Drug targets beyond HMG-CoA reductase: Why venture beyond the statins?

In this review, we aim to convey a brief, select history of the development of cholesterol-lowering therapies. We focus particularly on the highly successful statins as well as setbacks that should serve as cautionary tales. We go on to preview recent developments that may complement, if not one day replace, the statins. Our focus is on pharmacological interventions, particularly those targeting the cholesterol biosynthetic pathway. Also, we examine therapies under current investigation that target the assembly of atherogenic lipoproteins (via apolipoprotein B or microsomal triglyceride transfer protein), the stability of the low-density lipoprotein-receptor (via PCSK9, proprotein convertase subtilisin kexin 9), or are designed to increase high-density lipoprotein-cholesterol (via inhibition of cholesteryl ester transfer protein).     

Hepatoselectivity of statins: design and synthesis of 4-sulfamoyl pyrroles as HMG-CoA reductase inhibitors

4-Sulfamoyl pyrroles were designed as novel hepatoselective HMG-CoA reductase inhibitors (statins) to reduce myalgia, a statin-induced adverse effect. The compounds were prepared via a [3+2] cycloaddition of a Münchnone with a sulfonamide-substituted alkyne. We identified compounds with greater selectivity for hepatocytes compared to L6-myocytes than rosuvastatin and atorvastatin. There was an inverse correlation of myocyte potencies and ClogP values. A number of analogs were effective at reducing cholesterol in acute and chronic in vivo models but they lacked sufficient chronic in vivo activity to warrant further development.     

Hepatic HMG-CoA reductase expression and resistance to dietary cholesterol

The premise that the intrinsic level of expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase determines the relative sensitivity to the serum cholesterol raising action of dietary cholesterol was examined in 9 strains of rat. For further comparison purposes, hamsters were also examined. The basal expression of hepatic HMG-CoA reductase, extent of feedback regulation by cholesterol, and changes in serum cholesterol levels and the hepatic low-density lipoprotein (LDL) receptor in response to cholesterol challenge were determined in these animals. The Sprague-Dawley, Wistar-Furth, Spontaneously Hypertensive, Lewis, and Wistar-Kyoto rats were all very resistant to dietary cholesterol and exhibited hepatic HMG-CoA reductase activities above 150 pmol / min(-1) / mg(-1). The Buffalo, Brown Norway, and Copenhagen 2331 rats had hepatic HMG-CoA reductase activities below 90 pmol / min(-1) / mg(-1) and had increases in serum cholesterol levels ranging from 12 to 33 mg/dl when given a 4-day, 1% cholesterol challenge. The extent of feedback regulation was reduced to only 3-fold in the Fisher 344 and Brown Norway rats that exhibited significant increases in serum cholesterol levels when given a cholesterol challenge. The Golden Syrian hamsters exhibited the largest increase (197 mg/dl) in serum cholesterol levels in response to dietary cholesterol and the lowest basal expression of hepatic HMG-CoA reductase (3.3 pmol / min(-1) / mg(-1)). Hepatic LDL receptor levels were not significantly decreased by dietary cholesterol in any of the animals. The data from these inbred rats and the hamsters strongly support the conclusion that the animals expressing the highest levels of hepatic HMG-CoA reductase are the most resistant to the serum cholesterol raising action of dietary cholesterol.     

Activation of HMG-CoA reductase by microsomal phosphatase

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.     

Rapid reverse phase-HPLC assay of HMG-CoA reductase activity

Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP⁺) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP⁺, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.HMG-CoA reductase (HMGR) is the enzyme that catalyze the four-electron reductive deacylation of HMG-CoA to CoA and mevalonate (Fig. 1) (1). This reaction is the controlling step in the biosynthesis of sterols and isoprenoids (2, 3); hence, a large number of studies on the modulation of HMGR activity are continuously performed in the effort of developing new drugs in the treatment of hypercholesterolemic disorders (1).Open in a separate windowFig. 1.Schematic representation of HMGR enzymatic reaction.HMGR activity is conventionally assayed using elaborate radiochemical assay (4–9), chromatographic techniques coupled with mass spectrometry (10–15), or spectrophotometrically by monitoring the decrease in the absorbance of cofactor NADPH at 340 nm (16).Herein, as an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions.     

Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 degrees C, deltaHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 microM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered K(M) and k(cat) values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.     

Measurement of human leukocyte microsomal HMG-CoA reductase activity

Methods were developed for determination of microsomal HMG-CoA reductase activity from freshly isolated human lymphocytes, monocytes, and granulocytes or cultured human lymphoid cells. Reductase activity in monocytes is approximately twice that in lymphocytes or granulocytes. The activity in cultured cells is approximately 34-fold greater than that in freshly isolated cells. Assay conditions were such as to preclude formation of HMG-CoA cleavage products. Leukocyte reductase activity was inhibited by dichloroacetate, a noncompetitive inhibitor of rat liver reductase and a serum cholesterol-lowering agent in man. Measurement of microsomal reductase activity from freshly isolated leukocytes may prove useful in assessing in vivo regulation of cholesterol synthesis in man.     

Binding effect and design of a competitive inhibitory peptide for HMG-CoA reductase through modeling of an active peptide backbone

This study presents an application of two approaches in the design of constrained and unconstrained peptides in an investigation of the peptide binding effect for HMG-CoA reductase (HMGR). In previous works, hypocholesterolemic peptides isolated from soybean were determined as competitive inhibitory peptides for HMGR. Based on the modeling of an active peptide backbone in the active site of HMGR, two peptide libraries for constrained and unconstrained peptides were designed using different amino acids varying in hydrophobicity and electronic properties. Active peptides were selected by the design parameter 'V' or 'Pr', which reflects the probability of active peptide conformations for constrained and unconstrained peptides, respectively. Using peptides designed as mimics of HMGR substrates, and a combination of in vitro test and circular dichroism study, it was found that: (1) peptide binding causes an ordering of secondary structure, reflecting an increase of alpha-helical content; (2) HMGR binds the peptide without closure of the active site; and (3) peptide binding induces the protein aggregation. The GFPDGG peptide (IC(50)=1.5 microM), designed on the basis of the rigid peptide backbone, increases the inhibitory potency more than 300 times compared to the first isolated LPYP peptide (IC(50)=484 microM) from soybean. The obtained data imply the possibility of designing a highly potent inhibitory peptide for HMGR and confirm that changes of the secondary structure in the enzyme play an important role in the mechanism of HMGR inhibition.     

HMG-CoA reductase inhibitors reduce adhesion of human monocytes to endothelial cells.

HMG-CoA reductase inhibitors (statins) are believed to reduce coronary heart disease by mechanisms in addition to their well-known cholesterol-lowering effect. We studied the effect of these drugs on monocyte cell adhesion to endothelium. Pretreatment of monocytic cells (U937, THP-1, human CD14(+) monocytes) with 0.01-10 microM concentrations of atorvastatin, cerivastatin, or simvastatin significantly reduced cell adhesion to endothelium. In contrast, pretreatment of endothelium with statins did not affect adhesion of monocytes. Adhesion of monocytes to vascular cell adhesion molecule-1-coated dishes was reduced by these drugs. Cerivastatin also reduced PMA induction of NF-kappaB. Since monocyte adhesion to endothelium is an early event in atherogenesis, treatment with statins in prevention of coronary heart disease may have additional salutary effects to lowering of plasma LDL cholesterol. Our results indicate that the reduction of monocyte adhesion by HMG-CoA reductase inhibitors may be considered as a class effect.     

HMG-CoA reductase kinase: measurement of activity by methods that preclude interference by inhibitors of HMG-CoA reductase activity or by mevalonate kinase

Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.     

Synthesis and biological evaluations of quinoline-based HMG-CoA reductase inhibitors

A series of quinoline-based 3,5-dihydroxyheptenoic acid derivatives were synthesized from quinolinecarboxylic acid esters by homologation, aldol condensation with ethyl acetoacetate dianion, and reduction of 3-hydroxyketone to evaluate their ability to inhibit the enzyme HMG-CoA reductase in vitro. In agreement with previous literature, a strict structural requirement exists on the external ring, and 4-fluorophenyl is the most active in this system. For the central ring, substitution on positions 6, 7, and 8 of the central quinoline nucleus moderately affected the potency, whereas the alkyl side chain on the 2-position had a more pronounced influence on activity. Among the derivatives, NK-104 (pitavastatin calcium), which has a cyclopropyl group as the alkyl side chain, showed the greatest potency. We found that further modulation and improvement in potency at inhibiting HMG-CoA reductase was obtained by having the optimal substituents flanking the desmethylmevalonic acid portion, that is, 4-fluorophenyl and cyclopropyl, instead of the usual isopropyl group.     

Inactivation of rat liver HMG-CoA reductase phosphatases by nucleotides

Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP caused a concentration-dependent inactivation of enzyme activities. The nucleotides of guanine, cytosine and uracil produced similar effects to those by the nucleotides of adenine for the same number of phosphates present in the molecules. The greater the number of phosphate groups in nucleotides, the higher was the inhibition in reductase phosphatases observed. Preincubation of phosphatases with ATP and subsequent dilution did not diminish the inactivation effect, showing that nucleotides inhibit the enzyme prior to their binding to the substrate. A relationship was observed between those concentrations of nucleotides which produce 50% inactivation and the logarithm stability constant of Mg or Mn salts of nucleotides. ATP-inactivated enzymes were reactivated by Mn++ and to a lesser proportion by Mg++, the conclusion being that HMG-CoA reductase phosphatases have the characteristics of metalloenzymes.     

Cell-type control of membrane biogenesis induced by HMG-CoA reductase

Quantitative increases in HMG-CoA reductase, the rate-limiting enzyme in sterol biosynthesis, induce membrane biogenesis in both yeast and mammalian cells. The subcellular organization of the resulting membrane differs in the two cell types: mammalian cells generate crystalloid endoplasmic reticulum whereas yeast cells assemble karmellae. We examined the consequences of heterologous expression of HMG-CoA reductase to distinguish features of this response that were cell-type specific from those that were isozyme-specific. This analysis demonstrated that membrane proliferation was induced in both mammalian and yeast cells by HMG-CoA reductase from either organism. However, the morphology of the induced membranes was determined by the cell type rather than the particular isozyme. Thus, both yeast and mammalian HMG-CoA reductase contained functional signals for membrane proliferation that were operational in either cell type, but the qualitative response to those signals was cell-type specific.