Ligand discovery and virtual screening using the program LIDAEUS.

This paper discusses advances in docking and scoring approaches with examples from the high-throughput virtual screening program LIDAEUS. We describe the discovery of small molecule inhibitors for the immunophilin CypA, the cyclin-dependent kinase CDK2 and the cyclapolin series of potent Polo-like kinase inhibitors. These results are discussed in the context of advances in massively parallel computing and in the development of annotated databases.

Two structures of cyclophilin 40: folding and fidelity in the TPR domains.

BACKGROUND: The "large immunophilin" family consists of domains of cyclophilin or FK506 binding protein linked to a tetratricopeptide (TPR) domain. They are intimately associated with steroid receptor complexes and bind to the C-terminal domain of Hsp90 via the TPR domain. The competitive binding of specific large immunophilins and other TPR-Hsp90 proteins provides a regulatory mechanism for Hsp90 chaperone activity. RESULTS: We have solved the X-ray structures of monoclinic and tetragonal forms of Cyp40. In the monoclinic form, the TPR domain consists of seven helices of variable length incorporating three TPR motifs, which provide a convincing binding surface for the Hsp90 C-terminal MEEVD sequence. The C-terminal residues of Cyp40 protrude out beyond the body of the TPR domain to form a charged helix-the putative calmodulin binding site. However, in the tetragonal form, two of the TPR helices have straightened out to form one extended helix, providing a dramatically different conformation of the molecule. CONCLUSIONS: The X-ray structures are consistent with the role of Cyclophilin 40 as a multifunctional signaling protein involved in a variety of protein-protein interactions. The intermolecular helix-helix interactions in the tetragonal form mimic the intramolecular interactions found in the fully folded monoclinic form. These conserved intra- and intermolecular TPR-TPR interactions are illustrative of a high-fidelity recognition mechanism. The two structures also open up the possibility that partially folded forms of TPR may be important in domain swapping and protein recognition.

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.

X-ray structures of small ligand-FKBP complexes provide an estimate for hydrophobic interaction energies.

A new crystal form of native FK506 binding protein (FKBP) has been obtained which has proved useful in ligand binding studies. Three different small molecule ligand complexes and the native enzyme have been determined at higher resolution than 2.0 A. Dissociation constants of the related small molecule ligands vary from 20 mM for dimethylsulphoxide to 200 microM for tetrahydrothiophene 1-oxide. Comparison of the four available crystal structures shows that the protein structures are identical to within experimental error, but there are differences in the water structure in the active site. Analysis of the calculated buried surface areas of these related ligands provides an estimated van der Waals contribution to the binding energy of -0.5 kJ/A(2) for non-polar interactions between ligand and protein.

An example of a protein ligand found by database mining: description of the docking method and its verification by a 2.3 A X-ray structure of a thrombin-ligand complex.

A computer program (SANDOCK) has been developed for the automated docking of small ligands to a target protein. It uses a guided matching algorithm to fit ligand atoms into the protein binding pocket. The protein is described by a modified Lee-Richard's dotted surface with each dot coded by chemical property and accessibility. Orientations of the ligand in the active site are generated such that a chemical and a shape complementary between the ligand and the active site cavity have to be fulfilled. The generated fits are evaluated with scoring functions which account for van der Waals, hydrophobic and hydrogen bonding interactions. This newly developed docking program can efficiently screen very large databases in a reasonable time and has been used to successfully identify novel ligands. The X-ray structure of a thrombin-ligand complex predicted by SANDOCK is described. The ligand binds to thrombin with a Kd of 65 microM and has an rmsd of 0.7 A for all ligand atoms from the predicted binding mode by SANDOCK.

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.

X-ray structure of a monomeric cyclophilin A-cyclosporin A crystal complex at 2.1 A resolution.

The crystal structure of a complex between recombinant human cyclophilin A (Cyp) and cyclosporin A (CsA) has been determined from a novel orthorhombic crystal form that contains only one monomer of complex per asymmetric unit rather than five in the previously determined tetragonal structure. The structure has been refined at 2.1 A resolution to a crystallographic R-factor of 16.7%. The conformation of Cyp is practically unchanged with respect to the tetragonal form. A certain number of previously undefined side-chains have been located in the electron density and a very detailed picture of the ordered solvent structure has been obtained. The interactions between CsA and Cyp are conserved. A network of the possibly conserved, water-mediated contacts is described. The structure of CsA in the monomeric complex is similar to that of the decameric complex, but shows a few small differences in the so-called effector domain of CsA, probably due to differences in crystal environment. The fact that this monomeric crystal form can be obtained shows that the formation of pentamer or decamer complexes is not a generally observed phenomenon and is not a prerequisite for biological activity.

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.

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.

The discovery of steroids and other novel FKBP inhibitors using a molecular docking program.

The molecular docking computer program SANDOCK was used to screen small molecule three-dimensional databases in the hunt for novel FKBP inhibitors. Spectroscopic measurements confirmed binding of over 20 compounds to the target protein, some with dissociation constants in the low micromolar range. The discovery that FK506 binding protein is a steroid binding protein may be of wider biological significance. Two-dimensional NMR was used to determine the steroid binding mode and confirmed the interactions predicted by the docking program.

Structures of redox enzymes.

There is an ever increasing flood of structural information and over 1,000 protein structures have been deposited in the Protein Data Base between January 1999 and January 2000. Major advances in the past year in the field of redox enzymes have included the structures of nitric oxide synthases in ligand-free and ligand-bound complexes, and the determination of the multi-subunit mitochondrial bc1 complex. The first,structures of flavocytochrome have also appeared providing insight into novel electron and proton pathways.

Modeling conformational changes in cyclosporin A.

NMR and X-ray structures for the immunosuppressant cyclosporin A (CsA) reveal a remarkable difference between the unbound (free) conformation in organic solvents and the conformation bound to cyclophilin. We have performed computer simulations of the molecular dynamics of CsA under a variety of conditions and confirmed the stability of these two conformations at room temperature in water and in vacuum. However, when the free conformation was modeled in vacuum at 600 K, a transition pathway leading to the bound conformation was observed. This involved a change in the cis MeLeu-9 peptide bond to a trans conformation and the movement of the side chains forming the dominant hydrophobic cluster (residues MeBmt-1, MeLeu-4, MeLeu-6, and MeLeu-10) to the opposite side of the plane formed by the backbone atoms in the molecular ring. The final conformation had a backbone RMS deviation from the bound conformation of 0.53 A and was as stable in dynamics simulations as the bound conformation. Our calculations allowed us to make a detailed analysis of a transition pathway between the free and the bound conformations of CsA and to identify two distinct regions of coordinated movement in CsA, both of which underwent transitions independently.

The X-ray structure of a tetrapeptide bound to the active site of human cyclophilin A.

Human cyclophilin A (165 residues) has peptidyl-prolyl cis-trans isomerase activity. Here we report a high-resolution three-dimensional X-ray structure of a substrate, ac-Ala-Ala-Pro-Ala-amc (ac, acetyl; amc, amidomethylcoumarin) bound to the active-site of cyclophilin. The structure consisting of a dimer of complexes and 135 water molecules was refined to a crystallographic R-factor of 17.7% for all data in the range 8 A-2.3 A.

Prediction of substrate-specific pockets in cyclosporin synthetase.

Amino acid sequence comparisons between domains of cyclosporin synthetase have been used to identify regions of the sequence which are responsible for the recognition and binding of the individual amino acids. Using a limited set of selection rules it was possible to identify three amino acid positions in the subdomain sequences which are responsible for amino acid specificity. Homology with the firefly luciferase protein shows that these three key residues are close to each other and line the surface of a putative specific substrate binding pocket located on the amino acyl-adenylation subdomain. These results allow us to predict a large number of cyclosporin synthetase mutants which could be used to synthesise alternative cyclosporin-like peptides.

Crystallisation and preliminary X-ray diffraction studies of cyclophilin-tetrapeptide and cyclophilin-cyclosporin complexes.

Recombinant human cyclophilin has been co-crystallised with a number of peptides to give crystals suitable for X-ray analysis. The crystal complexes for which heavy-atom derivatives have been prepared and X-ray data collected are: cyclophilin with N-acetyl-Ala-Ala-Pro-Ala-amidomethyl-coumarin (I) which crystallises in space group P2(1)2(1)2(1) with a = 108.2, b = 123.0, c = 35.8 A, and cyclophilin with cyclosporin (II) which crystallises as tetragonal plates in space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 94.98, c = 278.55 A.

Crystallization and preliminary X-ray crystallographic study of interleukin-8.

Interleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 A. The crystals belong to the space group P3(1)21 or P3(2)21 and have unit cell dimensions a = b = 40.9 A, c = 90.3 A.

The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi.

A structure of residues 1-177 of the cyclophilin domain of a large divergent cyclophilin from the filarial nematode parasite Brugia malayi has been crystallised and solved in two different crystal forms. The active site has a similar structure to that of human cyclophilin A. Two of the 13 residues important in forming the human cyclophilin A/cyclosporin A complex are altered in the B. malayi cyclophilin and explain the relatively poor inhibition of peptidyl prolyl isomerase activity by cyclosporin A.

Cyclosporin A-cyclophilin complex formation. A model based on X-ray and NMR data.

The previously determined 3D NMR solution structure of cyclophilin-bound cyclosporin A (CsA) was docked onto the X-ray crystal structure of cyclophilin. Intermolecular nuclear Overhauser effects (NOE) between CsA and cyclophilin were used as constraints in a restrained energy minimization to generate a model of the complex which satisfied all the NOE distance constraints. The model shows that the residues 9 to 11 and 1 to 5 of the cyclic CsA molecule are in contact with cyclophilin. Comparing the model of the CsA-cyclophilin complex to the X-ray crystal structure of a complex of cyclophilin with a substrate for peptidyl-proline cis-trans isomerase activity, i.e. the linear tetrapeptide substrate ac-Ala-Ala-Pro-Ala-amc (ac, acetyl; amc, amidomethylcoumarin), one notices that the contacting peptide segments in the two ligands are oriented in opposite directions, and that the side chain of MeVal-11 of CsA superposes rather precisely with the position of the prolyl residue in ac-Ala-Ala-Pro-Ala-amc.

Centrally acting alpha 1-adrenoceptor agonists based on hexahydronaphth[2,3-b]-1,4-oxazines and octahydrobenzo[g]quinolines.

Centrally acting alpha 1-agonists may be of therapeutic value in dementias and other CNS disorders characterized by symptoms of noradrenergic insufficiency. Therefore, on the basis of known peripherally acting alpha 1-agonists two new groups of centrally acting alpha 1-agonists with improved lipophilicity, the hexahydronaphth[2,3-b]-1,4-oxazines type A and the octahydrobenzo[g]quinolines type B were designed. The N-methylated derivatives 14 and 33 demonstrate potent, direct agonistic activity at postjunctional alpha 1-receptors. Ring substituent alterations in compounds of type A and B change the potency of compounds on the rabbit ear artery by over 3 orders of magnitude (pD2 = 5.35-8.40). The efficacy of these compounds varies from 42 to 110%. Those alpha 1-agonists which were selective in the pithed rat increase vigilance in rats. Compound 14 was found to be a centrally acting alpha 1-agonist with good tolerability in different animal species and in healthy volunteers. Furthermore, 14 selectively stimulates the breakdown of phosphatidylinositol in rat cerebral cortex slices. In vivo, the compound reverses behavioral deficits in animals which received noradrenergic lesions following DDC or DPS4 treatment. Oxazine 14 and its close derivatives are by far more lipophilic than commonly known alpha 1-agonists. This is demonstrated in a ClogP-PROBIS plot.

Orbital osteoma in Gardner's syndrome.

A 30-year-old woman developed proptosis secondary to a left ethmoidal compact osteoma. At age 29 years, a mandibular eburnated (ivory) osteoma was excised. At age 25 years, multiple adenomatous polyps of the colon were resected. Her father, age 61 years, had multiple intestinal polyps and bilateral mandibular osteoma. A 24-year-old sister had an osteoma of the forehead. Gardner's syndrome is an autosomal dominantly inherited disorder characterized by intestinal polyposis, various skin and soft tissue tumors, and osteomas of the bony skeleton. Orbital osteomas occur rarely.