An alkaloid initiates phosphodiesterase 3A–schlafen 12 dependent apoptosis without affecting the phosphodiesterase activity

The promotion of apoptosis in tumor cells is a popular strategy for developing anti-cancer drugs. Here, we demonstrate that the plant indole alkaloid natural product nauclefine induces apoptosis of diverse cancer cells via a PDE3A-SLFN12 dependent death pathway. Nauclefine binds PDE3A but does not inhibit the PDE3A’s phosphodiesterase activity, thus representing a previously unknown type of PDE3A modulator that can initiate apoptosis without affecting PDE3A’s canonical function. We demonstrate that PDE3A’s H840, Q975, Q1001, and F1004 residues—as well as I105 in SLFN12—are essential for nauclefine-induced PDE3A-SLFN12 interaction and cell death. Extending these molecular insights, we show in vivo that nauclefine inhibits tumor xenograft growth, doing so in a PDE3A- and SLFN12-dependent manner. Thus, beyond demonstrating potent cytotoxic effects of an alkaloid natural product, our study illustrates a potentially side-effect-reducing strategy for targeting PDE3A for anti-cancer therapeutics without affecting its phosphodiesterase activity.

immunoprecipitation and western blotting. Representative data is shown from two independent experiments (with n=3 wells each time) as mean + SD, and the p values from two-tailed unpaired Student's t-tests are indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.
(a and b) mCherry or mCherry-SLFN12 vectors were transfected into SLFN12-KO HeLa cells for 36 hours. Cell morphology was determined using confocal microscopy (n=3 independent experiments) (a) and cell viability was assessed by measuring ATP levels (b) (n=3 independent experiments, with 4 replicates each time). Scale bars, 10 µM. (c and d) Dox (1 µg/mL) was added to drive expression of Flag-SLFN12 cells for the indicated times (c) or the indicated concentrations of Dox were added for 48 hours (d). Cell viability was determined by assessing ATP levels (n=4 wells). Expression of SLFN12 was analyzed using an anti-Flag antibody. experiments (mean + SD), and the p values from two-tailed unpaired Student's t-tests are indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant (e) Nauclefine (Nauc) decreased protein level of Bcl-2 24 hours after treatment in a PDE3A dependent manner. (f) Dox induced expression of SLFN12 decreased protein level of Bcl-2. The western blotting results were repeated twice.
Supplementary Figure 10. HeLa-toxic nauclefine did not affect mice body weight.
(A) HeLa cells stably expressing a luciferase gene. The luciferase substrate was added to facilitate assessment of the number of HeLa-luc cells, and cell luminescence was determined.
(g) Expression of PDE3A was knocked down in MCF-7, EKVX, and H4 cells. Nauclefine (500 nM) was used to treat the MCF-7 and EKVX cells for 36 hours, and to treat the H4 cells for 24 hours. Protein expression was analyzed by western blotting with antibodies against the PDE3A and SLFN12 proteins. GAPDH was used as an internal control. Experiments were repeated The indolizinone natural products nauclefine, angustine, and 20-bromonauclefine were constructed using a common cascade cyclization strategy. This process involves a key hydroamination of an internal alkyne followed by lactamization. The internal alkyne was prepared from the Sonogashira coupling of iodobenzen 19A/B and 2-ethynyl-3-aminoethylindole 19C. The resulting 20-bromonauclefine product from cyclization was transferred to angustine via Stille cross-coupling of a vinyl tin substrate. The subditine (16) was synthesized from oxidative cleavage of the vinyl substrate of 15, which was generated from the Stille crosscoupling of vinyl tin and bromo-methylpyridine 17. This key cyclization intermediate 17 was constructed from intramolecular cyclization of enamide 24, which was the condensation product of 5-bromo-6-methylnicotinoyl chloride 23 and imine 22.

Supplementary note
Nauclefine was synthesized starting from Di-t-butyloxycarboryl protected 2-ethynyl tryptamine. After Sonogashira coupling with methyl 4-iodonicotinate and subsequent deprotection, we obtained the cascade reaction precursor, of which the amino was engaged into the crucial nucleophilic addition to the alkynyl in Cs2CO3/MeOH with excellent regioselectivity (exo-type) followed by concomitant cyclization to generate C ring of nauclefine 3. We also employed the same strategy to accomplish the synthesis of Angustine (14). Prior to the cascade cyclization to afford 20-bromonauclefine (21), the precursor 20B was produced by Sonogashira coupling from 19B and 19C. Finally, the vinyl group was introduced by PPh3-catalyzed Stille coupling to yield Angustine (14) (scheme 1). For the synthesis of subditine, the C ring of the subditine skeleton was established by referring to Lavilla's method30.
As shown in scheme 1 in the supplementary note, the condensation of compound 22 with 5bromo-6-methylnicotinoyl chloride 23 gave 24 accompanied by the isomerization of 1-cyclic olefinic bond, which then underwent 6 electric cyclization at 190 oC in vacuo to afford 17 in 48% yield. Subsequently, we also installed a vinyl group into the pyridine ring by Stille coupling reaction (15), the structure of which was verified by X-ray single crystal diffraction.
Further dihydroxylation and oxidation (in one pot) under strict temperature control yielded the target subditine ( Supplementary Fig.12).
Oxygen-and moisture-sensitive reactions were carried out under nitrogen atmosphere.
Under N2 atmosphere, at 0 oC, to the solution of 23 (250 mg, 1.1 mmol, 1.0 eq.) in CH2Cl2 (10 mL) was added Et3N (6 ml) dropwise, the reaction was then raised to RT and stirred for additional 1 h. After that, 22 (250 mg, 1.34 mmol, 1.2 eq.) was added to the reaction and then stirred at 45 oC for 2 h, which was then evaporated in vacuo and purified by silica gel column chromatography (15% ethyl acetate-petroleum ether) to afford crude 24(230mg, about 50%, decomposed quickly at room temperature)as a yellow solid. Rf = 0.63 (30% ethyl acetatepetroleum ether).