We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, ,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to ,E-geranyl diphosphate or ,Zneryl diphosphate yielding ,E,Z-farnesyl diphosphate and ,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K m values of 124 M for isopentenyl diphosphate, 38 M for geranyl diphosphate, and 16 M for neryl diphosphate. Inhibitors of the Zfarnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of ,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).Isoprenyl diphosphate synthases catalyze the condensation of an allylic diphosphate with isopentenyl diphosphate (IPP, 1 C 5 ) via an electrophilic alkylation reaction to produce longer allylic diphosphates (1, 2). Chain elongation continues until a physiologically appropriate chain length is reached, at which time the molecule may undergo further modifications (dephosphorylation, cyclization, or head-to-head condensation reactions). Polyprenyl phosphate (Pol-P) is formed by dephosphorylation of an allylic prenyl diphosphate chain. The predominant form of prokaryotic Pol-P is ,diE,polyZ-undecaprenyl phosphate 2 (C 55 ); however, there are documented exceptions in Paracoccus denitrificans (3) and in Mycobacterium sp. (4 -8). M. smegmatis contains heptaprenyl diphosphate (8) (C 35 , four saturated, three Z double bonds) and decaprenyl diphosphate (5) (C 50 , , one E, and eight Z double bonds), whereas M. tuberculosis contains only decaprenyl phosphate (6). Although the stereochemistry of decaprenyl phosphate from M. tuberculosis has not been determined, our enzymatic studies suggest that it has similar stereochemistry to decaprenyl phosphate from M. smegmatis (9).Pol-P is central to prokaryotic cell wall synthesis as a sugar carrier, and it has been reported that the levels of Pol-P may be rate-limiting for in vivo cell wall synthesis (10 -13). Our laboratory has shown that Pol-P is instrumental in the synthesis of each component of the covalently linked peptidoglycan-arabinogalactan-mycolic acid cell wall core of mycobacteria, and other noncovalently associated macromolecules such as lipomannan and lipoarabinomannan (5,14,15). The importance of Pol-P is also demons...

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10-to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes ,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes ,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the ,E,Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the ,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, ,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.Polyprenyl phosphates (Pol-P) are involved in the biosynthesis of bacterial cell walls (14), and their availability is rate limiting for several aspects of cell wall synthesis in Staphylococcus aureus (15) and Bacillus spp. (2). It has also been suggested that the rate of synthesis of lipid I (in peptidoglycan synthesis) of Escherichia coli may be dependent on the pool level of Pol-P (26), and Baddiley (4) reported that Pol-P levels could regulate the rate of bacterial cell wall synthesis in vivo.Mycobacterium smegmatis is known to contain two forms of Pol-P that are covalently attached to mannose (25). These are structurally unusual in that the decaprenyl phosphate contains one -isoprene unit, one E-isoprene unit, and eight Z-isoprene units (mono-E, poly-Z) (28) and the heptaprenyl phosphate consists of either four saturated isoprene units on the omega end of the molecule, two E-isoprene units, and one Z-isoprene unit (6) or four saturated and three Z-isoprene units (27) (Fig. 1A) (the stereochemical configuration of the isoprene units is always listed starting at the omega end of the molecule). It appears that Mycobacterium tuberculosis may be more typical than M. smegmatis, as a single predominant Pol-P (decaprenyl phosphate) was identified in this species, however; the stereochemistries of the individual isoprene units were not determined (24).The fact that both forms of Pol-P in M. smegmatis are glycosylated suggested that both could be involved in the synthesis of cell wall polysaccharides. Our laboratory has shown that M. smegmatis utilizes its unusual...

A deficiency in apoptosis is one of the key events in the proliferation and resistance of malignant cells to antitumor agents; for these reasons, the search for apoptosis-inducing drugs represents a valuable approach for the development of novel anticancer therapies. In this study we report the first example of conformationally restrained analogues of ceramide (compounds 1-4), where the polar portion of the molecule has been replaced by a thiouracil (1, 3) or uracil (2, 4) ring. The evaluation of their biologic activity on CCRF-CEM human leukemia cells demonstrated that the most active was compound 1 followed by compound 2 (mean 50% inhibition of cell proliferation [IC(50)] 1.7 and 7.9 microM, respectively), while compounds 3 and 4 were inactive, as were uracil, thiouracil, and 5,6-dimethyluracil, the pyrimidine moieties of compounds 1-4. For comparison, the IC(50) of the reference substance, the cell-permeable C2-ceramide, was 31.6 microM. Compounds 1 and 2 and C2-ceramide were able to trigger apoptosis, as shown by the occurrence of DNA and nuclear fragmentation, and to release cytochrome c from treated cells. The treatment of female CD-1 nu/nu athymic mice bearing a WiDr human colon xenograft with the most active compound 1 at 2, 10, 50, and 200 mg/kg ip daily for 10 days resulted in an antitumor effect that was equivalent at 50 mg/kg or superior (200 mg/kg) to that of cyclophosphamide, 20 mg/kg ip daily, delivered on the same schedule, with markedly lower systemic toxicity. In conclusion, the present study demonstrates that the new ceramide analogues 1 and 2 are characterized by in vitro and in vivo antitumor activity and low toxicity.

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