Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus.

Type 2 diabetes mellitus is a severe disease with large economic consequences, which is significantly under-diagnosed and incompletely treated in the general population. Control of blood glucose levels is a key objective in treating diabetic patients, who are most often prescribed one or more oral hypoglycaemic agents in addition to diet and exercise modification as well as insulin. In spite of the availability of different classes of hypoglycaemic drugs, treatment regimens are often unable to achieve an intensive degree of glucose control known to most effectively reduce the incidence and severity of diabetic complications. Hepatic glucose output is elevated in type 2 diabetic patients and current evidence indicates that glycogenolysis (release of monomeric glucose from the glycogen polymer storage form) is an important contributor to the abnormally high production of glucose by the liver. Glycogen phosphorylase is the enzyme that catalyses this release and recent advances in new inhibitors of this structurally and kinetically well studied enzyme have enabled work which further delineate the pharmacological and physiological consequences of inhibiting glucose production by this pathway. Most notably, these agents lower glucose in diabetic animal models, both acutely and chronically, appear to affect both gluconeogenic and glycogenolytic pathways and demonstrate potential for a beneficial effect on cardiovascular risk factors. Cumulatively, this information has bolstered interest and promise in glycogen phosphorylase inhibitors (GPIs) as potential new hypoglycaemic agents for treatment of type 2 diabetes mellitus.

X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity.

Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.

Human liver glycogen phosphorylase inhibitors bind at a new allosteric site.

BACKGROUND: Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate for glycolysis. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. RESULTS: The binding site in human liver glycogen phosphorylase (HLGP) for a class of promising antidiabetic agents was identified crystallographically. The site is novel and functions allosterically by stabilizing the inactive conformation of HLGP. The initial view of the complex revealed key structural information and inspired the design of a new class of inhibitors which bind with nanomolar affinity and whose crystal structure is also described. CONCLUSIONS: We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.

Lead interaction with human protamine (HP2) as a mechanism of male reproductive toxicity.

During spermatogenesis, histones are replaced by protamines, which condense and protect sperm DNA. In humans, zinc contributes to sperm chromatin stability and binds to protamine P2 (HP2). Chemical interactions with nuclear protamines, which prevent normal sperm chromatin condensation, may induce changes in the sperm genome and thus affect fertility and offspring development. Since lead has a high affinity for zinc-containing proteins, we investigated lead interactions with HP2 as a novel mechanism of its toxicity to sperm. UV/vis and CD spectroscopy results indicated that HP2 binds Pb(2+) at two different sites, causing a conformational change in the protein. They also provided evidence that thiol groups are primarily involved in Zn(2+) and Pb(2+) binding to HP2 and that HP2 may have additional binding sites for Pb(2+) not related to Zn(2+). HP2 affinities for Pb(2+) and Zn(2+) were very similar, suggesting that Pb(2+) can compete with or replace Zn(2+) in HP2 in vivo. This interaction of lead with HP2 resulted in a dose-dependent decrease in the extent of HP2-DNA binding, although lead interaction with DNA also contributed to this effect. Therefore, the ability of lead to decrease the level of HP2-DNA interaction may result in alterations to sperm chromatin condensation, and thus in reduced fertility.

A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure.

In the crystal structure of uncomplexed native chymosin, the beta-hairpin at the active site, known as 'the flap', adopts a different conformation from that of other aspartic proteinases. This conformation would prevent the mode of binding of substrates/inhibitors generally found in other aspartic proteinase complexes. We now report the X-ray analysis of chymosin complexed with a reduced bond inhibitor CP-113972 ¿(2R,3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexy l-2-hydroxybutanoate¿ at 2.3 A resolution in a novel crystal form of spacegroup R32. The structure has been refined by restrained least-squares methods to a final R-factor of 0.19 for a total of 11 988 independent reflections in the resolution range 10 to 2.3 A. The extended beta-strand conformation of the inhibitor allows hydrogen bonds within the active site, while its sidechains make both electrostatic and hydrophobic interactions with residues lining the specificity pockets S4-->S1. The flap closes over the active site cleft in a way that closely resembles that of other previously determined aspartic proteinase inhibitor complexes. We conclude that the usual position and conformation of the flap found in other aspartic proteinases is available to native chymosin. The conformation observed in the native crystal form may result from intermolecular interactions between symmetry-related molecules in the crystal lattice.

Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo.

An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, [R-(R*, S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)pr opyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50 of 0.13 microM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 microM) and in primary human hepatocytes (2.1 microM IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with 14C-glucose to label liver glycogen, CP-91149 administration reduced 14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.

The three-dimensional structure at 2.4 A resolution of glycosylated proteinase A from the lysosome-like vacuole of Saccharomyces cerevisiae.

The crystal structures of glycosylated native proteinase A, an aspartic proteinase found in the vacuole of Saccharomyces cerevisiae, and its complex with a difluorostatone-containing tripeptide have been determined by molecular replacement to 3.5 A and 2.4 A resolutions, respectively. Superposition of the bound and native forms gave an r.m.s. difference of 0.6 A largely reflecting the poor resolution of the native crystal structure. The secondary and tertiary structures are highly similar to those found in porcine pepsin and lysosomal cathepsin D; superposition of the structure of proteinase A bound to the difluorostatone inhibitor on those of pepsin and cathepsin D gave pairwise r.m.s. differences for C(alpha) atoms of 1.36 A and 0.88 A. Most differences occur in loop regions. Comparison of the structure of the proteinase A-difluorostatone complex with that of endothiapepsin bound with the same inhibitor shows that the conformation and hydrogen bond interactions of the inhibitor in the active site are very similar, even though the enzymes have only 27% sequence identity. Electron density for the crystal structure of the proteinase A complex reveals five residues of the oligosaccharide structure attached to Asn67: Man-(1 --> 2)-alpha-Man-(1 --> 3)-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4)-beta-GlcNAc-Asn-67. The first three residues of the oligosaccharide cover the same region of the protein surface as those of the oligosaccharide attached to the equivalent position in cathepsin D. The second carbohydrate attachment site is disordered beyond the first carbohydrate residue in both enzymes.

Discovery of inhibitors of human renin with high oral bioavailability.

Knowledge of the sequence of a bioactive protein (angiotensinogen) and the availability of a natural product inhibitor lead (pepstatin) were the starting point for discovery of potent penta- and hexapeptide renin inhibitors. Study of the metabolism and disposition of these substances forced the discovery of simpler inhibitors leading to the discovery of oral activity in Terlakiren (22). Modification of physical properties led to the synthesis of aminopiperidine 30, which was identified by oral efficacy profiling. Structural modification to give enzymatic stability produced the bioavailable benzylsuccinate inhibitor 34. Its bioactive monomethylamine metabolite (35, CP-108,671) was subsequently found to have uniformly high oral bioavailability and activity in various species including primates.

Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes.

The crystal structures of rhizopuspepsin complexed with two oligopeptide inhibitors have been determined. CP-69,799, an azahomostatine dipeptide isostere, had previously been associated with a displacement of the C-terminal subdomain of endothiapepsin [Sali, A., Veerapandian, B., Cooper, J. B., Foundling, S. I., Hoover, D. J., & Blundell, T. L. (1989) EMBO J. 8, 2179-2188]. Here, we report the measurement of two data sets, one from crystals soaked in the inhibitor and the other from protein crystallized in the presence of excess inhibitor. In neither case is there any significant movement of the C-terminal subdomain of the rhizopuspepsin. The data suggest that the energy associated with any conformational change is small and is overcome by the crystal packing forces. The second inhibitor, a hydrated difluorostatone, was examined in a search for transition-state analogs that could cast further light on the mechanism of action [Suguna, K., Padlan, E. A., Smith, C. W., Carlson, W. D., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7009-7013]. The gem-diol provides a set of contact distances with the enzyme that mimic the interactions with the tetrahedral intermediate of the substrate during catalysis. These data provide support for the suggestion that the polarization of the keto group of the peptide substrate is enhanced by a hydrogen bond from the OD1 of Asp 35 (Suguna et al., 1987).

Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.

Androgen metabolism by porcine granulosa cells during the process of luteinization in vitro: identification of 19-oic-androstenedione as a major metabolite and possible precursor for the formation of C18 neutral steroids.

The present studies were conducted to define the pathway(s) by which androstenedione is metabolized in porcine granulosa cells (pGC) and determine whether metabolism of this steroid is affected by in vitro luteinization. pGC isolated from large preovulatory follicles were cultured for up to 2 days in the presence of 5 microM unlabeled or [4-14C]-labeled androstenedione. Metabolism of androstenedione was assessed by HPLC, using in-line liquid scintillation detection. Metabolite identification was confirmed by gas chromatography-mass spectrometry of HPLC fractions isolated from medium conditioned by granulosa cells (pGCCM) cultured for 48 h in the presence of unlabeled androstenedione. The metabolites identified were 19-oic-androstenedione (3,17-dioxo-4-androsten-19-oic acid), 19-hydroxytestosterone, 19-hydroxyandrostenedione, 19-nor-testosterone, an estrenolone of as yet unproven stereoisomerism, 5(10)-estrene-3 beta, 17 beta-diol, 17 beta-estradiol, testosterone, and 19-nor-androstenedione. Results indicate that 19-nor-androstenedione is artifactually derived from 19-oic-androstenedione as a result of degradation in storage and during isolation. After metabolite identification, studies of the time course of androstenedione metabolism by pGC during in vitro luteinization were conducted. 17 beta-Estradiol and 19-oic-androstenedione were the predominant metabolites, and accumulation of these steroids was virtually identical. Production of these metabolites was maximal during the first 12 h of culture. The accumulation of 5(10)-estrene-3 beta,17 beta-diol and 19-nor-testosterone was maximal at 48 h of culture, with 5(10)-estrene-3 beta,17 beta-diol consistently accumulating in greater concentrations than 19-nor-testosterone. Aromatase activity of pGC was negligible from 36-48 h of culture, as demonstrated by minimal accumulation of 17 beta-estradiol during this period of culture. The accumulation of 19-oic-androstenedione, 5(10)-estrene-3 beta,17 beta-diol, and 19-nor-testosterone was also negligible during this latter time period, suggesting that their formation is associated with aromatase. From these results, pGC from preovulatory follicles undergoing luteinization in vitro lose the ability to convert androstenedione to estrogens. The formation of 19-oic-androstenedione, shown here for the first time, parallels the formation of 17 beta-estradiol, and this acidic steroid is proposed to be a product of aromatase. As reported in previous studies, pGC do produce C18 neutral steroids from exogenous androstenedione. The production of these steroids requires an active aromatase to produce their immediate precursor, which is here hypothesized to be 19-oic-androstenedione. However, their maximal production does not commence until aromatase activity has declined, and it is hypothesized that their production depends on modifications in steroid metabolism associated with luteinization.

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.

Changes in inhibin activity, and progesterone, oestrogen and androstenedione concentrations, in rat follicular fluid throughout the oestrous cycle.

Follicular fluid was aspirated from all visible surface follicles of rats at selected times of the oestrous cycle. Fluids from a pair of rat ovaries were pooled and assayed for inhibin activity by the rat anterior pituitary cell culture assay. Serum LH, FSH and progesterone as well as follicular fluid progesterone, total oestrogens and androstenedione were also measured. Follicular fluid inhibin activity was relatively constant throughout the oestrous cycle (30.7 +/- 3.4% inhibition of FSH per 0.1 microliter follicular fluid) except for a well defined surge at pro-oestrus (09:00-16:00 h, peak at 14:00 h = 84.0 +/- 7.2% inhibition of FSH per 0.1 microliter follicular fluid). The follicular fluid was not treated with charcoal before assay because a pilot experiment showed that such treatment did not alter the inhibin activity of follicular fluid. Steroids in follicular fluid were generally lowest on the afternoon of oestrus and the morning of dioestrus I and generally elevated during pro-oestrus.

Pharmacokinetics of erythromycin in foals and in adult horses.

The pharmacokinetic parameters of erythromycin in foals were determined following intravenous administration of 5.0 mg/kg to animals aged 1, 3, 5 and 7 weeks. The distribution of the drug was described by a two-compartment open model, and no significant differences were observed between coefficients on which the parameters were based. Pharmacokinetic values were also determined for four mares given 5.0 mg/kg intravenously and for six 10-12-week-old foals given 20.0 mg/kg intravenously. The half-life of erythromycin for all groups of animals (foals less than 7 weeks, mares, foals 10-12 weeks) was 1.0-1.1 h; the apparent volume of distribution was between 2.3 and 7.2 l/kg, and the clearance of the drug from the body was between 1.9 and 5.0 mg/kg/h. No drug could be detected in the serum following oral administration of 5.0 mg/kg erythromycin estolate; detectable levels were found for 5 h in mares given 12.5 mg/kg, and for 8 h in foals given 20.0 mg/kg orally. Peak levels in foals given the drug orally were 0.42 micrograms/ml at 120 min after administration. Foals given 10.0 mg/kg of erythromycin base intramuscularly had serum concentrations detectable 12 h later; the peak level achieved was 1.44 micrograms/ml serum 90 min after administration and concentrations exceeded 0.25 micrograms/ml for 6 h. In the mares the milk concentrations were approximately twice those in serum. Recommendations were made for drug dosage to be used in the treatment of Corynebacterium equi pneumonia of foals.