A novel series of non-nucleoside inhibitors of inosine 5'-monophosphate dehydrogenase with immunosuppressive activity.

Inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) are effective immunosuppressive drugs that may also have additional potential applications as antitumour and antimicrobial agents. The clinical value of the most potent and specific inhibitor of IMPDH, mycophenolic acid, is limited by its rapid metabolism in vivo to an inactive glucuronide derivative. There is, therefore, a considerable incentive to develop structurally novel, preferably non-nucleoside, inhibitors with greater metabolic stability than mycophenolic acid. Here, we describe a high throughput screen for inhibitors of IMPDH, which facilitated the discovery of a single novel non-nucleoside inhibitor from a collection of approximately 80,000 compounds. The inhibitor is a pyridazine, which, like mycophenolic acid, exerts uncompetitive inhibition of IMPDH. Analysis of the enzyme kinetics suggests that the inhibitory action of the pyridazine is similar to that of mycophenolic acid, which involves trapping of a covalent intermediate formed during the conversion of IMP to xanthosine monophosphate. Chemical modification of the lead compound resulted in pyridazine derivatives with enhanced potency against IMPDH and guanine nucleotide synthesis in cultured cells in vitro and also against guanine nucleotide synthesis in the mouse spleen in vivo. One of the compounds was available in sufficient quantity to demonstrate highly effective immunosuppressive activity in a model of delayed type hypersensitivity in mice. To our knowledge, the novel pyridazines described in this report represent the first non-nucleoside uncompetitive inhibitors of IMPDH with immunosuppressive activity since the discovery of the inhibitory activity of mycophenolic acid and its derivatives thirty years ago.

Kinetic characteristics of ZENECA ZD5522, a potent inhibitor of human and bovine lens aldose reductase.

Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522 follows uncompetitive kinetics with respect to glucuronate (K(ii) = 39 nM), indicating a selectivity greater than 5-fold for bovine aldose reductase relative to aldehyde reductase.

Epidermal growth factor receptor tyrosine kinase. Investigation of catalytic mechanism, structure-based searching and discovery of a potent inhibitor.

Inhibition of tyrosine kinases is a possible approach for the treatment of cancer. We have investigated the catalytic mechanism of the epidermal growth factor receptor tyrosine kinase (EGF-RTK) in order to obtain information for use in structure-based searching for inhibitors. Initial rate studies imply that EGF-RTK forms a ternary complex together with ATP and peptide substrate. Investigation of pH and temperature dependence suggests that the kinase reaction requires the ionised form of a carboxylate (pK = 6.3) and the protonated form of another group (pK = 9.1). These characteristics are consistent with a mechanism where the carboxylate of Asp813(pK = 6.3) facilitates deprotonation of the tyrosyl hydroxyl of the peptide substrate, activating it as a nucleophile to attack the gamma-phosphorus of ATP which interacts with a protonated enzyme side-chain (pK = 9.1), possibly the guanidinium group of Arg817. This proposed catalytic mechanism was used to define a query when searching for inhibitors in a database of predicted three-dimensional structures. The procedure involved searching for compounds that mimic the ATP gamma-phosphate, tyrosyl hydroxyl and the tyrosyl aromatic ring, all of which seem to interact strongly with the enzyme during catalysis. This search allowed identification of inhibitors of EGF-RTK which were used to define queries for two-dimensional searching of a larger database, leading to the discovery of 4-(3-chloroanilino)quinazoline (CAQ) which is a potent inhibitor (Ki = 16 nM) of the enzyme. The compound is believed to be the first representative from a new structural class of anilinoquinazoline tyrosine kinase inhibitors. It follows competitive kinetics with respect to ATP and noncompetitive kinetics when the peptide is varied, implying that it functions as an analogue of ATP. CAQ is a novel and potent lead in the search for tyrosine kinase inhibitors as potential agents for the treatment of cancer.

Inhibition of aldose reductase by (2,6-dimethylphenylsulphonyl)nitromethane: possible implications for the nature of an inhibitor binding site and a cause of biphasic kinetics.

Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.

(2,6-Dimethylphenylsulphonyl)nitromethane: a new structural type of aldose reductase inhibitor which follows biphasic kinetics and uses an allosteric binding site.

Many of the complications of diabetes seem to be due to aldose reductase (aldehyde reductase 2, ALR2) catalysing the increased conversion of glucose to sorbitol. Therapy with aldose reductase inhibitors (ARIs) could, therefore, decrease the development of diabetic complications. (2,6-Dimethylphenylsulphonyl)nitromethane (ICI 215918) is an example from a newly discovered class of ARIs, and we here describe its kinetic properties. Preparations of bovine lens ALR2 exhibit biphasic kinetics with respect to glucose and various inhibitors including ICI 215918. The inhibitor sensitive form (ALR2S) has a higher affinity for glucose than does the inhibitor insensitive form (ALR2I). Only ALR2S was characterized in detail because ALR2I activity is very low at physiological levels of glucose and is difficult to measure with accuracy. Aldehyde reductase (ALR1) is the most closely related enzyme to ALR2. Inhibition of ALR1 was, therefore, investigated in order to assess the specificity of ICI 215918. The values of Ki and Kies (dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for ICI 215918 with bovine kidney ALR1 and bovine lens ALR2S have been determined. When glucose is varied, the compound is an uncompetitive inhibitor of ALR2S (Kies = 0.10 microM and Ki is much greater than Kies), indicating that ICI 215918 associates with an allosteric site on the enzyme. These kinetic characteristics would cause a decrease in the concentration required to give 50% inhibition when glucose levels rise during hyperglycaemia. ICI 215918 is a mixed noncompetitive inhibitor of ALR1 (Ki = 10 microM and Kies = 1.8 microM) when glucuronate is varied. Thus, the compound has up to 100-fold specificity in favour of ALR2S relative to ALR1. Therapeutic interest has now centred upon at least three distinct structural types of ARIs: spirohydantoins, acetic acids and sulphonylnitromethanes. Using one representative of each type, we have demonstrated kinetic competition for inhibition of ALR2S. This observation strongly suggests that the different inhibitors use overlapping binding sites.

Application of three-dimensional computer reconstruction from computerized tomography to intracranial tumors in children.

A three-dimensional surface reconstruction algorithm was devised from contour information based upon anatomic structures identified by computed tomographic (CT) scanning. The triangular 'tiling' method from which the algorithm was derived accurately reconstructs the surfaces between contours of both supra- and infra-tentorial anatomic sites. Volume and surface area of the lateral ventricles and tumor site were calculated from the surface reconstruction algorithm in four children with intracranial mass lesions. Reconstruction of three-dimensional surfaces from CT head scans could be utilized to plan the surgical approach to supra-tentorial and posterior fossa lesions. The incorporation of this technique may greatly assist neurosurgeons and neuro-oncologists in planning multi-modality therapy and also for localization of small and relatively inaccessible lesions which may not be adequately assessed by conventional methods. Furthermore, the efficacy of radiation therapy and/or chemotherapy in brain tumors can accurately be assessed.

Three-dimensional computer reconstructions of brain lesions from surface contours provided by computed tomography: a prospectus.

We present a prospectus on the use of computer graphics for the three-dimensional reconstruction and visualization of brain lesions from computed tomographic head examinations, including an algorithm that utilizes surface contour information to reconstruct and display three-dimensional anatomical sites. We provide examples of the use of this algorithm. We offer an algorithm for estimation of the volume and surface area of anatomical sites. The advantages and disadvantages for the clinical use of these algorithms are discussed.

Femoral mechanics in the lesser bushbaby (Galago senegalensis): structural adaptations to leaping in primates.

One method used to examine the relationship between behavioral strategies and anatomical adaptation is to study the results of mechanical stress associated with a given behavior and compare this with skeletal adaptations to other behaviors. This comparative approach is appropriate for highlighting combinations of features that are specializations to specific types of behavior. The purpose of this paper is to compare femoral mechanics in Galago senegalensis with previously collected data for macaques and humans as a basis for discussing structural adaptations in the primate hindlimb to leaping. The stiffness and load carrying capabilities of the femoral diaphyses of 27 G. senegalensis were analyzed using the SCADS computer program. The data suggest that the galago femur is well adapted to sustain large sagittal plane compressive loads rather than large bending loads. The straightness of the femoral shaft and large midshaft area moments of inertia prevent buckling from these large compressive loads. Calculations indicate that the ratio of critical buckling load to body weight in galago is 31 times that in macaques and 55 times that in humans. The femur of this saltatory primate is morphologically adapted to resist buckling when subjected to large compressive loads, while those of macaques and humans are better adapted to resist bending moments caused by ground reaction forces acting on the extended limb. The differences between galago on the one hand and macaques and humans on the other suggest that relatively smaller moments about the hip and relatively larger moments about the knee accompany more quadrupedal and bipedal walking, while habitual leaping is associated with relatively larger moments about the hip. These data reinforce the apparent similarity of the mechanical effects of quadrupedal and bipedal locomotion on the femur and dissimilarity with femoral mechanics in habitually saltatory primates.

Quantitative morphological analysis of interatrial muscle cells in the ferret heart.

Cells located in the interatrial septum of the ferret heart were examined and mean cell volume, surface area, length, width, as well as cell length/width and surface area/volume ratios were obtained. The muscle cells were from two different regions. One region was the area of the middle internodal tract while the other was from the area where the anterior and middle internodal tracts intermingled. Based on the data obtained, at least two different subpopulations of interatrial muscle cells could be defined. The larger cells had a mean cell length of 109.7 micrometer, a mean cell width of 13.1 micrometer, a length/width ratio of 8.61, a mean cell surface area of 5,057.6 micrometer2, a mean cell volume of 5960.8 micrometer3, and a surface area/volume ratio of 0.87. The smaller cells had a mean cell length of 58.0 micrometer, a mean cell width of 12.2 micrometer, a length/width ratio of 4.85, a mean cell surface area of 2494.1 micrometer2, a mean cell volume of 2553.6 micrometer3, and a surface area/volume ratio of 1.00. The large cell population had cells that were myofibril rich and also others that were myofibril poor. These quantitative data indicate that the regions of internodal pathways are not composed of a single specialized cell type, but rather are composed of at least two, if not more, cell types that intermingle with each other.

The use of a computerized algorithm to determine single cardiac cell volumes.

Single cardiac muscles cell volume data have been difficult to obtain, especially because the shape of a cell is quite complex. With the aid of a surface reconstruction method, a cell volume estimation algorithm has been developed that can be used on serial of cells. The cell surface is reconstructed by means of triangular tiles so that the cell is represented as a polyhedron. When this algorithm was tested on computer generated surfaces of a known volume, the difference was less than 1.6%. Serial sections of two phantoms of a known volume were also reconstructed and a comparison of the mathematically derived volumes and the computed volume estimations gave a per cent difference of between 2.8% and 4.1%. Finally cell volumes derived using conventional methods and volumes calculated using the algorithm were compared. The mean atrial muscle cell volume derived using conventional methods was 7752.7 +/- 644.7 micrometers3, while the mean computerized algorithm estimated atrial muscle cell volume was 7110.6 +/- 625.5 micrometers3. For AV bundle cells the mean cell volume obtained by conventional methods was 484.4 +/- 88.8 micrometers3 and the volume derived from the computer algorithm was 506.0 +/- 78.5 micrometers3. The differences between the volumes calculated using conventional methods and the algorithm were not significantly different.

Three-dimensional computer reconstruction from surface contours for head CT examinations.

A three-dimensional surface reconstruction algorithm based on contour information from anatomic sites as identified on computed tomography examinations is presented. This algorithm efficiently reconstructs the surfaces between the contours using a triangular "tiling" method. A three-dimensional perspective view of the reconstructed surfaces is provided by a gray scale cathode-ray tube computer graphics system. Volume and surface area estimations are provided as a direct results of the surface reconstruction algorithm. Three cases are presented to illustrate the three-dimensional surface reconstruction algorithm.

The use of computer imaging techniques to visualize cardiac muscle cells in three dimensions.

Atrial muscle cells and atrioventricular bundle cells were reconstructed using a computer-assisted three-dimensional reconstruction system. This reconstruction technique permitted these cells to be viewed from any direction. The cell surfaces were approximated using triangular tiles, and this optimization technique for cell reconstruction allowed for the computation of cell surface area and cell volume. A transparent mode is described which enables the investigator to examine internal cellular features such as the shape and location of the nucleus. In addition, more than one cell can be displayed simultaneously, and, therefore, spatial relationships are preserved and intercellular relationships viewed directly. The use of computer imaging techniques allows for a more complete collection of quantitative morphological data and also the visualization of the morphological information gathered.