Novel compounds related to 2-(cyclohexylthio)-3,4-dihydro-5-methyl-6-(3-methylbenzyl)-4-ox opyrimidine (3c, MC 639) have been synthesized and tested as inhibitors of human immunodeficiency virus type-1 (HIV-1). Reaction of thiourea with ethyl arylmethylacetoacetates furnished 5-alkyl-6-(arylmethyl)-3,4-dihydro-2-mercapto-4-oxopyrimidines which were then alkylated at the sulfur atom to afford the required 2-alkylthio or 2-cycloalkylthio derivatives (S-DABOs). Chemical modifications at N-3, C-4, and C-6 of the pyrimidine ring were attempted with the aim of improving antiretroviral activity. In particular, replacement of the benzyl group with the 1-naphthylmethyl moiety enhanced the activity of S-DABOs, whereas N-3 alkylation and C=O transformation into C=S at position 4 of the pyrimidine ring led to compounds devoid of anti-HIV-1 activity. Lower activity was generally observed when 1-naphthylmethyl was replaced by the isomeric 2-naphthylmethyl moiety. The most active compounds showed activity in the low micromolar range with EC50 values comparable to that of nevirapine.

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Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefoldhigher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC 50 ] range, 1.4 to 40 M) were comparable to those of antiprion reference compounds (EC 50 range, 0.6 to 10 M). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture they show important differences in cholesterol metabolism, CEM being less efficient at synthesizing cholesterol and having a lower activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase. To investigate further the relationship between regulation of intracellular cholesterol metabolism at various steps and rate of cell growth, cholesterol synthesis, esterification and efflux were evaluated in CEM and MOLT4 cells at different times during exponential and stationary growth in vitro. It was shown that, although CEM cells have a lower rate of cholesterol synthesis, they grow at a faster rate than MOLT4 cells. However, CEM cells exhibit an increased capacity to esterify cholesterol associated with a decreased efflux of newly synthesized cholesterol into the medium. These results provide evidence for an association between the capability to synthesize and retain cell cholesterol esters and the growth rate potential.

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum-induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin-1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58-035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum-induced DNA synthesis by accumulation of VSMCs in the G1 phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down-regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up-regulation of caveolin-1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.