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Biomedical subjects

J Kallen

Publications and source records attributed to J Kallen.

At least 19 recordsLinked to original sources
PubMed2001

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.

Allosteric Site↗
PubMed1999

Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain.

The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.

Amino Acid Sequence↗
PubMed1999

X-ray structure and conformational dynamics of the HIV-1 protease in complex with the inhibitor SDZ283-910: agreement of time-resolved spectroscopy and molecular dynamics simulations.

Based on the X-ray structure of the human immunodeficiency virus type-1 (HIV-1) protease in complex with the statine-derived inhibitor SDZ283-910, a 542 ps molecular dynamics trajectory was computed. For comparison with the 805 ps trajectory obtained for the uncomplexed enzyme, the theoretical fluorescence anisotropy decay of the unliganded protease and the inhibitor complex was calculated from the trajectories of the Trp6A/Trp6B and Trp42A/Trp42B transition dipole moments. This enabled us to directly compare the simulated data with the experimental picosecond time-resolved fluorescence data. Fitting both experimental and simulated data to the Kohlrausch-Williams-Watts (KWW) function exp(-t/tauk)beta revealed a very good agreement for the uncomplexed protease as well as for the SDZ283-910 complex. Binding of the inhibitor induced a faster decay of both the experimental and the computed protease fluorescence anisotropy decay. By this integrative approach, the atomic detail of inhibitor-induced changes in the conformational dynamics of the HIV-1 protease was experimentally verified and will be used for further inhibitor optimisation.

Anti-HIV Agents↗
PubMed1999

Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate.

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.

Alanine Transaminase↗
PubMed1999

Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. II. Structure elucidation, stereochemistry and physico-chemical properties.

A novel class of macrolides, the sanglifehrins, was discovered by screening of actinomycete strains with a cyclophilin-binding assay. The chemical structures and absolute stereochemistries of the sanglifehrins A, B, C and D were determined unambiguously by NMR-techniques and by X-ray crystallography of the complex with cyclophilin A. Sanglifehrin A consists of a 22-membered macrocycle containing a tripeptide subunit and features in position 23 a chain of nine carbon atoms bearing a spirocyclic substituent. Sanglifehrins A and B are genuine metabolites whereas sanglifehrins C and D are artefacts.

Anti-Bacterial Agents↗
PubMed1998

X-ray structures and analysis of 11 cyclosporin derivatives complexed with cyclophilin A.

Eight new X-ray structures of different cyclophilin A/cyclosporin-derivative complexes are presented. These structures, combined with the existing three published cyclosporin complexes, provide a useful structural database for the analysis of protein-ligand interactions. The effect of small chemical differences on protein-ligand hydrogen-bonding, van der Waals interactions and water structure is presented.

Cyclosporine↗
PubMed1998

Conformational differences of an immunosuppressant peptolide in a single crystal and in a crystal complex with human cyclophilin A.

The crystal structure of (Thr2, Leu5, d-Hiv8, Leu10)-cyclosporin (cyclic peptolide SDZ 214-103) has been determined as the unbound crystal form and as a complex with human cyclophilin A. This pair of structures provides an example of a significant difference in conformation between free and bound ligand in crystals. The conformation of the unbound form is unlike that of both free and bound conformations of cyclosporin A (with the amide bond between residues 3 and 4 in the cis conformation), while the bound conformation is similar to that of CsA bound to cyclophilin. The cyclophilin-bound conformations of both ligands are similar, though this involves a significantly different waterellipsisligand hydrogen-bonding structure, which compensates for the chemical differences between the two ligands.

Cyclosporins↗
PubMed1998

Rational design, synthesis, and X-ray structure of selective noncovalent thrombin inhibitors.

We have designed, synthesized, and tested in vitro a novel class of noncovalent thrombin inhibitors. The main feature of these inhibitors is a 6,5-fused bicyclic core structure that fills the S2 pocket of the active site of thrombin. The bicycle introduces conformational constraint into the ligand and locks the Xaa-Pro amide bond into the desired trans configuration. Among the known ring systems, we selected by molecular modeling the 7-thiaindolizidinones (BTD) as our basic template. The influence of several structural features was analyzed: the length of the argininal side chain, the stereochemistry at C6, and the importance of making optimal use of the S3 pocket. Finally, an X-ray crystal structure of inhibitor 15 bound to thrombin was obtained at a resolution of 2.3 A. These designed thrombin inhibitors, which were prepared by an efficient synthesis, showed high selectivity over trypsin and other serine proteases. Further derivation based on the information obtained by X-ray crystallography should certainly allow to improve the potency.

Animals↗
PubMed1998

Demonstration of a cell-specific isomerization of invertebrate neuropeptides.

Neurohemal organs of the lobster Homarus americanus contain isoforms of the crustacean hyperglycemic hormone, which differ by the third amino acid (phenylalanyl) residue that is either in the L- or in the D-configuration. Polyclonal antisera have been raised in rabbit against synthetic octapeptides with the sequence corresponding to the N-terminal part of the L- or D-phenylalanine-containing isoforms. Their specificity was shown by immunoassays, indicating that they discriminate the isoforms of the lobster hyperglycemic neuropeptides. It was demonstrated that the two major forms of the crayfish Orconectes limosus hyperglycemic hormone also correspond to peptide isomers containing the L- or D-phenylalanyl residue. The cellular distribution of the isoforms among the neurosecreting cells of the major neuroendocrine complex in lobster and crayfish has been studied by immunohistochemistry. Every hyperglycemic hormone-containing cell was labelled with the anti-L antisera while only some of them were visualized with the anti-D antisera. These results constitute the first observation of peptide isomerization at the cellular level and suggest that the isomerization process occurs in specialized neuroendocrine cells.

Amino Acids↗
PubMed1997

Conformational control of cyclosporin through substitution of the N-5 position. A new class of cyclosporin antagonists.

Cyclosporin A (CsA) can be regiospecifically alkylated at the NH of Val-5 with reactive bromides in the presence of phosphazene-base P4-t-Bu to yield derivatives 2-5. These are devoid of immunosuppressive activity in vitro but they have binding affinity for cyclophilin A (CypA) similar to that of CsA and thus represent a new class of cyclosporin antagonists. 1H NMR (DMSO-d6) studies have shown that the compounds exist in a single, all trans conformation. A comparison of this NMR data with X-ray crystallographic analysis of a CypA/CsA derivative complex demonstrates that the solution structure does not correspond to the bioactive conformation.

Cyclosporine↗
PubMed1996

Conformational polymorphism in peptidic and nonpeptidic drug molecules.

Macrolide ligands that bind FK506 binding proteins and cyclosporins that a bind cyclophilins are chemically dissimilar but can share a number of structural and biological properties. Both families of ligands have very different conformations in the free state compared to those adopted when complexed with their binding protein. These transformations involve twisting from cis to trans about specific amide bonds, which result in significant changes in the hydrogen-bonding capabilities of the molecular surfaces. The three-dimensional structure of a new cyclosporin-like ligand (SDZ214 - 103) is described in the free crystalline state and bound to cyclophilin, and is shown to have a very different conformation from cyclosporin A in the free crystal, but a very similar conformation when bound to cyclophilin.

Amino Acid Isomerases↗
PubMed1995

Structure of octreotide, a somatostatin analogue.

Octreotide, a synthetic somatostatin analogue, is an octapeptide with one disulfide bridge. Crystals of octreotide are orthorhombic, space group P2(1)2(1)2(1), a = 18.458 (5), b = 30.009 (7), c = 39.705 (27) A, with three molecules of octapeptide, one ordered oxalate dianion and 52 water molecules in the asymmetric unit. Complete protonation of the NH(2) groups (as assumed in the refinement) would require three oxalate dianions in the asymmetric unit for charge neutrality; a chemical analysis indicated that four are present. In either case they are so disordered that they cannot be distinguished from the water molecules. The 18 951 unique reflections (R(sym) = 0.026) used for structure solution and refinement were recorded with the EMBL imaging-plate scanner using synchrotron radiation. The structure was solved by Patterson interpretation, locating the three disulfide bridges, followed by tangent phase expansion and E-Fourier recycling. The anisotropic refinement against all F(2) data between 1.04 and 10.0 A resolution by blocked restrained full-matrix least-squares techniques converged to a conventional R index based on F of 0.084 [I > 2a(I) and 10.0 > d > 1.04 A] and wR2, the weighted R-index on F(2), of 0.246 (for all data). One peptide molecule adopts a flat beta-sheet structure; the other two possess different irregular backbone conformations, but are similar to each other. All three molecules have a distorted type II' beta-turn around the D-Trp-Lys region, but exhibit different side-chain conformations. The crystal structure is stabilized by a network of inter- and intramolecular hydrogen bonds.

Journal Article↗
PubMed1995

Comparison of different X-ray data-collection systems using the crystal structure of octreotide.

The octapeptide octreotide crystallizes with three peptide molecules and about 20% water in the asymmetric unit, and in many ways possesses diffraction properties similar to those of a 'mini-protein' consisting of 24 amino-acid residues. It diffracts to about 1.0 A but data in the range 1.4-1.0 A are weak. It provides a suitable test of different macromolecular X-ray data-collection techniques, especially of their ability to measure weak reflections accurately. In contrast to typical proteins it is possible to perform a full anisotropic refinement, that we believe provides a more objective test of the quality of the data than the internal consistency of equivalent reflections. We have collected a total of six data sets. The X-ray sources included synchrotron radiation, Cu Kalpha rotating anodes and Mo Kalpha sealed tubes; position-sensitive two-dimensional detectors from four manufacturers and a four-circle diffractometer with scintillation counter were employed. Two of the six data sets were collected at low temperature. Reasonable anisotropic refinement was possible with all area-detector data sets, although significant differences in the precision of the final model were observed. In addition we tested the ability of automated Patterson interpretation to solve the structure using the six independent data sets. The structure solution was only successful using the synchrotron or rotating-anode data sets, i.e. for the more intense sources. It appears that for structure solution the maximum resolution of the data is critical, whereas for refinement the accuracy of the data is more important.

Journal Article↗
PubMed1995

Three-dimensional structure and actions of immunosuppressants and their immunophilins.

The use of the immunosuppressant drug cyclosporin A (CsA) as a biochemical tool to study the signal transduction pathway in T cells has led to the discovery of a first family of immunosuppressant-binding proteins or "immunophilins," the cyclophilins (Cyp). Another, chemically unrelated immunosuppressant molecule, FK506, was then found to be related to a second class of immunophilins, the FK506-binding proteins (FKBPs). This paper reviews the existing structural information on these immunophilins in the context of present knowledge of the biochemical mechanisms for immunosuppression. The formation of Cyp-CsA and FKBP-FK506 complexes, and the subsequent specific interaction of these complexes with the serine/threonine phosphatase calcineurin (CN), are key steps in the cascade of events that result in the desired immunosuppression. Knowledge of the conformation of the Cyp-CsA-CN and FKBP-FK506-CN ternary complexes is of significant biomedical interest, because mimics of the composite contact surfaces of, for example, Cyp-CsA or FKBP-FK506, could provide immunosuppressant drugs with improved pharmacological profiles.

Amino Acid Isomerases↗
PubMed1994

The molecular replacement solution and X-ray refinement to 2.8 A of a decameric complex of human cyclophilin A with the immunosuppressive drug cyclosporin A.

The X-ray structure of a decameric form of a complex of human cyclophilin A (CypA) with the immunosuppressive drug cyclosporin A (CsA) has been determined. The crystals of space group P43212 with cell dimensions a = b = 95.2 A, c = 280.0 A have five copies of the cyclophilin A/cyclosporin A complex in the asymmetric unit. The structure was solved by molecular replacement techniques, using a known cyclophilin A model. Procedures were developed to construct a self-rotation function using the results of cross-rotation searches. The comparison of experimental and constructed self-rotation maps was an important aid in selecting the correct rotation function solution. The translation functions revealed the presence of a cyclic pentamer. A crystallographic dimer axis passes through the non-crystallographic 5-fold rotation axis of the pentameric asymmetric unit, and generates a decameric "sandwich" of CypA/CsA heterodimers that has 52 symmetry. The five CypA/CsA protomers were refined independently using all data to 2.8 A giving a final crystallographic R-factor of 15.7%. Despite the constraints due to the packing arrangement within the decamer, the CypA and CsA conformations are similar to other CypA/CsA structures determined by X-ray crystallography and NMR spectroscopy. The hydrophobic CsA molecules are embedded in the middle of the decameric sandwich with only 20% of their surface exposed to solvent. The binding loop of CsA (residues 1 to 3 and 9 to 11) comprising 42% of the CsA surface, is buried in the peptidyl-prolyl-cis-trans isomerase active site of the cognate binding partner CypA, while the effector loop (residues 4 to 8) packs in the core of the decamer making hydrogen-bonding and van der Waals contacts with three neighbouring molecules. The environment of CsA in the decamer has been analysed and may provide a mimic for the interactions likely to occur between the CypA/CsA complex and its biological target calcineurin. There is no evidence to suggest that the decameric sandwich itself plays a role in immunosuppression by inhibiting calcineurin. However, the chaperone/foldase activity of CypA could require oligomer formation for its biological function.

Amino Acid Isomerases↗
PubMed1994

Conformational polymorphism of cyclosporin A.

BACKGROUND: Cyclosporin A (CsA) is a cyclic undecapeptide fungal metabolite with immunosuppressive properties, widely used in transplant surgery. It forms a tight complex with the ubiquitous 18 kDa cytosolic protein cyclophilin A (CypA). The conformation of CsA in this complex, as studied by NMR or X-ray crystallography, is very different from that of free CsA. Another, different conformation of CsA has been found in a complex with an antibody fragment (Fab). RESULTS: A detailed comparison of the conformations of experimentally determined structures of protein-bound CsA is presented. The X-ray and NMR structures of CsA-CypA complexes are similar. The Fab-bound conformation of CsA, as determined by X-ray crystallography, is significantly different from the cyclophilin-bound conformation. The protein-CsA interactions in both the Fab and CypA complexes involve five hydrogen bonds, and the buried CsA surface areas are 395 A2 and 300 A2, respectively. However, the CsA-protein interactions involve rather different side chain contacts in the two complexes. CONCLUSIONS: The structural results presented here are consistent with CypA recognizing and binding a population of CsA molecules which are in the required CypA-binding conformation. In contrast, the X-ray structures of the Fab complex with CsA suggest that in this case there is mutual adaptation of both receptor and ligand during complex formation.

Amino Acid Isomerases↗