[Paraneoplastic ophthalmopathies]. / Ophtalmies paranéoplasiques.

Ocular paraneoplastic syndromes are rare conditions that can affect any part of the eye at any age. Thus, every ophthalmologist should be familiar with their management, as some of them may reveal severe, life-threatening conditions. These consist overwhelmingly of neuro-ophthalmological manifestations, affecting the optic nerve (paraneoplastic optic neuritis), retina (paraneoplastic retinopathy) or neurological pathways generating eye movements (saccadic intrusion, oculomotor palsy, nystagmus...); occasionally, they involve the anterior segment, orbit or uveal tract. As some of these manifestations appear to be quite common and non-specific, any systemic or especially neurologic comorbidities should increase suspicion. Treatment relies first on oncologic management, and then often more targeted therapy for the associated immune involvement.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

[Pseudopolymorphism of NO-SPA, 1-(3,4-diethoxy-benzyl)-6,7-diethoxy-3,4-dihydro-isoquinoli ne hydrochloride]. / A No-Spa, 1-(3,4-dietoxibenzil)-6,7-dietoxi-3,4-dihidroizokinolin hidroklorid, pszeudopolimorfiájáról.

The spasmolytic agent No-Spa, 1-(3,4-diethoxybenzyl)-6,7-diethoxy-3,4-dihydroisoquinoli ne hydrochloride (1) is a synthetic analogue of the naturally occurring alkaloid papaverine. (1) is prone to form stoichiometric crystalline solvates with a number of solvents causing technological and stability problem. Present paper describes the X-ray structure determination of the hemiethanol, hemibenzene and hemihydrochloride structures being the most important and interesting from both practical and theoretical point of view. The solvate formation is facilitated by the presence of the flexible ethoxy group encapsulating the solvent and by the quasi perpendicular position of the isoquinoline and phenyl group.

Structure determination and refinement of the Al3+ complex of the D254,256E mutant of Arthrobacter D-xylose isomerase at 2.40 A resolution. Further evidence for inhibitor-induced metal ion movement.

The structure of the D254.256E double mutant of Arthrobacter xylose isomerase with Al3+ at both metal-binding sites was determined by the molecular replacement method at a conventional R-factor of 0.179. Binding of the two Al3+ does not alter the overall structure significantly. However, there are local rearrangements in the octahedral co-ordination sphere of the Al3+. The inhibitor molecule moves somewhat away from the active site. Furthermore, evidence was revealed for metal ion movement from site 2(1) to site 2(2) upon double mutation. Xylose isomerase requires two divalent metal cations for activation. The catalytic metal ion is translocated 1.8 A away from its initial position during the catalytic reaction. The fact that both activating and inactivating metals (including Al3+) were found exclusively at a single location in the double mutant was an indication that the consequently missing shuttle may account for the crippled catalytic efficiency.

The three-dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease.

Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, A., Szilágyi, L., Hegyi, G., Pintér, K., Náray-Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA 85, 4961-4965] has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural-functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate-specific serine protease action.

[Synthesis and reactivity of debenzorutaecarpine derivatives]. / Debenzorutekarpin származékok szintézise és reaktivitása.

A series of 7,12-dihydropyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-ones was prepared by Fischer indolization of 9-arylhydrazono-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one s. Quantumchemical calculations (ab initio and AM1) indicate that position 3 of 7,12-dihydro-pyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-one can be involved in electrophilic substitutions, while position 2 is sensitive towards nucleophilic attack. Bromination of 6-methyl-7,12-dihydropyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-o ne (16) with bromine afforded 3-bromo derivative (25), which was reacted with cyclic amines to give 2-amino-7,12-dihydro-pyrimido[1',2';1,2]pyrido[3,4-b]indol-4(6H)-ones (26-30) in an addition-elimination reaction. Vielsmeier-Haack formylation of compound (16) give 12-formyl (31) and 3,12-diformyl (32) derivatives (an N-formyl-1-aza derivative of nauclefidine alkaloid (34) at 60 degrees C and 100 degrees C, respectively. 3,12-dformyl compound (32) was oxidized to 3-carboxyl derivative (33). The quaternary salt (35), obtained from compound (16) with dimethyl sulphate, suffered a ring opening on the action of aqueous sodium hydroxide. The new compounds have been characterized by elemental analyses uv, 1H nmr and in some cases by 13C ruler, CD spectra and X-ray investigations.

Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and spectroscopic studies.

The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 A resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3"-(beta-chloroethyl)-2",4"-dioxo-3, 5"-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.

Prolyl oligopeptidase: an unusual beta-propeller domain regulates proteolysis.

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

[Application of protein crystallography in drug design]. / Fehérje-krisztallográfia alkalmazása a gyógyszerkutatásban.

An overview of the application of protein crystallography in drug design is given in the paper. First a few basic principles of the systematic target based drug design discussed. Secondly are some problems encountered in the process of the protein crystallographic analysis are mentioned when it is applied to cases of pharmaceutical interest.

Crystallization and preliminary X-ray analysis of porcine muscle prolyl oligopeptidase.

Prolyl oligopeptidase from pig muscle has been crystallized in complex with an inhibitor, using PEG 8000 and calcium acetate as precipitants. The crystals are orthorombic and the space group is P212121 with cell dimensions a = 111.8, b = 101.8, c = 72.4 A. The asymmetric unit contains a single chain of prolyl oligopeptidase, corresponding to a specific volume of 2.55 A3 Da-1 and a solvent content of 52%. The observed diffraction pattern extends to 2.3 A resolution and the native crystals are well suited for structural analysis by X-ray diffraction methods.

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E.

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 A resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.

Crystallization and preliminary diffraction analysis of Ca(2+)-calmodulin-drug and apocalmodulin-drug complexes.

Ca(2+)-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3"-(beta-chloroethyl)-2",4"-dioxo-3,5"- spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4- di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 A and differ in unit cell parameters from each other as well as from crystals of Ca(2+)-calmodulin or Ca(2+)-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca(2+)-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca(2+)-free calmodulin crystals diffracting-beyond 2.5 A resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca(2+)-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies.

Ipriflavone, an antiostheophorotic agent.

The antiostheophorotic agent ipriflavone [7-(1-methyl-ethoxy)-3-phenyl-4H-1-benzopyran-4-one, C18H16O3] is an important member of the isoflavone family. The structure of the molecule is dominated by the dihedral angle (50 degrees) between the planes of the phenyl and benzo-pyran moieties. A structural comparison with other related published structures is represented.

Microbial transformation of beta-sitosterol and stigmasterol into 26-oxygenated derivatives.

The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.

Novel 26-oxygenated products in microbial degradation of ergosterol.

In order to investigate the effect of the different stereochemistry of C-24 on the microbial C-26 oxidation of sterol side-chain the genetically modified Mycobacterium sp. BCS 396 strain was used to transform erogsterol. Ergosterol was converted to 3-oxo-4,22-ergostadien-26-oic acid methyl ester, 3-oxo-1,4,22-ergostatrien-26-oic acid methyl ester, and 3-oxo-1,4,22-ergostatrien-26-oic acid, the structures of which have been determined by IR, 1H NMR, 13C NMR, and mass spectroscopy. The X-ray structure of 3-oxo-4,22-ergostadien-26-oic acid methyl ester revealed that oxidation at C-26 of the ergostane side-chain generates a chiral center with S-configuration at C-25 as a result of chiral induction of the C-24 center.

Two antithrombotic quinazolone derivatives.

The structures of two antithrombotic quinazolone derivatives, 1,2,3,5-tetrahydro-2-benzylimidazo[5,1-b]-quinazolin-5-one, (I), and 3-benzyl-2-[1-(2,5-xylidino)-ethyl]quinazolin-4(3H)-one, (II), display significant differences in the bond lengths in one region of the quinazolone moiety.

Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography.

The principal protein excreted in male rat urine, urinary alpha 2-globulin and the homologous mouse protein, major urinary protein, have been well characterized, although their functions remain unclear. Male rat urine affects the behaviour and sexual response of female rats, leading to the proposal that rodent urinary proteins are responsible for binding pheromones and their subsequent release from drying urine. Urinary alpha 2-globulin is also involved in hyaline droplet nephropathy, an important toxicological syndrome in male rats resulting from exposure to a number of industrial chemicals and characterized by the accumulation of liganded urinary alpha 2-globulin in lysosomes in the kidney, followed by the induction of renal cancer. We now report the three-dimensional structures of mouse major urinary protein (at 2.4 A resolution) and rat urinary alpha 2-globulin (at 2.8 A resolution). The results corroborate the role of these proteins in pheromone transport and elaborate the structural basis of ligand binding.

[Study of selegiline and related compounds with x-ray diffraction]. / Selegilin és rokon vegyületek vizsgálata röntgendiffrakcióval.

Selegiline and its parent compounds were studied by X-ray diffraction. It was established that the racemates of primary and secondary amines (p-fluoro-amphetamine, methamphetamine, p-fluoro-methamphetamine) hydrochloride do not form racemic compounds but crystalline as conglomerates, at the same time tertiary amines like selegiline and p-fluoro-selegiline hydrochlorides do. The crystalline structure of five enantiomeric hydrochlorides were determined, the CPhe-C-C-N torsion angle is anti-periplanar in all cases but in p-fluoro-amphetamine where it is gauche.

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.