Dennis J. Hoover

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.

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.

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.

A simple method for the formation of cyclopropylamines: the first synthesis of tricyclopropylamine

Abstract A mild, one-step method to cyclopropylate amines is described. Treatment of a variety of secondary and primary amines with [(1-ethoxycyclopropyl)oxy]trimethylsilane and sodium cyanoborohydride in methanol gave mono- and dicyclopropylamines in good yield. Sterically hindered di- and tricyclopropylamines, including the previously unreported tricyclopropylamine, were prepared using this method. The pKas of some mono-, di- and tricyclopropylamines were measured showing a reduction of ∼1–2 pKa unit per added cyclopropyl group.

Structure-Activity Analysis of the Purine Binding Site of Human Liver Glycogen Phosphorylase

Human liver glycogen phosphorylase (HLGP) catalyzes the breakdown of glycogen to maintain serum glucose levels and is a therapeutic target for diabetes. HLGP is regulated by multiple interacting allosteric sites, each of which is a potential drug binding site. We used surface plasmon resonance (SPR) to screen for compounds that bind to the purine allosteric inhibitor site. We determined the affinities of a series of compounds and solved the crystal structures of three representative ligands with K(D) values from 17-550 microM. The crystal structures reveal that the affinities are partly determined by ligand-specific water-mediated hydrogen bonds and side chain movements. These effects could not be predicted; both crystallographic and SPR studies were required to understand the important features of binding and together provide a basis for the design of new allosteric inhibitors targeting this site.

Three-Dimensional Quantitative Structure-Activity Relationship Analysis of Human CYP51 Inhibitors

CYP51 fulfills an essential requirement for all cells, by catalyzing three sequential mono-oxidations within the cholesterol biosynthesis cascade. Inhibition of fungal CYP51 is used as a therapy for treating fungal infections, whereas inhibition of human CYP51 has been considered as a pharmacological approach to treat dyslipidemia and some forms of cancer. To predict the interaction of inhibitors with the active site of human CYP51, a three-dimensional quantitative structure-activity relationship model was constructed. This pharmacophore model of the common structural features of CYP51 inhibitors was built using the program Catalyst from multiple inhibitors (n = 26) of recombinant human CYP51-mediated lanosterol 14α-demethylation. The pharmacophore, which consisted of one hydrophobe, one hydrogen bond acceptor, and two ring aromatic features, demonstrated a high correlation between observed and predicted IC50 values (r = 0.92). Validation of this pharmacophore was performed by predicting the IC50 of a test set of commercially available (n = 19) and CP-320626-related (n = 48) CYP51 inhibitors. Using predictions below 10 μM as a cutoff indicative of active inhibitors, 16 of 19 commercially available inhibitors (84%) and 38 of 48 CP-320626-related inhibitors (79.2%) were predicted correctly. To better understand how inhibitors fit into the enzyme, potent CYP51 inhibitors were used to build a Cerius2 receptor surface model representing the volume of the active site. This study has demonstrated the potential for ligand-based computational pharmacophore modeling of human CYP51 and enables a high-throughput screening system for drug discovery and data base mining.

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.

Establishment of Correlation between in Vitro Enzyme Binding Potency and in Vivo Pharmacological Activity: Application to Liver Glycogen Phosphorylase a Inhibitors

In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic Kd for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro Kd determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.

Crystallization and preliminary X-ray analysis of complexes of peptide inhibitors with human recombinant and mouse submandibular renins.

Inhibitor-complexed crystals of mouse and human renins suitable for X-ray analysis have been prepared. The mouse renin is complexed with a non-hydrolysable decapeptide analogue of rat angiotensinogen containing a hydroxyethylene isostere in place of the scissile bond. The crystals are monoclinic, space group P2(1) with cell dimensions a = 78.3 A, b = 117.8 A, c = 85.9 A, beta = 101.18 degrees containing four molecules per asymmetric unit. The human renin is fully glycosylated and complexed with a tetrapeptide containing norstatine. The complex crystallises in the cubic space group P2(1)3 with a = 143.1 A and has two molecules in the asymmetric unit. The rotation function of the mouse renin complex indicates pseudo 222 symmetry while that of human renin indicates a pseudo 2-fold axis. Full structural analyses of the two complexes are underway.

Crystal structures of rhizopuspepsin/inhibitor complexes.

The crystal structures of the aspartic proteinases have been extensively studied over the past fifteen years (Hsu et al., 1977; Subramanian et al., 1977; reviewed by Davies, 1990). After the initial determinations of the native structures uncomplexed with inhibitors, a few complexes with pepstatin (Bott et al., 1982) and with a fragment of pepstatin (James et al., 1982) were reported. These studies showed that these inhibitors bound in the deep groove that separates the N- and C-terminal domains of the enzyme. Accompanying the binding of the inhibitor was a displacement of the “flap” region of the molecule, a hairpin loop that closes down on the inhibitor, the extent of the displacement depending on the initial location of the flap (James et al., 1982; Bott et al., 1982; Suguna et al., 1987 and Cooper et al., 1987). Since no other major conformational changes were observed, the crystals of these proteinases offered a convenient vehicle for examining a number of bound inhibitor conformations. Other factors facilitating the examination of these inhibitors were the availability of large numbers of renin inhibitors, and the fortunate ease of access to the combining site in several of the crystal forms, thus enabling the inhibitors to be soaked into the crystals.