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V Mikol

Publications and source records attributed to V Mikol.

At least 19 recordsLinked to original sources
PubMed2001

The use of 3D structural data in the design of specific factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a critical role in the blood coagulation process and qualifies as an attractive target for finding new antithrombotics. In the case of fXa several structure based drug design strategies have been followed because of the difficulty in growing fXa co-crystals routinely. This has led to the use of surrogate proteins such as trypsin. Factor Xa inhibitors for which the binding mode has been determined experimentally or modeled are described in this review. The inhibitors are divided into three fragments: a P1 group, a central scaffold and a P4 group. In this review, interactions in each sub-site of fXa with various inhibitor fragments have been examined at the molecular level and were shown to bind, in most cases, independently of the rest of the molecule. Knowledge of the 3D structure of the binding mode of ligands to target proteins has been successfully applied in designing fXa inhibitors with enhanced specificity, affinity and has provided hints to modulate the physico-chemical properties of the small molecule ligand.

Animals↗
PubMed2000

Crystal structures of human factor Xa complexed with potent inhibitors.

Involved in the coagulation cascade, factor Xa (FXa) is a serine protease which has received great interest as a potential target for the development of new antithrombotics. Although there is a great wealth of structural data on thrombin complexes, few structures of ligand/FXa complexes have been reported, presumably because of the difficulty in growing crystals. Reproducible crystallization conditions for human des-Gla1-45 coagulation FXa have been found. This has led to an improvement in the diffraction quality of the crystals (about 2.1 A) when compared to the previously reported forms (2.3-2.8 A) thus providing a suitable platform for a structure-based drug design approach. A series of crystal structures of noncovalent inhibitors complexed with FXa have been determined, three of which are presented herein. These include compounds containing the benzamidine moiety and surrogates of the basic group. The benzamidine-containing compound binds in a canonical fashion typical of synthetic serine protease inhibitors. On the contrary, molecules that contain surrogates of the benzamidine group do not make direct hydrogen-bonding interactions with the carboxylate of Asp189 at the bottom of the S1 pocket. The structural data provide a likely explanation for the specificity of these inhibitors and a great aid in the design of bioavailable potent FXa inhibitors.

Benzamidines↗
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

Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases.

Human immunodeficiency virus (HIV) integrase is the enzyme responsible for insertion of a DNA copy of the viral genome into host DNA, an essential step in the replication cycle of HIV. HIV-1 integrase comprises three functional and structural domains: an N-terminal zinc-binding domain, a catalytic core domain and a C-terminal DNA-binding domain. The catalytic core domain with the F185H mutation has been crystallized without sodium cacodylate in a new crystal form, free and complexed with the catalytic metal Mg2+. The structures have been determined and refined to about 2.2 A. Unlike the previously reported structures, the three active-site carboxylate residues (D,D-35-E motif) are well ordered and both aspartate residues delineate a proper metal-binding site. Comparison of the active binding site of this domain with that of other members from the polynucleotidyl transferases superfamily shows a high level of similarity, providing a confident template for the design of antiviral agents.

Binding Sites↗
PubMed1998

Crystal structure of the cyclophilin-like domain from the parasitic nematode Brugia malayi.

Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.

Amino Acid Sequence↗
PubMed1996

Structure of the fab fragment of SDZ CHI621: a chimeric antibody against CD25.

A specific drug targeted to the IL-2 receptor on activated T lymphocytes could limit acute immunological rejection during organ transplantation. A high-affinity monoclonal antibody directed against the alpha-chain of the IL-2 receptor (CD25) was chimerized with the constant regions of the human IgG1 heavy and k light chain resulting in SDZ CHII621 [Amlot, Rawlings, Fernando, Griffin, Heinrich, Schreier, Castaigne, Moore & Sweny (1995). Transplantation, 60, 748-756]. The Fab fragment of SDZ CHI621 has been purified and crystallized (P2(l), a = 39.58, b = 59.76, c = 102.09 A, beta = 99.98 degrees ). Its structure has been determined by molecular replacement and refined at 2.6 A to a crystallographic R factor of 19.7%. The protein exhibits the typical immunoglobulin fold. The complementary determining regions (CDR's) 1 and 2 of both heavy and light chains show similar conformations to other known reported structures whereas the CDR3 from the light chain seems to adopt a novel type of conformation. There is a network of interactions which maintain the CDR3 of both chains together and limit their solvent accessibility. The interaction between V(L) and C(L) has been strengthened by the chimerization whereas that between V(H) and C(H)1 has been weakened.

Journal Article↗
PubMed1996

Crystal structures of apolipoprotein(a) kringle IV37 free and complexed with 6-aminohexanoic acid and with p-aminomethylbenzoic acid: existence of novel and expected binding modes.

Kringles are protein modules found within a wide variety of fibrinolytic and coagulation-related proteins that show binding affinity for lysine, lysine analogs and for fibrin. We report here the crystal structures of apolipoprotein(a) kringle IV37 (apo(a) K4(37)) in its free state and in separate complexes with two omega-amino acids, 6-aminohexanoic acid (6AHA) and p-aminomethylbenzoic acid (PAMBA). The structures of the unliganded form and of both complexes have been determined and refined by restrained least-squares methods to about 2.0 angstrom. The overall kringle architecture is essentially identical with that determined in other kringles but it shows some small significant structural changes in the lysine binding site. Ther is virtually no difference in conformation between the unliganded and complexed forms, suggesting that apo(a) K4(37) does not undergo any conformational rearrangement upon binding. The 6AHA molecule binds to apo(a) K4(37) in a completely different way from that observed with the kringle 4 of plasminogen (PGK4). Its amino group makes an ion pair interaction with the two aspartate residues (Asp55/Asp57) of the anionic center and its carboxylate group faces out into the solvent making water-mediated contacts with the protein. The mode of binding of PAMBA resembles more that decribed for 6AHA when bound to PGK4. The PAMBA molecule is bound by ion pair interactions with the two aspartate residues (Asp55/Asp57) and with Arg71 from the cationic center and by van der Waals contacts. The relative importance of the cationic center from kringles for binding zwitterionic ligands is discussed.

4-Aminobenzoic Acid↗
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

Crystal structure of the SH2 domain from the adaptor protein SHC: a model for peptide binding based on X-ray and NMR data.

Src homology 2 domains (SH2) are protein molecules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl (pY)-containing protein sequences. We report here for crystal structure of the SH2 domain from the adaptor protein SHC (Shc), which has been refined by restrained least-squares methods to an R-factor of 17.3% to 2.7 A. The overall Shc architecture is essentially similar to that determined in other SH2 domains but it shows significant differences in a number of loops, thus providing a molecular surface with no obvious secondary pocket. Based on the knowledge of the crystal structure of the protein a model for a low affinity Shc-bound peptide has been generated from nuclear magnetic resonance data in solution using transferred nuclear Overhauser enhancements as intramolecular distance restraints. The model shows that the tyrosine moiety binds Shc in a rather similar way to that observed for other SH2-peptide complexes, but that the residue in position +3 does not seem to make specific contact with the protein. An intermolecular crystallographic interaction occurs between the pY-binding site and the C-terminal residues of a symmetry-related molecule. This crystal packing interaction suggests how inhibitory regulation could play a role in SHC activity.

Amino Acid Sequence↗
PubMed1995

The role of water molecules in the structure-based design of (5-hydroxynorvaline)-2-cyclosporin: synthesis, biological activity, and crystallographic analysis with cyclophilin A.

Analysis of the contact surface of the cyclophilin A (CypA)/cyclosporin A (CsA, 1) crystal structure delineates a unique cavity between both molecules in the vicinity of the Abu-2 side chain atoms of 1 (Abu pocket). Therefore, (5-hydroxynorvaline)-2-cyclosporin (2) was designed and prepared as a CsA derivative which could mediate additional interactions within the pocket. The X-ray crystal structure of the CypA/2 complex at 1.76 A resolution shows that 1 and 2 have identical backbone conformations and that the introduced hydroxypropyl chain makes indeed the expected supplemental interactions with CypA. However, 2 has 8-9-fold lower binding affinity than 1 for CypA. This results from a presumed unfavorable free energy change associated with the displacement of one of the tightly bound water molecules within the pocket and a change in prebinding equilibria. The role of the later was assessed by comparing the conformation distribution of 1 and 2 to that of norvaline-2-cyclosporin (3) and norvaline-2-(D-MeSer)-3-cyclosporin (4).

Amino Acid Isomerases↗
PubMed1995

The crystal structures of the SH2 domain of p56lck complexed with two phosphonopeptides suggest a gated peptide binding site.

Src homology-2 (SH2) domains are protein modules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl-containing protein sequences. In order to develop SH2 inhibitors that contain phosphotyrosyl analogues resistant to cellular phosphatases, we have solved the crystal structures of the SH2 domain of p56lck in separate complexes with two high-affinity p-(phosphonomethyl)phenylalanine-containing peptides. The structures have been determined at 2.3 A and 2.25 A, and refined to crystallographic R-factors of 19.2% and 18.5%, respectively. The conformation of the SH2 domain of p56lck is essentially similar to that observed in Src and Lck complexed with a phosphotyrosine-containing peptide except in some loops and especially in the loop that connects the second and third beta-strands. This loop, which was involved in hydrogen-bond interactions with the phosphotyrosine moiety, has moved away in the phosphonopeptide complexes as a rigid body by about 7 A on two hinges leaving the tyrosine phosphate mimetic moiety accessible to the solvent. Some intramolecular hydrogen bonds with other residues of the third and fourth beta-strands stabilize an open conformation of the lid, suggesting a flap mechanism for peptide binding.

Amino Acid Sequence↗
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

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↗
PubMed1994

Crystal and molecular structure of allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate.

The alpha-(2-->8)-linked Kdo disaccharide derivative allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate C19H28O15Na2.H2O, M(r) = 542.32, is orthorhombic, P2(1)2(1)2(1) with a = 9.229(1), b = 12.036(1), c = 21.671(1) A, and Z = 4. The structure was solved by direct methods and refined to R = 0.040 for 2677 observed reflections. The torsion angles about the (2-->8)-glycosidic bond are stabilized by an intramolecular hydrogen bond between the carboxylate group at the anomeric carbon atom of the terminal Kdo residue and the hydroxyl group O-17 of the second Kdo moiety.

Carbohydrate Conformation↗
PubMed1994

Human cyclophilin C: primary structure, tissue distribution, and determination of binding specificity for cyclosporins.

A complementary DNA (cDNA) for human cyclophilin C (Cyp-C) was isolated from a human kidney cDNA library. Northern blot experiments with several human tissues and cell lines revealed that Cyp-C is less abundant than Cyp-A. The amount of Cyp-C mRNA was 10-fold lower than that of Cyp-A in kidney. Expression of human Cyp-C in the kidney is not significantly elevated compared to pancreas, skeletal muscle, heart, lung, and liver. This argues against a previously postulated specific role for Cyp-C in the nephrotoxic effects of CsA in humans, based on the studies of its relative abundance in murine kidney. It is present in extremely low concentrations in brain and in the Jurkat T cell line. The binding of recombinant human Cyp-A, -B, and -C to cyclosporin A (CsA) was studied by immunochemical methods. The relative affinity of Cyp-C for CsA is lower by a factor of 2 than that of Cyp-A, which itself is 10-fold lower than that of Cyp-B. Cross-reactivity studies with a series of Cs derivatives showed that Cyp-C binds CsA with a fine specificity similar to that of Cyp-A and Cyp-B. Cs amino acid residues 1, 2, 10, and 11 seemed essential for the interaction with all three Cyp subtypes. However, Cyp-C tolerates a greater variety of structures on Cs at position 2 than Cyp-A does, suggesting that this residue of CsA might not be in tight contact with Cyp-C. This was confirmed by modeling of human Cyp-C on the structure of the complex formed by Cyp-A and CsA. The knowledge of the fine specificity of human Cyps for CsA and of their expression levels may provide better insights into how CsA acts on its different target proteins in vivo.

Amino Acid Isomerases↗
PubMed1994

Crystallization of the complex between cyclophilin A and cyclosporin derivatives: the use of cross-seeding.

With ammonium sulfate as the precipitant, different crystal forms of the complex between cyclosporin A (CsA) and cyclophilin A (CypA) have been crystal- lized [Zurini, Kallen, Mikol, Pflügl, Jansonius & Walkinshaw (1990). FEBS Lett. 276, 63-66]. All have large unit cells and contain a pentamer or a decamer in the asymmetric unit. Using a more water soluble CsA analog, orthorhombic crystals containing only one molecule per asymmetric unit could be grown. They diffract to significantly higher resolution (2.1 A). In this crystal form, CsA has no packing interactions with neighbouring molecules and these crystals could be used to cross-seed other CypA/CsA analog complexes. Nine different CypA/ CsA analog complexes could be crystallized using this technique, most of them yielding highly diffracting crystals, quickly solvable by Fourier difference methods.

Journal Article↗