conferring Resistance to Cefepime.

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance are reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical E. coli isolates obtained from a patient. The CMY-2-producing E. coli (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli (CMY-33-Ec) was detected in bronchoalveolar lavage. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime.CMY-33-Ec and CMY-2-Ec were identical by rep-PCR, being of hyperepidemic ST131, but showed different β-lactam MICs (e.g., cefepime 16 vs. ≤0.5 μg/ml). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but cefepime MICs were higher for CMY-33 than CMY-2 (8 vs. 0.25 μg/ml). The kcat/Km or kinact/KI (μM(-1) s(-1)) indicated that CMY-33 possesses an ESBL-like spectrum compared to CMY-2 (cefoxitin: 0.2 vs. 0.4; ceftazidime: 0.2 vs. not measurable; cefepime: 0.2 vs. not measurable; tazobactam 0.0018 vs. 0.0009). Using molecular modeling, we show that a widened active site (∼4 Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide, therefore awareness that cefepime treatment may select for resistant isolates is critical.

Pseudodominant Inheritance of Goitrous Congenital Hypothyroidism Caused by TPO Mutations: Molecular and in Silico Studies

Context and Objective: Most cases of goitrous congenital hypothyroidism (CH) from thyroid dyshormonogenesis 1) follow a recessive mode of inheritance and 2) are due to mutations in the thyroid peroxidase gene (TPO). We report the genetic mechanism underlying the apparently dominant inheritance of goitrous CH in a nonconsanguineous family of French Canadian origin. Design, Setting, and Participants: Two brothers identified by newborn TSH screening had severe hypothyroidism and a goiter with increased (99m)Tc uptake. The mother was euthyroid, but the father and two paternal uncles had also been diagnosed with goitrous CH. After having excluded PAX8 gene mutations, we hypothesized that the underlying defect could be TPO mutations. Results: Both compound heterozygous siblings had inherited a mutant TPO allele carried by their mother (c.1496delC; p.Pro499Argfs2X), and from their father, one brother had inherited a missense mutation (c.1978C-->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). The thyroid gland of one uncle who is a compound heterozygote for TPO mutations (p.Phe653Valfs15X/p.Gln660Glu) was removed because of concurrent multiple endocrine neoplasia type 2A. Immunohistochemistry revealed normal TPO staining, implying that Gln660Glu TPO is expressed properly. Modeling of this mutant in silico suggests that its three-dimensional structure is conserved, whereas the electrostatic binding energy between the Gln660Glu TPO and its heme group becomes repulsive. Conclusion: We report a pedigree presenting with pseudodominant goitrous CH due to segregation of three different TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the high frequency of TPO mutations carriers may lead to pseudodominant inheritance.