Georgianna Harris

Inhibition of rat α-reductases by finasteride: Evidence for isozyme differences in the mechanism of inhibition

The mechanism of inhibition of the rat types 1 and 2 5alpha-reductase by finasteride was investigated using recombinantly expressed enzymes. These studies revealed that finasteride is a potent, reversible inhibitor of the rat type 1 5alpha-reductase with Ki=10.2+/-1.3 nM. Finasteride is a potent inhibitor of the rat type 2; however, in this case the compound binds to the type 2 isozyme-NADPH complex to form a ternary complex with Ki=1.19+/-0.10 nM, which then rearranges to a high affinity complex (E:I) with a pseudo first order rate constant of 1.62+/-0.22 x 10(-3)/s. The second order rate constant is k3/Ki=1.37+/-0.31 x 10(6) M/s. Heat denaturation of the (type 2 enzyme:inhibitor) complex releases dihydrofinasteride and presumably the NADP+-adduct previously identified with the human 5alpha-reductases. The effects of finasteride were also studied in intact COS cells transiently expressing the rat types 1 and 2 5alpha-reductase. Results with whole cell assays confirm differences in mechanism of inhibition of rat types 1 and 2 5alpha-reductase by finasteride.

Glucose-lowering in a db/db mouse model by dihydropyridine diacid glycogen phosphorylase inhibitors.

The synthesis of a series of novel dihdyropyridine diacid glycogen phosphorylase inhibitors is presented. SAR and functional assay data are discussed, along with the effect of a single inhibitor on blood glucose in a diabetic animal model.

Steroid 5α-reductase inhibitors in androgen-dependent disorders

Abstract Inhibition of the steroid 5α-reductases shows promise in the treatment of a number of androgen-dependent disorders, such as benign prostatic hyperplasia, male pattern baldness, and acne. The design of potent and isozyme-selective inhibitors has provided biologists and clinicians with important tools for elucidating complex androgen physiology, and has already resulted in the development of one marketed drug.

Skeletal muscle: a dual system to measure glucocorticoid-dependent transactivation and transrepression of gene regulation

The use of chronic glucocorticoid (GC) therapy for the treatment of inflammatory diseases is limited by associated metabolic side effects, including muscle atrophy. Therefore, selective glucocorticoid receptor-(GR)-binding ligands that maintain anti-inflammatory activity and demonstrate diminished side-effect profiles would have great therapeutic utility. In this work, we use Taqman PCR and ELISA methods to show that GCs can inhibit basal, and lipopolysaccharide (LPS)-stimulated levels of cytokines IL-6 and TNFalpha, and also the chemokine MCP-1 in a non-inflammatory system such as primary human skeletal muscle cells. In the murine C2C12 skeletal muscle cell line we observe a similar effect of GCs on IL-6 and MCP-1; however, in contrast to previous reports, we observe a time-dependent repression of TNFalpha. Furthermore, in skeletal muscle cells, concomitant with cytokine repression, GCs transcriptionally induce glutamine synthetase (GS), a marker for muscle wasting, in an LPS independent manner. Similarly, administration of dexamethasone to mice, previously administered LPS, results in an increase in GS and an inhibition of TNFalpha and MCP-1 in skeletal muscle tissue. Thus, skeletal muscle cells and tissues present a novel system for the identification of selective GR-binding ligands, which simultaneously inhibit cytokine expression in the absence of GS induction.

MK386 : a potent, selective inhibitor of the human type 1 5α-reductase

Abstract Steroid 5α-reductase is required for the conversion of testosterone to dihydrotestosterone. Localization of type 1 5α-reductase in the sebaceous gland of skin offers the possibility for selective inhibition of this isozyme as a treatment for acne. The goals of these studies are to demonstrate the mechanism of inhibition of MK386 and its selectivity for type 1 5α-reductase. The apparent potency of MK386 differed depending on the source of the enzyme (i.e. recombinant vs. native), yet selectivity for type 1 5α-reductase was unchanged. Our results indicate that the apparent potency of MK386 is modulated by the membrane concentration of the assay. These results suggest that MK386 has a high affinity for the lipid-rich membrane environment of 5α-reductase. MK386 was also found to be a slow binding inhibitor of type 1 5α-reductase. However, the cause of this time-dependent inhibition is unrelated to partitioning of the inhibitor into the membrane because similar studies with type 2 5α-reductase indicate that MK386 is a reversible, competitive inhibitor. A number of counterscreens were developed to demonstrate that MK386 is a poor inhibitor of other steroid metabolizing enzymes.

Selective glucocorticoid receptor nonsteroidal ligands completely antagonize the dexamethasone mediated induction of enzymes involved in gluconeogenesis and glutamine metabolism.

Glucocorticoids (GCs) are vital multi-faceted hormones with recognized effects on carbohydrate, protein and lipid metabolism. Previous studies with the steroid antagonist, RU486 have underscored the essential role of GCs in the regulation of these metabolic pathways. This article describes the discovery and characterization of novel GRalpha selective nonsteroidal antagonists (NSGCAs). NSGCAs 2 and 3 are spirocyclic dihydropyridine derivatives that selectively bind the GRalpha with IC(50s) of 2 and 1.5 nM, respectively. Importantly, these compounds are full antagonists of the induction by dexamethasone (Dex) of marker genes for glucose and glutamine metabolism; the tyrosine amino transferase (TAT) and glutamine synthetase (GS) enzymes, respectively. In contrast, GC-dependent transcriptional repression of the collagenase 1 (MMP-1) enzyme, an established GRalpha responsive proinflammatory gene; is poorly antagonized by these compounds. These NSGCAs might have useful applications as tools in metabolic research and drug discovery.

Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-Reductase: evidence for mechanism-Based inhibition by finasteride

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus SaRs shared 94% and 96% identity with the human type 1 and 2 isozymes, respectively. Despite a four amino acid insertion at the N-terminal region of rhesus 5aR1, the biochemical properties of rhesus and human homologs are very similar with respect to pH optimum, Km values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expected, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays time dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M(-1) s(-1) and 5.2 x 10(5) M(-1)s(-1). Inhibition of rhesus 5aR2 with 3H-finasteride resulted in 3H bound to the enzyme which is not released by dialysis. Heat denaturation of the [rhesus SaR2:inhibitor] complex releases dihydrofinasteride, a breakdown product presumably related to the NADP+-adduct previously identified with the human SaRs (Bull et al., Mechanism-based inhibition of human steroid 5alpha-reductase by finasteride: Enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J. Amer. Chem. Soc., 1996, 118, 2359-2365). Taken together, these results provide good evidence that the rhesus macaque is a suitable model to evaluate the pharmacological properties of finasteride and other 5aR inhibitors.

Design and synthesis of novel antibacterial agents with inhibitory activity against DNA polymerase III.

4-Substituted 2-amino-6-(anilino)pyrimidines have been found to be selective inhibitors of DNA polymerase III, a replicative enzyme known to be essential in the DNA synthesis of Gram-positive bacteria. Among the analogues, 18 displayed an IC(50) of 10 microM against DNA polymerase III from Staphylococcus aureus.

4-methyl-3-oxo-4-aza-5α-androst-1-ene-17β-n-aryl-carboxamides: an approach to combined androgen blockade [5α-reductase inhibition with androgen receptor binding in vitro]

Abstract 4-Aza-5α-androstan-3-one 17β-(N-substituted carboxamides) are potent human type 2 5α-reductase (5aR) inhibitors with generally poor binding to the human androgen receptor (hAR). When the 17-amide N-substituent included an aromatic residue, potent dual inhibitors of both type 1 and 2 5aR are produced, but hAR binding remained poor. Tertiary-substituted-17-amides have reduced inhibition of both 5aR isozymes. The addition of an N4-methyl substitutent to the A-ring profoundly increased hAR affinity and the addition of unsaturation to the A-ring (Δ1) modestly augmented hAR binding. The unsubstituted carbanilides in the Δ1-N4-methyl series show some selectivity for type 1 5aR over the type 2 isozyme, whereas addition of aryl substituents, particularly at the 2-position, increased type 2 5aR binding to provide dual inhibitors with excellent hAR binding, e.g. N-(2-chlorophenyl)-3-oxo-4-methyl-4-aza-5α-androst-1-ene-17β-carboxamide (9c). Compounds of this type exhibit low nanomolar IC50s for both human 5aR isozymes as well as the human androgen receptor. Kinetic analysis confirms that the prototype 9c displays reversible, competitive inhibition of both human isozymes of 5aR with Ki values of less than 10 nM. Furthermore, this compound binds to the androgen receptor with an IC50 equal to 8 nM. Compounds in this series are projected to be powerful antagonists of testosterone and dihydrotestosterone action in vivo, with potential utility in the treatment of prostatic carcinoma (PC).

Discovery of betamethasone 17α-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.