Q2. What is the cytotoxic activity of the inhibitors?
Incubation in the presence of 2-13C-acetate leads to the incorporation of 13C atoms into the cholesterol molecules biosynthesized during the incubation period, and allows us to distinguish newly synthesised cholesterol from unlabelled matrix cholesterol that was present in the cells before incubation, by GC-MS.
Q3. What was the ozonolysis reaction of intermediate 2?
Since the stereocenters at both C-3a and C-4 were to be destroyed in the subsequent step, intermediate 2 was purified only superficially over a short silica column.
Q4. What is the effect of the oxidosqualene cyclase inhibitor?
The screening results of this first, relatively small collection of inhibitors gave first insights into structure-activity-relationships, showing that the size and charge of the side chain have significant impact on activity, selectivity, and cytotoxicity of the compounds.
Q5. What did the BIBX 79 inhibitors show?
At a fixed concentration of 1 µM 4 and 6 led to a decrease of overall cholesterol biosynthesis of >90% compared to untreated control samples.
Q6. What is the cytotoxicity of the inhibitors?
N-Oxide 9 showed moderate cytotoxicity (IC50 = 20 M), what makes it less attractive for further drug development compared to its tertiaryamino analogue 6, which is, like all other primary and secondary amines investigated here, devoid of cytotoxicity.
Q7. What is the effect of the inhibitors on cholesterol biosynthesis?
The compounds were tested in vitro for inhibition of five different oxidosqualene cyclases, among which four lanosterol synthases (from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Homo sapiens), and one cycloartenol synthase (from Arabidopsis thaliana).
Q8. What is the effect of the inhibitors on the cholesterol biosynthesis?
In a whole cell assay on a human cell line compounds 4 and 6 showed strong inhibition of total cholesterol biosynthesis, indicating that this class of inhibitors has proper physicochemical properties for being active in living cells.
Q9. What is the concept of combining a significant partial structure of the steroid backbone?
The concept of combining a significant partial structure of the steroid backbone with functional groups which are able to mimic cationic high energy intermediates, is promising for further evaluation.
Q10. What was the first screening for cytotoxic activities of the compounds?
A first screening for cytotoxic activities of the compounds was performed in a MTT assay according to the method of Mosmann [32] on human leukaemia HL-60 cells.
Q11. What was the purpose of the assay?
4 and 6, which showed significant inhibition of human OSC in the in vitro screenings (Table 1), were subjected to a whole cell assay on human HL-60 cells in order to investigate their activity at the cellular level.
Q12. What is the ketone that was selected?
The piperidine substituent was selected since it might additionally mimic the ring A of the sterol, the N-allylN-methyl motif was selected, since it can be found in the benzophenone-type OSC inhibitor Ro-48-8071 (Fig. 1), which had recently been co-crystallized with human OSC and shown to bind to the enzyme in a manner like the pro-C-4 HEI A (Figure 2) does [5].
Q13. What is the IC50 value of the inhibitors?
Among the target enzymes, cycloartenol synthase from the plant A. thaliana appeared to be the most sensitive to the inhibitors, with IC50 values ≤1 µM for five of the six compounds assayed (Table 1).
Q14. What is the purpose of this paper?
This paper describes the design, synthesis, and biological evaluation of a series of aminopropylindenes as a new chemotype of oxidosqualene cyclase (OSC) inhibitors.
Q15. What is the IC50 of the inhibitors?
IC50 [M]_________________________________________________________________Cisplatin 5 Ro 48-8071 8 BIBX 79 > 50 4 >100 5 >100 6 >100 7 >100 8 3.5 9 20________________________________________________________________Compounds
Q16. What is the difference between the two species?
The oxidosqualene cyclases of the other species tested are more sensitive to inhibitors with small substituents at the nitrogen, and show no or only week inhibitory effects if bulky residues were chosen.