William F. Heath

Amelioration of Vascular Dysfunctions in Diabetic Rats by an Oral PKC β Inhibitor

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the β isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC β1 and β2, with a half-maximal inhibitory constant of ∼5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

Inhibition of protein kinase C by calphostin C is light-dependent

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C.

Protein kinase C inhibitors

Protein kinase C (PKC) is a family of serine-threonine protein kinases that are involved in signal transduction pathways that regulate growth factor response, proliferation, and apoptosis. Its central role in these processes, which are closely involved in tumor initiation, progression, and response to antitumor agents, makes it an attractive therapeutic target in cancer. Despite initial activity seen in melanoma (bryostatin and UCN-01), non-Hodgkin’s lymphoma (ISIS 3521, bryostatin, and UCN-01), and ovarian carcinoma (ISIS 3521 and bryostatin) in phase I studies, single-agent activity in those phase II studies reported to date has been limited. Preclinical data highlight a role for PKC in modulation of drug resistance and synergy with conventional cytotoxic drugs. A randomized phase III study of ISIS 3521 in combination with carboplatin and paclitaxel, compared with chemotherapy alone, in advanced non-small-cell lung cancer is underway. This paper reviews the rationale for using PKC inhibitors in cancer therapy, the challenges for clinical trial design, and the recent clinical experience with modulators of PKC activity.

Glucose Transporter Levels in Tissues of Spontaneously Diabetic Zucker fa/fa Rat (ZDF/drt) and Viable Yellow Mouse (Avy/a)

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose < 11 mM) and obese, and severely diabetic (blood glucose > 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25–55%, and GLUT2 in liver was increased 30–40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state. In both rodent models, similar alterations in GLUT2 and/or GLUT4 levels were observed under comparable glycemia, but quite distinct insulin levels, consistent with the possibility that glucose itself may be an important contributing regulator of glucose transporter expression.

LY290181, an Inhibitor of Diabetes-Induced Vascular Dysfunction, Blocks Protein Kinase C—Stimulated Transcriptional Activation Through Inhibition of Transcription Factor Binding to a Phorbol Response Element

Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181. LY290181 prevented glucose-induced increases in blood flow and permeability in rat granulation tissue and corresponding vascular changes in the retina, sciatic nerve, and aorta of diabetic rats. Tested for its ability to inhibit PKC-regulated processes, LY290181 inhibited phorbol ester–stimulated plasminogen activator activity in a dose-dependent manner in bovine retinal endothelial cells and in human dermal fibroblasts. In addition, LY290181 inhibited phorbol ester–stimulated activation of the porcine urokinase plasminogen activator (uPA) promoter (−4600/+398) linked to the chloramphenicol acetyltransferase (CAT) reporter gene (p4660CAT). More detailed analysis of the uPA promoter revealed that LY290181 inhibited phorbol ester–stimulated activation of the uPA phorbol response element (−2458/−2349) located upstream of the thymidine kinase promoter (puPATKCAT). LY290181 appears to inhibit uPA promoter activation by blocking phorbol ester–stimulated binding of nuclear proteins to the uPA PEA3/12-O-tetradecanoylphorbol 13-acetate responsive element (TRE). These results suggest that LY290181 may inhibit diabetes-induced vascular dysfunction by inhibiting transcription factor binding to specific PKC-regulated genes involved in vascular function.

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.

Synthesis of bisindolylmaleimide macrocycles

Abstract The synthesis of a novel class of N-N′-macrocyclic bisindolylmaleimides is reported. The key step involves a remarkably efficient intramolecular cyclization reaction. The method was further developed to provide an efficient synthesis of this type of macrocycle through an intermolecular alkylation with subsequent intramolecular cyclization.

Biosynthesis and Processing of the gag and pol Polyproteins of Human Immunodeficiency Virus Type 1 in Escherichia coli

The three major structural genes of human immunodeficiency virus type 1 (HIV-1) are arranged in the viral genome in the order of 5’ gag-pol-env 3’. The gag gene encodes four group specific antigens, MA(p17), CA(p24), NC(p7) and p6, while the pol gene codes for protease, reverse transcriptase/ribonuclease H (RT/RH, 66 kDa) and integrase (IN, 32 kDa) (Chassagne et al.., 1986; Henderson et al., 1988; Kramer et al., 1986; Lightfoot et al., 1986; Lillehoj et al., 1988). The primary translational product of the gag gene is a 55 kDa polyprotein (pr55gag) (Kalyanarman et al., 1984; Sarngadharan et al., 1985), and that of the pol gene is a gag-pol fusion protein 160 kDa in size (pr160gag-Pol) achieved by ribosomal frameshift near the 3’ end of the gag gene (Gendelman et al., 1987; Jacks et al., 1988). They are processed to the individual viral proteins by the protease encoded by the pol gene after the enzyme cleaves itself from the pr160gag-Pol precursor by autocatalysis (Farmerie et al., 1987; Graves et al., 1988). There are at least seven protease cleavage sites in the gag and pol polyprotein precursors (Darke et al., 1988), as depicted in Figure 1. The numbering of amino acid residues at the cleavage sites is based on the amino acid sequence predicted from the nucleotide sequence of clone HXB2 of HIV-1 isolate, HTLV HIB (Ratner et al., 1985).